Efficient crop improvement depends on the application of accurate genetic information contained in diverse germplasm resources. Here we report a reference-grade genome of wild soybean accession W05, with a final assembled genome size of 1013.2 Mb and a contig N50 of 3.3 Mb. The analytical power of the W05 genome is demonstrated by several examples. First, we identify an inversion at the locus determining seed coat color during domestication. Second, a translocation event between chromosomes 11 and 13 of some genotypes is shown to interfere with the assignment of QTLs. Third, we find a region containing copy number variations of the Kunitz trypsin inhibitor (KTI) genes. Such findings illustrate the power of this assembly in the analysis of large structural variations in soybean germplasm collections. The wild soybean genome assembly has wide applications in comparative genomic and evolutionary studies, as well as in crop breeding and improvement programs.
Genomic information is the essential foundation of current crop improvement programs. Accurate information is required for effective tracking of genomic variations, mapping important quantitative trait loci (QTLs), and discovering novel alleles. These tasks are intrinsically dependent on the availability of a range of genetic resources and, most crucially, high-quality reference genomes1. Wild germplasm contributes a significant proportion of the genetic resources of major crop species2,3. Although reference genomes are currently available for two soybean cultivars, the Williams 82 (Wm82) genome has been most commonly used for a range of applications4,5. However, these approaches alone have limitations and cannot fully address questions regarding large structural variations or complex genomic rearrangements. A high-quality reference genome from wild soybean is also a crucial tool for use in such studies, because it increases the precision of population genetic analysis of complicated genomes. For example, genomic information is the essential foundation for understanding domestication-related events that involved wild germplasms6,7,8.
We have previously reported whole-genome sequencing data for wild and cultivated soybeans, and demonstrated the high genome diversity in wild soybean populations compared with cultivated soybean9. The genomic diversity of wild soybean has been confirmed and elaborated in reports by ourselves and others6,7,10,11,12,13. Despite several previous attempts at whole-genome assembly in wild soybeans6,7,8, a high-quality reference genome has remained elusive. In resolution of this important issue, we report here a high-quality genome for the wild soybean accession W05. W05 has previously been employed to identify several agronomically important QTLs, together with the identification of the causal gene conferring salt tolerance in wild soybean8,14. In this study, we not only demonstrate the power of W05 reference genome but also highlight its applicatibity in a wide range of comparative genomic and evolutionary studies, using a range of examples, such as the identification of large structural variations, QTLs, genes, and alleles. The advantages of combining high-quality reference genomes and optical mapping (OM) in studying structural variations among multiple accessions is also described.
State-of-the-art whole-genome sequencing technologies were used to assemble a high-quality reference genome for wild soybean accession W05 with long contigs and high sequence fidelity. PacBio subreads (85.5 Gb) were error-corrected and de novo assembled into primary contigs (Supplementary Figure 1a). Sequences were then polished with PacBio subreads and Illumina paired-end reads (101.3 Gb) (Supplementary Figure 1b, Supplementary Table 1). The polished contigs are 989.7 Mb in length and are composed of 2281 sequences with a contig N50 (50% of the genome covered by contigs above this length) of 2.0 Mb. Details for assembly procedures can be found in Methods section.
To anchor polished contigs onto chromosomes with high accuracy, two complementary technologies: OM (Supplementary Table 2) and Hi-C sequencing (Supplementary Table 1) were employed. Based on the optical contigs generated with the nickases Nt.BspQI and Nb.BssSI (Supplementary Figure 1c), two-enzyme hybrid scaffolding was performed to generate OM-sequence hybrid scaffolds (hybrid scaffolds) (Supplementary Figure 1d). The hybrid scaffolds comprised 1438 sequences with a total length of 1019.8 Mb and a scaffold N50 of 13.9 Mb (Supplementary Figure 1d). In addition, Hi-C contact frequency derived from Hi-C sequencing was used to order and orient the polished contigs into Hi-C scaffolds (Supplementary Figure 1e). The resulting Hi-C scaffolds comprised 1161 sequences, with a total length of 989.8 Mb and a scaffold N50 of 48.5 Mb (Supplementary Figure 1e). Superscaffolds were generated by merging hybrid scaffolds and Hi-C scaffolds (Supplementary Figure 1f).
After gap filling and polishing, the final assembly for W05 is 1013.2 Mb in length, with 988.6 Mb unambiguous bases (Supplementary Figure 1g and Table 1). In total, 95.7% of sequences are anchored to 20 superscaffolds, corresponding to 20 chromosomes, whereas 43.6 Mb in 1098 contigs remain unplaced. The contig N50 of the final assembly is 3.3 Mb (Table 1). The longest contig of the W05 final assembly is 23.2 Mb in length, spanning 47.7% of chromosome 6. The contiguity of the W05 assembly is approximately a 17-fold improvement over the current reference genome Wm82_v2 and of similar quality as the recently published Chinese cultivated soybean reference genome of ZH135 (Supplementary Table 3). Contig N50, scaffold N50, and total assembled genome size of other soybean genome assemblies6,7,11,15 were compared (Supplementary Table 3), but these genomes were not included in subsequent analysis, because they are highly fragmented.
The completeness of this genome assembly was examined using the Benchmarking Universal Single-Copy Orthologs (BUSCO) evaluation score16. The completeness of W05 is comparable to the Wm82_v2 and ZH13 reference genomes (Supplementary Table 4). The mapping rate of the PacBio Isoform Sequencing (IsoSeq) full-length transcripts was 97.7% (Supplementary Table 5). Cent91/92 soybean-specific centromeric repeats17 were found in 19 chromosomes, except chromosome 1 (Fig. 1e). In addition, the Arabidopsis-type telomeric tandem repeat array18 (CCCTAAA/TTTAGGG repeats) was found at both distal ends for 13 chromosomes and at a single distal end for the remaining 7 chromosomes (Fig. 1e and Supplementary Table 6). In contrast, only 9 and 7 chromosomes of Wm82_v2 and ZH13, respectively, contain telomeric tandem repeats at both distal ends. The completeness of telomeres in the W05 genome is therefore improved compared with that of Wm82_v2 and ZH13 (Supplementary Table 6). GC content along each chromosome was calculated (Fig. 1c). Furthermore, using in silico PCR, 874 conserved, unique-site soybean simple sequence repeat markers were mapped to the W05 genome, which are evenly distributed on the 20 chromosomes (Fig. 1d and Supplementary Data 1).
Protein-coding genes and alternative spliced isoforms were annotated by combining evidence generated from RNA-seq/PacBio IsoSeq transcript mapping, homology-based protein mapping, and ab initio prediction. In total, 234.7 Gb of Illumina RNA-seq reads were collected from 31 samples at various development and physiological stages (Supplementary Data 2). PacBio IsoSeq libraries were constructed in order to generate 414,750 full-length and non-chimeric transcripts (Supplementary Table 7). In total, 89,477 protein-coding transcripts were annotated for 55,539 gene loci, with 69,455 transcripts (77.6%) having 5′-untranslated region (UTR) and 71,271 transcripts (79.7%) having 3′-UTR (Supplementary Table 8). In addition, 82,567 transcripts (92.3%) encode proteins that contain predicted functional domains (Table 1). Features of the annotated transcripts in W05 are similar to those of Wm82_v2 and ZH134,5 (Fig. 1b and Supplementary Table 8). BUSCO evaluation shows that completeness of the W05 annotated gene set was comparable to that of Wm82_v2 and ZH13 (Supplementary Table 9). A total of 288 microRNAs (miRNA), 1988 small nuclear RNAs (snRNA), and 147 ribosomal RNAs (rRNA) were identified in the wild soybean genome (Table 1). In addition, 892 transfer RNAs (tRNA) were identified, representing anti-codons for all 20 types of amino acids.
One major application of the wild soybean reference genome is the identification of QTLs, genes, and alleles. We previously constructed a recombinant inbred (RI) population by crossing W05 to a cultivated soybean Union (C08)8, which shared the same recurrent parent with Wm82. To demonstrate that W05 can be used effectively as a reference genome for QTL mapping, we make use of our previous published phenotypic data together with new data of seed size and sequencing reads of 96 core RI lines8 to construct binmaps, and map QTLs using W05 genome or Wm82_v2 genome as reference. The relative genomic location and span of QTLs are comparable when either genome was used as the reference, with very few discrepancies (Supplementary Data 3). For example, the growth period QTL on chromosome 11 spans a 3.55 Mb region in the Wm82_v2 genome, but only 500 Kb in the W05 genome. This may be due to the low-quality assembly of Wm82_v2 in this region, as an ~3.8 Mb sequence within this region, which was originally present in Wm82_v1, was not anchored to the chromosome in Wm82_v2 (Supplementary Data 3).
The quality of the assembly down to the nucleotide level was assessed by examining known traits associated genes. For Ncl8,19, Rj2/Rfg120, and G21,22 loci, the alleles in W05 match the observed phenotypes (Table 2, Supplementary Data 3 and 4). In addition, we have also identified known polymorphisms and additional alleles of the causal genes in the QTLs controlling growth period, flower color, seed coat color, and pubescence color in W05 (Table 2, Supplementary Data 3, 4).
To investigate a more complex case, we examined the I locus on chromosome 8, which determines the pigmentation of the seed coat23, a major trait that was selected during domestication21. It was reported that the dominant allele in cultivated soybeans contains an inverted repeat of the chalcone synthase (CHS) gene cluster, which triggers posttranscriptional gene silencing (PTGS) and inhibits the expression of CHS gene family members in the seed coat; hence, resulting in colorless seed coat and yellow seeds24. Deletion that disrupts the inverted repeat CHS gene cluster in a revertant soybean accession resulted in seed coat color transition from colorless to pigmented23.
W05 has a pigmented seed coat, whereas C08 has a colorless seed coat. A seed coat color QTL that overlaps with the known I locus was mapped (Supplementary Data 3). The W05 reference genome possesses the same inverted repeat of the CHS gene cluster as Wm82 (Fig. 2a), indicating that the inverted repeat is not sufficient to explain the seed coat color change during domestication.
To identify the genetic variation that caused the seed coat color change during domestication, the W05 genome was compared with the two published bacterial artificial chromosomes of Wm82 that were previously used in I locus studies23,25 (the I locus region is poorly assembled in Wm82_v2), as well as the recently published ZH13 genome5. This comparative analysis reveals a complex structural rearrangement next to the CHS gene cluster in Wm82 and ZH13, which includes both inversion and gene duplication (Fig. 2a and Supplementary Figure 2). The inversion brought the promoter and the first four exons of a subtilisin gene (Glysoja.08G020214 in W05) to a position next to the CHS gene cluster in Wm82 and ZH13 (Fig. 2a and Supplementary Figure 2). This finding indicates that the subtilisin promoter may drive the expression of a chimeric transcript that reads through the subtilisin gene fragment and anti-CHS1 gene region to cause PTGS of the CHS genes. This is in agreement with the previous speculation based on Expressed Sequence Tag data26. To validate this hypothesis, the subtilisin-anti-CHS1 chimeric transcript was successfully amplified with PCR primers specific to both the subtilisin gene fragment and the anti-CHS1 genes using complementary DNA from developing seed coat of Wm82 in a strand-specific manner (Fig. 2b). The subtilisin-anti-CHS1 chimeric transcript was able to form double-stranded RNA with the sense CHS mRNAs, hence causing PTGS to inhibit the expression of CHS genes (Fig. 2c). Deletion of the CHS gene cluster B adjacent to the subtilisin gene fragment will disrupt PTGS and lead to the transition from a colorless seed coat to a pigmented seed coat, which is in agreement with the previous report23 (Fig. 2c).
Transposable elements (TEs) are repeated DNA sequences that make up of a significant proportion of plant genomes27. They are important source of variations for natural and artificial selection. Combining homology-based searches and de novo prediction, we identified 546.4 Mb of repeat elements (53.9% of the total assembled W05 genome) (Table 1, Fig. 1a). The most abundant type of TEs in the W05 genome is the long terminal repeat (LTR) retrotransposon element class, comprising 34.1% of the genome. The predominant LTR type is LTR/gypsy family.
Through whole-genome sequence comparisons of W05 with Wm82_v2 and ZH13, we have identified ~2300–3000 TE insertions for each accession (Supplementary Table 10). To identify Wm82- or ZH13-specific TE insertions, W05 was used as the reference genome. To identify W05-specific TE insertions, Wm82_v2 or ZH13 was used as the reference. In total, 361 and 350 W05 genes were found to contain TE insertions in Wm82_v2 and ZH13 genome, respectively. In contrast, 419 and 400 genes from Wm82_v2 and ZH13, respectively, were found to have TE insertions in W05 genome.
Gene Ontology (GO) enrichment analysis identified 29 and 8 enriched GO terms among the Wm82_v2 and ZH13 TE-affected genes, respectively. The enriched GO terms in both genomes mainly fall into the category of biological processes related to metabolism (Supplementary Data 5). Both sets contain GO:0044238 (primary metabolic process) and GO:0043170 (macromolecule metabolic process). For W05-specific TE-affected genes, there are three and nine GO terms enriched relative to ZH13 and Wm82, respectively. Unlike the two cultivated soybean references, the enriched GO terms mainly fall into the category of binding-related molecular functions (Supplementary Data 5) including GO:0036094 (small molecule binding), GO:0000166 (nucleotide binding), and GO:1901265 (nucleoside phosphate binding).
High-quality reference genomes also allow confident genome-wide detection of large structural variations, which cannot be achieved unambiguously solely by re-sequencing analysis. When comparing the genome sequence of W05 with Wm82_v2 and ZH13, good chromosome-to-chromosome collinearity relationships were found (Supplementary Figure 3). However, we also identified large structural variations ( >100 Kb inversions, intra-chromosomal translocations, and inter-chromosomal translocations) among the reference genomes (Supplementary Table 11). Compared with W05, there are 32 and 12 large structural variations in Wm82_v2 and ZH13, respectively (Supplementary Table 11). Nine of these variations are shared between Wm82_v2 and ZH13.
The largest structural variation in W05 relative to Wm82 and ZH13 is the inter-chromosomal reciprocal translocation in W05 between chromosomes 11 and 13. Previously, this translocation was detected in some Glycine soja accessions by fluorescent in situ hybridization (FISH)28. Analysis of the assembled genomes reveals this translocation event in W05 in comparison with Wm82 and ZH13 (Supplementary Figures 3 and 4). The structure of W05 chromosomes 11 and 13 is supported by Hi-C contact frequency matrix data, which shows higher intra-chromosomal contact frequency than inter-chromosomal contact frequency (Supplementary Figure 5). Consistent translocation breakpoints were identified when comparing W05 with ZH13 or Wm82_v1 but not with Wm82_v2 (Supplementary Figure 4). The discrepancy is most likely to be due to mis-assembly of Wm82_v2 in this region. Sequence comparisons revealed that the translocation breakpoint is located around 34.38 Mb on Chr11 and 27.06 Mb on Chr13 in the W05 genome (Supplementary Figure 4).
We also found that the QTLs controlling trailing growth, seed number per plant, and pod number per plant are located within the translocated regions (Supplementary Data 3). Therefore, the casual gene(s) are located on chromosome 13 in Wm82 and on chromosome 11 in W05 and some other soybeans (see below). If only Wm82 was used as the reference genome, these QTLs will be mis-placed to chromosome 13 in W05. Therefore, we have provided an important information for marker-assisted breeding or map-based cloning of the casual gene(s) for these QTLs.
Although comparisons of the W05 and other reference genomes allows identification of many structural variations, these variations could be accession-specific. Therefore, we expanded our scope to analyze more soybean accessions from diverse origins, including cultivated soybeans popularized in China, the United States, and Japan, as well as wild soybeans originating from China and Korea (Supplementary Data 6). OM technology that can be used to study long-range genomic DNA up to 1 Mb in length was employed together with an in silico map based on our high-quality W05 genome to detect structural variations in diverse soybean accessions. We can thus demonstrate that the W05 reference genome enhances genome comparisons at the kilobase scale. As proof-of-concept, several loci exhibiting different structural features among soybean accessions were investigated, including inversion in the I locus, the translocation between chromosomes 11 and 13, a previously reported region specific to cultivated soybean on chromosome 157, and gene copy number variation in a Kunitz trypsin inhibitor (KTI) gene cluster. Only soybean accessions with sufficient coverage and depth of optical contigs in the target regions were employed for comparative analysis (Supplementary Data 6).
For the I locus, comparative analysis revealed that soybean accessions exhibiting a pigmented seed coat have no inversion (similar to W05), whereas those with colorless seed coats share the same inversion as Wm82 and ZH13 (Fig. 3a). To further confirm the OM results, the inversion junction that creates the subtilisin-anti-CHS1 chimeric transcript was amplified from genomic DNA of different soybean accessions by PCR. Consistent with the OM results, inversion junction could be amplified from all soybean accessions with colorless seed coats but not from those with pigmented seed coats (Supplementary Figure 6).
Using Wm82_v1, ZH13, and W05 as references, the translocation event between chromosomes 11 and 13 were also detected. One (W01) out of three (W02, K02) wild soybean accessions share the same chromosome topology with W05. In contrast, all cultivated soybean accessions shared the same topology with the Wm82_v1 and ZH13 in silico maps, regardless of their origin whether they were popularized in China, the United States, or Japan (Fig. 3b). Furthermore, the regions at the junction of the translocation are reasonably well conserved among different accessions, which suggests that the translocation was a single evolutionary event.
Previously, a region specific to cultivated soybean (Gm15 46.0–46.5 Mb) was identified in a pan-genome study by mapping short reads of seven wild soybeans onto the Wm82_v1 genome7. We revisited this region and compared the optical contigs of different soybean accessions. Interestingly, the targeted region appears to have diversified among the different cultivated accessions that we have tested from China, the United States, and Japan, whereas only Wm82 possesses such a large insertion (Fig. 3c). This previously reported Glycine max-specific region was confirmed as Wm82 specific.
Comparisons of multiple reference genomes, together with the OM data, allow identification of gene copy number variations. KTI are anti-nutritional factors and are hence not desirable traits for selection in breeding purposes. Marker-assisted breeding has been used to introduce a null allele of KTI from an exotic soybean germplasm into two commercialized lines29. A KTI gene cluster was identified on chromosome 8 of W05 that contains 17 KTI genes. The optical signaling patterns in this region are similar for cultivated soybean accessions and diversify for wild soybean accessions. Wild soybean accessions W05, W02, and K01 possess long fragments, whereas W01 and all of the cultivated accessions tested (including Wm82) exhibited reduced size in this genomic region (Fig. 3d). In the annotated Wm82_v2 and ZH13 reference genomes, there are only 13 and 11 KTI genes, respectively, in this region (Supplementary Figure 7), implying that copy number variations of KTI genes might have occurred through artificial selection.
The availability of multiple high-quality reference genomes from diverse genetic backgrounds is a prerequisite for effective mining of crop genomes30, especially in studies involving wild germplasm. The current Wm82_v2 reference genome alone cannot provide conclusive answers to questions regarding large structural variations and complex genomic rearrangement. A new version of Wm82 genome is under construction (www.soybase.org), which will improve the quality over the current version. However, comparative genomic analysis using only the reference genome of Wm82 may still fail to uncover wild-specific genetic variations, as these may have been lost during domestication or artificial selection7. Here we report the assembly of a high-quality reference genome of a wild soybean accession W05, which has many distinguishing features compared with the two existing reference genomes of cultivated soybeans (see Results).
Wild soybeans exhibit agronomic traits that are different to cultivated soybean (e.g., smaller seed size, higher pod number, and seed number per plants, etc.). We have identified QTLs related to key yield components. A seed size QTL was reported and the proper chromosomal locations of the QTLs regulating trailing growth, pod number per plant, and seed number per plant were assigned. These findings demonstrate the value of the W05 reference genome. The combination of this genomic information and a genetic population that includes wild soybeans will result in the step change required for future soybean breeding.
Several examples demonstrate the unique strength of the W05 reference genome. In partcular, an inversion associated with seed coat color was identified at the I locus by comparing W05 with Wm82 and ZH13 genomes. The PCR results and OM data presented here demonstrate that the inversion is the source of PTGS (Figs 2, 3a). Furthermore, major structural changes were identified between the W05 genome and those of two cultivated soybeans. One example is the reciprocal translocation between chromosomes 11 and 13 (Supplementary Figures 3 and 4, Supplementary Table 11). Our findings successfully identify the translocation breakpoints, refining previous results obtained using FISH technology28. Together with the high base-to-base fidelity, the W05 genome provides an important tool for future investigations of the variations in genes and alleles in wild soybeans, which may not exist in modern cultivars due to domestication bottlenecks and poorly informed selection criteria9,10,11,12,13.
TEs are major drivers of plant evolution27. They occupy more than half of the genome sequences of many crop species27. Accession-specific TE insertion events can be effectively identified by comparing de novo genome assembles, as repeated regions could cause ambiguities in re-sequencing approaches. Comparisons of the W05 genome with those of two cultivated soybeans revealed that genes with TE insertions that are found in the two cultivated soybean genomes are concentrated in metabolic pathways (Supplementary Data 5). Conversely, similar patterns were not observed in W05-specific TE-affected genes. As metabolic changes lead to variations that are important for soybean production and quality, we may speculate that TE-affected metabolic genes were unintentionally selected during domestication. However, more wild and domesticated germplasm should be tested to further explore this hypothesis. The highlighted importance of variations in metabolic genes during domestication is consistent with our previous observations showing that fixed single-nuclotide polymorphisms (SNPs) are concentrated in metabolic genes in the cultivated soybeans, compared with wild soybeans31.
The data presented here also demonstrate the value of the W05 reference genome and the optical contigs of other soybean accessions. Together, these results provide a powerful tool that can be used to uncover large structural variations in soybean germplasm collections (Fig. 3). We present evidence showing that the inversion found in the I locus (Fig. 3a) and the translocation between Chromosomes 11 and 13 (Fig. 3b) are events that are found in multiple soybean genomes. Moreover, such comparisons reveal accession-specific genomic regions, e.g., a region on chromosome 15 that was previously reported as a feature of the cultivated soybean7 is in fact unique to Wm82 (Fig. 3c).
The strategy of mapping optical contigs to high-quality reference genomes will have a significant impact in soybean breeding programs. For example, new cultivars generated from radiation-driven mutations often contain major structural genomic changes and these structural variations could be effectively identified using this approach. This strategy also provides an elegant means to detect copy number variations, which are the basis of a number of phenotypic traits in humans, animals, and plants32. We specifically investigated a region containing multiple trypsin inhibitor genes as an example of how the genome can be interrogated to identify genes that influence seed nutritional quality. Trypsin inhibitors are considered to have evolved as a protective measure against herbivores and bacterial infections. Trypsin inhibitor proteins account for 2–6% total protein of soybean seeds, which is the highest value found in a range of legumes33, reducing the nutritional value of the beans as food and/or animal feed. The genomic analysis reported here reveals that a genomic region on chromosome 8, which presumably contains a KTI gene cluster, has shrunk in cultivated soybeans from China and other countries compared with most of the wild soybeans from China and Korea (Fig. 3d). This finding suggests that domestication may have involved selection for a reduced copy number of KTI genes.
In summary, this study provides information regarding the wild soybean that cannot be easily inferred from the reference genomes of the cultivated soybeans. As wild accessions are important genetic resources for crop improvement, the wild soybean genome reported here will be a valuable, if not indispensable, tool for use in a wide range of applications by legume researchers for comparative genomic and evolutionary studies, and soybean breeders for crop improvement programs.
W05 is a Chinese wild soybean accession originally collected in Henan Province. Union (C08) is a cultivated soybean bred in the United States by crossing Williams and SL12, and then backcrossing five times with Williams. W05 and C08 was used to generate a RI population for the identification of important QTLs and the causal gene of a major QTL for salt tolerance8,9,14. Information of other soybean accessions used in this study is presented in Supplementary Data 6.
For Illumina sequencing, seeds of W05 were germinated on 0.8% water agar in sterile magenta box at 28 °C in dark. Hypocotyls and radicals were collected 3–4 days after germination and snap frozen in liquid nitrogen. DNA was extracted from the hypocotyls and radicals of young W05 seedlings using DNeasy Plant Mini Kit (Qiagen, Hilden, Germany, Cat. 69104). The DNA sample was sent to BGI-Shenzhen (Shenzhen, China) for library construction and sequencing on the Illumina HiSeq2000 platform (Illumina, San Diego, CA).
For Pacbio sequencing, W05 was grown in greenhouse on regular soil. Trifoliate leaves were collected and snap frozen in liquid nitrogen. Nuclei were isolated from trifoliate leaves of W05 following a published protocol34 with minor modification. Frozen leaves were ground in liquid nitrogen. The powder was suspended in 200 mL nuclei isolation buffer (10 mM Tris HCl pH9.5, 10 mM EDTA, 100 mM KCl, 500 mM sucrose, 4 mM spermidine, 1 mM spermine, and 0.2% (v/v) β-mercaptoethanol) with 0.6% (v/v) Triton X-100 and filtered through 41 nm and 20 nm nylon mesh sequentially. The lysate was centrifuged at 1200 × g for 10 min at 4 °C to collect the nuclei. The nuclei was washed twice with nuclei isolation buffer with Triton X-100 and once with nuclei isolation buffer only. Nuclei DNA was extracted using modified cetyltrimethylammonium bromide (CTAB) method35. The DNA sample was sent to BGI-Shenzhen and Wuhan Institute of Biotechnology (Wuhan, HuBei, China) for library construction and sequencing. In total, 6 SMRTbell libraries with size selection using BluePippin (Sage Science, Beverly, MA) were constructed and sequenced using 72 SMRT cells with P6-C4 chemistry on PacBio RS II platform (Pacific Biosciences, Menlo Park, CA).
The Dovetail Hi-C library was prepared and sequenced by Dovetail Genomics (Santa Cruz, CA)36. Briefly, chromatin in the nucleus of soybean young seedlings was fixed with formaldehyde and extracted. Fixed chromatin was digested with DpnII and sticky ends were filled in with biotinylated nucleotides and ligated. Crosslinks were then reversed and DNA was purified. Purified DNA was treated to remove biotin that was not internal to ligated fragments. The DNA was then sheared to ~350 bp and sequencing libraries were constructed using NEBNext® Ultra™ DNA Library Prep Kit (Illumina, Cat. E7370S). Biotin-containing fragments were enriched through streptavidin bead pulldown before PCR amplification of the library. The library was sequenced on Illumina HiSeq X platform (Illumina).
For Bionano OM, young leaves of all the soybean germplasm used in this study were collected from 7- to 10-day-old seedlings grown in greenhouse and high-molecular weight (HMW) DNA was extracted following the Bionano IrysPrep® High Polysaccharides Plant Tissue DNA Isolation User Guide (Bionano Document Number: 30128). Extracted HMW DNA molecules were fluorescently stained using Nick-Label-Repair-Stain (NLRS) enzymatic reactions following the Bionano Prep™ Labeling - NLRS Protocol (Bionano Document Number: 30024). Briefly, single-strand breaks were introduced to DNA molecules by nicking enzyme Nb.BssSI or Nt.BspQI to generate sequence motif-specific patterns. Nicked sites were labeled with fluorescent nucleotides and repaired. Molecule backbones were also fluorescently stained with YOYO-1 to visualize the full lengths.
NLRS reaction products were then run on Bionano Saphyr (Bionano Genomics, San Diego, CA) for W05 or Irys system (Bionano Genomics) for the rest of the soybean germplasms, where DNA molecules were automatically stretched and imaged within nanochannel arrays. Distances between fluorescently labeled nicking sites form patterns as basis for alignment and assembly by Bionano AutoDetect software (v2.1.4).
Embryos of W05 were collected 24 h after germination in distilled water, in a dark incubator. Cotyledons and hypocotyl were collected 3 days, whereas root, apical buds, and stems were collected 10 days after sowing in vermiculite with 70% water content and growing in a greenhouse. Flower, 7-day pods, 14-day pods, 14-day seeds, 40-day pods, and 40-day seeds were collected at reproductive stages from soybean plant grown in the greenhouse. Total RNA was extracted using RNAiso Plus reagent (TaKaRa, Kyoto, Japan, Cat. 9108). Nodules and remaining roots were collected 28 days post inoculation with Sinorhizobium fredii strain CCBAU4543637. Total RNA was extracted from nodules and the remaining roots were extracted using TRIzol® reagent (Thermo Fisher Scientific, Waltham, MA, Cat. 15596018).
RNA samples were sent out to BGI-Shenzhen for both RNA sequencing (RNA-seq) and PacBio IsoSeq. For RNA-seq, stranded RNA-seq libraries were constructed for each RNA sample and sequenced on Illumina HiSeq4000 platform (Illumina). For PacBio IsoSeq, equal amount of RNA from different tissues were pooled. Four SMRTbell libraries were constructed with size selection of 1–2 Kb, 2–3 Kb, 3–6 Kb, and 5–10 Kb using the BluePippin (Sage Science), and sequenced with 4, 4, 2, 2 SMRT cells with P6-C4 chemistry on PacBio RS II platform (Pacific Biosciences).
Before de novo assembly, low-quality PacBio subreads with a read length shorter than 500 bp or a quality score lower than 0.8 were filtered out. The remaining clean PacBio subreads were error-corrected and assembled into contigs with MECAT38 (version 1.0). The MECAT de novo assembler is composed of three major steps: (i) mecat2pw: all vs. all alignment between PacBio subreads were performed; (ii) mecat2cns: high-quality consensus reads were generated with parameter setting: -a 2000 -c 4 -l 2000; and (iii) mecat2canu: 40 × longest high-quality consensus reads were assembled into primary contigs with parameter setting: genomeSize = 1100000000 ErrorRate = 0.013.
All clean PacBio subreads were mapped to the assembled contigs using pbalign (SMRTLink package release 4.0.0.190159). The primary contigs were polished with mapped PacBio subreads using Quiver implementation in variantCaller (SMRTLink package release 4.0.0.190159).
Illumina short reads with insert size of 250, 500, and 800 bp were used to correct residual errors in the polished contigs. Reads contain adapter sequences or 5% Ns, or with low quality, or derived from PCR artifacts were filtered.
Remaining clean reads were mapped to polished contigs using Burrows-Wheeler Aligner (BWA) mem39 (version 0.7.15-r1142) with default parameters. Duplicated reads were tagged using Picard MarkDuplicates implementation (https://broadinstitute.github.io/picard/, version 2.9.0–1-gf5b9f50), with default parameters. Residual errors in the polished contigs were corrected with mapped next generation sequencing (NGS) reads using Pilon40 (version 1.22), with the following parameter setting: –diploid–fix snps,indels,local. Polished contigs were used as input for scaffolding analysis.
For scaffolding, HiRiseTM pipeline (Dovetail Genomics) was used for scaffolding41. Hi-C reads and NGS clean reads with insert size of 250, 500, and 800 bp were mapped to polished contigs using modified Scalable Nucleotide Alignment Program (SNAP) mapper (http://snap.cs.berkeley.edu). Repeats in the contigs were masked, based on NGS read mapping depth. The order and orientation of contigs within Hi-C scaffolds were determined based on contact frequency calculated from mapped Hi-C read pairs.
Low-quality optical molecules with molecule length < 150 Kb or molecule signal intensity > 0.6, or molecule label number < 9 were removed. Optical molecules digested with Nt.BspQI or Nb.BssSI were de novo assembled into optical contigs using Bionano SolveTM (v3.0.1 release v06082017, parameter settings: Iteration:5; minlen:150 kb; minsites:9; initialAssembly: 1.00E − 10; extendRefine: 1.00E − 11; merge: 1.00E − 15). Two-enzyme hybrid scaffolding were performed using Bionano SolveTM (v3.0.1 release v06082017, default parameters) with polished contig sequences and two set of de novo assembled optical contigs as input to generate hybrid scaffolds.
Superscaffolds were generated by merging Hi-C scaffolds and hybrid scaffolds. Linkages between adjacent contigs within superscaffolds were classified into three categories based on supporting evidence: (i) both: linkage supported by both Hi-C scaffolds and hybrid scaffolds; (ii) map: linkage supported only by hybrid scaffolds; and (iii) Hi-C: linkage supported only by Hi-C scaffolds.
To fill and close gaps, PacBio subreads were mapped to superscaffolds using BLASR42 (version 1.3.1.142244). Gaps within the superscaffolds were filled with consensus sequences generated from PacBio subreads that span or flank gaps using PBJelly243 (PBSuite_15.8.24). After gap filling, another round of polishing was performed with PacBio subreads and Illumina short reads to eliminate sequence errors in the filled sequences.
Gaps within the superscaffolds were closed by searching for gap-flanking sequences that were identical to each other in a head-to-tail way using BLAST44 (version 2.2.31, default parameter). Illumina mate-pair reads with insert size of 2, 6, and 10 Kb, and optical molecules were used to distinguish redundant sequences from true tandem repeated sequences. Illumina mate-pair reads with different insert sizes were mapped to superscaffolds with BWA45 aln implementation (version 0.7.15-r1142, parameter settings: -a 50000). Optical molecules were aligned to the in silico digested map of superscaffolds with OMBlast46 (version 1.4a) and visualized with OMTools47 (version 1.4a). For gaps with identical flanking sequences, insert size for mate-pair reads and signal pattern for optical molecules that span or flank these gaps were manually inspected, to identify redundant sequences that were then trimmed to close the gaps. Another round of sequence polishing was performed, with Illumina short reads to eliminate residual errors.
The gene completeness of soybean assemblies was evaluated at the contig level using both the PacBio IsoSeq full-length reads and the 1440 conserved BUSCOs. PacBio IsoSeq full-length reads were mapped to genome assemblies using BLAT48 (version 35) with default parameters. Only one best hit was retained for each query sequence. The BLAT alignment hits were further filtered based on mapping identity ( ≥95%) and query coverage (≥50% or ≥90%). BUSCO16 (version 3.0.2, lineage dataset embryophyta_odb9) was used to identify conserved BUSCO genes in the genome assemblies. Telomeric repeats were identified by tandem repeat finder (TRF)49 (version 4.0.4). Centromeric repeats were identified by BLAST44 (version 2.2.31, E-value < 1e − 5) search Cent91/92 sequences against the genome sequence17. Primer sequences of soybean genetic map were downloaded from SoyBase (https://www.soybase.org/dlpages/#geneticmap). In silico PCR was performed using isPCR (https://github.com/bowhan/kent/tree/master/src/isPcr/isPcr), e-PCR50 (version 2.3.9, parameters: D = 30–1000 N = 2 G = 2 T = 4), and BLAST44 (version 2.2.31, E-value < 1e − 5). Only primer pairs that mapped to the genome with proper orientation (forward-reverse or reverse-forward) and proper insert size (< 1000 bp) were retained. Primer pairs that have multiple best hits in the genome were filtered.
Tandem repeats in the genome assembly was identified using TRF49 (version 4.0.4). Well-characterized TEs were identified by searching against W05 genome assembly at DNA level and protein level using RepeatMasker (version 4.0.7) (http://www.repeatmasker.org/) and ProteinRepeatMask (version 4.0.7) (http://www.repeatmasker.org/) with RepBase51 (release 20.04) as the query library. To identify TEs that were absent in the RepBase library, a de novo repeat library was constructed using Repeatmodeler (version 1.0.10) (http://www.repeatmasker.org/). RepeatMasker was run against the genome assembly again, with de novo repeat library as the query library.
For homology-based evidence generation, the sequences of proteins from Arabidopsis thaliana (araport11), Lotus japonicas (3.0, ftp://ftp.kazusa.or.jp/pub/lotus/lotus_r3.0/), G. max (Phytozome release 12), Vitis vinifera (Phytozome release 12), Medicago truncatula (Phytozome release 12), Prunus persica (Phytozome release 12), and Populus trichocarpa (Phytozome release 12) were downloaded. For each species, only the longest protein sequences were retained as the representative for each gene locus. The representative protein sequences were mapped to G. soja W05 genome using splice-site-aware aligner Exonerate52 (version 2.4.0) with the following parameters: –model protein2genome–showalignment–showtargetgff–refine region. Only the best alignment with the highest score was retained for each mapped gene locus for each species.
For expression-based evidence generation, low-quality RNA-seq reads were filtered and trimmed using trim_galore (version 0.4.1) (http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/). Trimmed reads with read length shorter than 80 bp were discarded. Stranded and non-stranded RNA-seq reads were assembled into stranded and non-stranded unigenes using Trinity53 (version 2.4.0) with default parameters, respectively. The assembled stranded and non-stranded unigenes were mapped to genome sequences and assembled into non-redundant transcripts using PASApipeline54 (version 2.2.0) with the following parameter setting: –MAX_INTRON_LENGTH 20000. Furthermore, consensus PacBio IsoSeq subreads were extracted, classified, and clustered to generate corrected consensus reads using SMRTanalysis package (smrtanalysis_2.3.0.140936). Corrected consensus sequences with low-quality score were further corrected with stranded RNA-seq reads using proovread55 (version 2.13.12, parameter setting: –mode sr). After correction, the consensus sequences were mapped to the genome assembly and assembled into non-redundant transcripts using PASApipeline54 (version 2.2.0) with parameters: –MAX_INTRON_LENGTH 20000.
For evidence synthesis and alternative spliced isoform annotation, transcripts that were well supported by both homology-based evidence and expression-based evidence were selected for parameter training for ab initio prediction software Augustus56 (version 3.2.3). All the generated transcript evidences mentioned above and trained Augustus parameters were fed into Maker57 (version 2.31.9) for evidence synthesis to generate primary protein-coding gene set. The primary gene set and expression-data-based evidence were fed into PASApipeline54 (version 2.2.0) for gene structure refinement and alternative spliced isoform annotation.
Potential function was assigned to each annotated protein sequence using InterProScan58 (version 5.29–68.0) by searching all available databases with corresponding utilities. Completeness of the annotated gene set was evaluated by BUSCO16 (version 3.0.2, lineage dataset embryophyta_odb9).
rRNA sequences of G. max were downloaded from NCBI GenBank database (GenBank ID: X15199.1, AJ009787.1, X02623.1, and AH001766.2). These rRNA sequences were mapped to G. soja W05 genome assembly using BLAST44 (version 2.2.31) with default parameters.
tRNAScan-SE59 (version 1.3.1) was used to search the G. soja W05 genome assembly for tRNAs genes with default parameters. Annotated tRNA genes that were classified as Pseudo were filtered.
To identify miRNA and snRNA genes, infernal60 (version 1.1.2, parameters: –cut_ga–rfam–nohmmonly) was used to search the G. soja W05 genome assembly based on covariance models deposited in Rfam61 database (release 13.0).
Binmap construction and QTL identification was performed with population sequencing and phenotype datasets generated in our previous studies8 using a slightly modified analysis pipeline. Briefly, short reads from the 96 RI lines were mapped to the genome sequence using BWA45 (version 0.7.15-r1140) with default parameters. Properly and uniquely mapped reads were extracted to identify SNPs using Samtools62 (version 1.2) with parameter setting: SNP quality value > 30 and < 3 SNPs were allowed in any 10 bp window. Heterozygous or non- parental line-derived SNPs were filtered. At least 20 RI lines were required to have SNPs at each SNP locus8. Recombination breakpoints were identified using a modified sliding window approach (window size: 15 SNPs, step size: 1 SNP)63. Adjacent 50 Kb intervals were merged into bins if no recombination events were identified from sequenced RI lines. Genetic distance among bins was calculated using R/qtl package64 (version 1.41.6, default parameters). QTLs for each agronomical trait were identified using QTL Cartographer (http://statgen.ncsu.edu/qtlcart/) (version 1.17j) with a 10 cM scanning window and a 0.5 cM step size. For each agronomical trait, logarithm of the odds (LOD) cutoff was determined by 1000 permutation of the phenotypes and genotypes with significance level of α < 0.05 using QTL Cartographer. Peaks having LOD value higher than the LOD cutoff were considered as significant. For green seed phenotype dataset, only RI lines of the green and yellow seeds were used. For seed size phenotype dataset, about 35 seeds from each RI line were measured using SmartGrain65 (version 1.1).
For the same QTL for each agronomical trait, sequence alignment was performed between two QTL regions (with referenced to different genomes) using the nucmer program from MUMmer package66 (version 4.0), with default parameter. Aligned block with length smaller than 1 Kb were filtered. The overall aligned region was defined as overlapped region.
Repeat elements in cultivated soybean genomes were annotated using the same pipeline as used for W05 genome repeat annotation. To identify TE insertions in Wm82_v2 and ZH13 genomes, the two genomes were aligned to W05. In reverse, the W05 genome was compared with those of Wm82 and ZH13 for W05-specific insertions. Genome comparisons were performed using nucmer from MUMmer package66 (version 4.0) with parameters: –mum–noextend. Adjacent alignment blocks with a gap length > 1000 bp in query genome and <100 bp in reference genome were identified as insertions in the query genome. If >80% of the inserted regions in the query genome were annotated as TE elements, the insertion was defined as a TE insertion and the corresponding alignment gap in the reference genome was defined as a TE insertion site. If a TE insertion site in the reference genome was located within a genic region or a 500 bp flanking genic region, then genes were defined as TE-affected genes in the query genome. GO enrichment analysis was performed for TE-affected genes using BINGO (version 3.0.3)67.
Whole-genome comparison between wild soybean W05 and two cultivated soybean genomes were performed using nucmer from MUMmer package66 (version 4.0) and visualized with mummerplot from MUMmer package (version 4.0). Large structural variations (>100 Kb) were identified based on synteny alignment blocks, with variation boundaries manually checked.
To enhance result quality, quality assessment using DataQualityCheck module47 was performed to exclude optical molecules from scans with possible anomalies. Throughput, signal-to-noise ratios and alignment rates in datasets were indentified using a reference in silico map. They were checked for deviations from the norm, which may indicate clogging of the nanochannel arrays.
OM de novo assembly was performed as follows. Filtered optical molecules were assembled into optical contigs using Bionano Solve (version 3.1) assembly pipeline (pipelineCL.py), with parameters optimized by Bionano (optArguments_nonhaplotype_irys.xml; -minlen 150, -minsites 9; merge: 1.00E − 15), with p-value cutoff thresholds for initialAssembly and extendRefine modified according to genome size to 1E − 8 and 1E − 9, respectively.
For better accuracy in downstream analysis, assembled optical contigs were preprocessed with the OMTools DataTools module before alignment. In consideration of the resolution of signal detection, signals within 1 Kb apart were merged for both optical molecules and optical contigs to improve alignment accuracy at regions of dense signals. Optical contigs containing only repetitive segments were removed by OMTools (version 1.4a) DataTools module with the lowden parameter. Processed optical molecules were aligned to retained optical contigs with OMblast (version 1.4a)46. Optical contigs with optical molecule coverage lower than 30 at the target region were excluded from subsequent comparative analysis.
To locate the coordinates of the optical contigs, sequence assemblies of W05, Wm82, and ZH13 were in silico digested for alignment. Signals on the in silico maps within 1 Kb were merged. For each region of interest, optical contigs or in silico maps were aligned to the W05 reference in silico map with OMBlast46. Pairwise alignment was performed between optical contigs that aligned to the target region. Multiple alignment of the optical contigs was then performed to obtain linkage information of genome segments using Optical Map Multiple Alignment package (https://github.com/TF-Chan-Lab/OMTools)47 (version 1.4a). Signal patterns and linkage information were compared, to characterize genomic structural variations among germplasms.
To verify the inversion in the I locus of soybeans with colorless seed coat, junction of the inversion that created the subtilisin-anti-CHS1 chimeric transcript were amplified from genomic DNA of selected soybean accessions. For the detection of subtilisin-anti-CHS1 chimeric transcript, RNA was extracted from the seed coat of developing seeds of Wm82 and W05 using Fruit-mateTM for RNA purification (Takara, Cat. 9192) and RNAiso Plus reagent (Takara, Cat. 9108). First-strand cDNAs and subsequent PCR were done using One-Step TB Green™ PrimeScript™ RT-PCR Kit II (Takara, Cat. RR086). In the reverse-transcription step, only the reverse primer specific for either the subtilisin-anti-CHS1 chimeric transcript or the GmACT11 gene68 (housekeeping gene) was added. Forward primers were then added for the subsequent PCR amplification. Primer information can be found in Supplementary Table 12.
Hi-C raw reads were mapped to W05 genome and Hi-C contact frequency between genomic loci was computed using Juicer69 (version 1.5) with window size of 100 Kb. The Hi-C reads contact frequency matrix was visualized using Juicebox70 (version 1.5.2).
Genome assembly and annotations data of G. soja W05 were deposited in the DDBJ/ENA/GenBank under accession QZWG00000000 [https://www.ncbi.nlm.nih.gov/assembly/GCA_004193775.1/]. The version described here is version QZWG01000000. All the raw sequencing reads were deposited in the NCBI Sequence Read Archive database under the accession SRP158454. The optical molecules and optical contigs of W05 were deposited as NCBI Supplementary Files under accession SUPPF_0000002760 [ftp://ftp.ncbi.nlm.nih.gov/pub/supplementary_data/bionanomaps.csv] and SUPPF_0000002761 [ftp://ftp.ncbi.nlm.nih.gov/pub/supplementary_data/bionanomaps.csv]. The optical contigs of other soybean accessions were deposited as NCBI Supplementary Files under accessions SUPPF_0000002797-SUPPF_0000002807 [ftp://ftp.ncbi.nlm.nih.gov/pub/supplementary_data/bionanomaps.csv]. W05 genome assembly and annotation are also available at wildsoydb database (www.wildsoydb.org/Gsoja_W05). Seeds of the sequenced wild soybean accession W05 cannot be freely distributed to scientists outside of China due to legal restrictions on the exchange of wild plant germplasms. However, seeds of the other parental line C08 and the derived recombinant inbreeding lines are available from Hon-Ming Lam (honming@cuhk.edu.hk) upon request. Data supporting the findings of this work are available within the paper and its Supplementary Information files. A reporting summary for this article is available as a Supplementary Information file. The datasets generated and analyzed during the current study are available from the corresponding author on reasonable request. The source data for Fig. 2b and Supplementary Figure 6 are provided as a Source Data file.
Journal Peer Review Information: Nature Communications thanks David Lightfoot and other anonymous reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.
Morrell, P. L., Buckler, E. S. & Ross-Ibarra, J. Crop genomics: advances and applications. Nat. Rev. Genet. 13, 85–96 (2012).
Munoz, N., Liu, A., Kan, L., Li, M. W. & Lam, H. M. Potential uses of wild germplasms of grain legumes for crop improvement. Int. J. Mol. Sci. 18, 328 (2017).
Zhang, H. Y., Mittal, N., Leamy, L. J., Barazani, O. & Song, B. H. Back into the wild-apply untapped genetic diversity of wild relatives for crop improvement. Evol. Appl. 10, 5–24 (2017).
Schmutz, J. et al. Genome sequence of the palaeopolyploid soybean. Nature 463, 178–183 (2010).
Shen, Y. et al. De novo assembly of a Chinese soybean genome. Sci. China Life Sci. 61, 871–884 (2018).
Kim, M. Y. et al. Whole-genome sequencing and intensive analysis of the undomesticated soybean (Glycine soja Sieb. and Zucc.) genome. Proc. Natl Acad. Sci. USA 107, 22032–22037 (2010).
Li, Y. H. et al. De novo assembly of soybean wild relatives for pan-genome analysis of diversity and agronomic traits. Nat. Biotechnol. 32, 1045–1052 (2014).
Qi, X. P. et al. Identification of a novel salt tolerance gene in wild soybean by whole-genome sequencing. Nat. Commun. 5, 4340 (2014).
Lam, H. M. et al. Resequencing of 31 wild and cultivated soybean genomes identifies patterns of genetic diversity and selection. Nat. Genet. 42, 1053–U1041 (2010).
Chung, W. H. et al. Population structure and domestication revealed by high-depth resequencing of Korean cultivated and wild soybean genomes. DNA Res. 21, 153–167 (2014).
Qiu, J. et al. Genome re-sequencing of semi-wild soybean reveals a complex Soja population structure and deep introgression. PLoS ONE 9, e108479 (2014).
Valliyodan, B. et al. Landscape of genomic diversity and trait discovery in soybean. Sci. Rep. 6, 23598 (2016).
Zhou, Z. K. et al. Resequencing 302 wild and cultivated accessions identifies genes related to domestication and improvement in soybean. Nat. Biotechnol. 33, 408–U125 (2015).
Li, M. W. et al. QTLs regulating the contents of antioxidants, phenolics, and flavonoids in soybean seeds share a common genomic region. Front. Plant Sci. 7, 854 (2016).
Shimomura, M. et al. The Glycine max cv. Enrei genome for improvement of Japanese soybean cultivars. Int. J. Genom. 2015, 358127 (2015).
Kaul, S. et al. Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature 408, 796–815 (2000).
Guan, R. X. et al. Salinity tolerance in soybean is modulated by natural variation in GmSALT3. Plant J. 80, 937–950 (2014).
Yang, S. M., Tang, F., Gao, M. Q., Krishnan, H. B. & Zhu, H. Y. R gene-controlled host specificity in the legume-rhizobia symbiosis. Proc. Natl Acad. Sci. USA 107, 18735–18740 (2010).
Song, J. et al. Identification and validation of Loci Governing Seed Coat Color by Combining Association Mapping and Bulk Segregation Analysis in Soybean. PLoS ONE 11, e0159064 (2016).
Wang, M. et al. Parallel selection on a dormancy gene during domestication of crops from multiple families. Nat. Genet. 50, 1435–1441 (2018).
Tuteja, J. H., Zabala, G., Varala, K., Hudson, M. & Vodkin, L. O. Endogenous, tissue-specific short interfering RNAs silence the chalcone synthase gene family in Glycine max seed coats. Plant Cell 21, 3063–3077 (2009).
Tuteja, J. H., Clough, S. J., Chan, W. C. & Vodkin, L. O. Tissue-specific gene silencing mediated by a naturally occurring chalcone synthase gene cluster in Glycine max. Plant Cell 16, 819–835 (2004).
Tuteja, J. H. & Vodkin, L. O. Structural features of the endogenous CHS silencing and target loci in the soybean genome. Crop Sci. 48, S49–S68 (2008).
Clough, S. J. et al. Features of a 103-kb gene-rich region in soybean include an inverted perfect repeat cluster of CHS genes comprising the I locus. Genome 47, 819–831 (2004).
Tian, Z. X. et al. Genome-wide characterization of nonreference transposons reveals evolutionary propensities of transposons in soybean. Plant Cell 24, 4422–4436 (2012).
Findley, S. D. et al. A fluorescence in situ hybridization system for karyotyping soybean. Genetics 185, 727–744 (2010).
Maranna S., et al. Introgression of null allele of Kunitz trypsin inhibitor through marker-assisted backcross breeding in soybean (Glycine max L. Merr.). BMC Genet. 17, 106 (2016).
Yao, W., Li, G. W., Zhao, H., Wang, G. W., Lian, X. M. & Xie, W. B. Exploring the rice dispensable genome using a metagenome-like assembly strategy. Genome Biol. 16, 187 (2015).
Zhao, S. C. et al. Impacts of nucleotide fixation during soybean domestication and improvement. BMC Plant Biol. 15, 81 (2015).
Dolatabadian, A., Patel, D. A., Edwards, D. & Batley, J. Copy number variation and disease resistance in plants. Theor. Appl. Genet. 130, 2479–2490 (2017).
Vagadia, B. H., Vanga, S. K. & Raghavan, V. Inactivation methods of soybean trypsin inhibitor - a review. Trends Food Sci. Tech. 64, 115–125 (2017).
Liu, Y. G. & Whittier, R. F. Rapid preparation of megabase plant DNA from nuclei in agarose plugs and microbeads. Nucleic Acids Res. 22, 2168–2169 (1994).
Doyle, J. J. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem. Bull. Bot. Soc. Am. 19, 11–15 (1987).
Tian, C. F. et al. Comparative genomics of rhizobia nodulating soybean suggests extensive recruitment of lineage-specific genes in adaptations. Proc. Natl Acad. Sci. USA 109, 8629–8634 (2012).
Xiao, C. L. et al. MECAT : fast mapping, error correction, and de novo assembly for single-molecule sequencing reads. Nat. Methods 14, 1072–1074 (2017).
Li H. Aligning sequence reads, clone sequences and assembly contigs with BWA-MEM. Preprint at https://arxivorg/abs/13033997 (2013).
Putnam, N. H. et al. Chromosome-scale shotgun assembly using an in vitro method for long-range linkage. Genome Res. 26, 342–350 (2016).
Chaisson, M. J. & Tesler, G. Mapping single molecule sequencing reads using basic local alignment with successive refinement (BLASR): application and theory. BMC Bioinformatics 13, 238 (2012).
Leung, A. K. Y. et al. OMBlast: alignment tool for optical mapping using a seed-and-extend approach. Bioinformatics 33, 311–319 (2017).
Leung, A. K. Y., Jin, N., Yip, K. Y. & Chan, T. F. OMTools: a software package for visualizing and processing optical mapping data. Bioinformatics 33, 2933–2935 (2017).
Kent, W. J. BLAT - the BLAST-like alignment tool. Genome Res. 12, 656–664 (2002).
Rotmistrovsky, K., Jang, W. & Schuler, G. D. A web server for performing electronic PCR. Nucleic Acids Res. 32, W108–W112 (2004).
Bao, W. D., Kojima, K. K. & Kohany, O. Repbase update, a database of repetitive elements in eukaryotic genomes. Mob. DNA 6, 11 (2015).
Slater, G. S. & Birney, E. Automated generation of heuristics for biological sequence comparison. BMC Bioinformatics 6, 31 (2005).
Grabherr, M. G. et al. Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat. Biotechnol. 29, 644–U130 (2011).
Haas, B. J. et al. Improving the Arabidopsis genome annotation using maximal transcript alignment assemblies. Nucleic Acids Res. 31, 5654–5666 (2003).
Hackl, T., Hedrich, R., Schultz, J. & Forster, F. proovread: large-scale high-accuracy PacBio correction through iterative short read consensus. Bioinformatics 30, 3004–3011 (2014).
Stanke, M., Diekhans, M., Baertsch, R. & Haussler, D. Using native and syntenically mapped cDNA alignments to improve de novo gene finding. Bioinformatics 24, 637–644 (2008).
Campbell, M. S., Holt, C., Moore, B. & Yandell, M. Genome annotation and curation using MAKER and MAKER-P. Curr. Protoc. Bioinformatics 48, 4.11.11–14.11.39 (2014).
Nawrocki, E. P. & Eddy, S. R. Infernal 1.1: 100-fold faster RNA homology searches. Bioinformatics 29, 2933–2935 (2013).
Kalvari, I. et al. Rfam 13.0: shifting to a genome-centric resource for non-coding RNA families. Nucleic Acids Res. 46, D335–D342 (2018).
Huang, X. H. et al. High-throughput genotyping by whole-genome resequencing. Genome Res. 19, 1068–1076 (2009).
Broman, K. W., Wu, H., Sen, S. & Churchill, G. A. R/qtl: QTL mapping in experimental crosses. Bioinformatics 19, 889–890 (2003).
Tanabata, T., Shibaya, T., Hori, K., Ebana, K. & Yano, M. SmartGrain: high-throughput phenotyping software for measuring seed shape through image analysis. Plant Physiol. 160, 1871–1880 (2012).
Marcais, G. et al. MUMmer4: a fast and versatile genome alignment system. PLoS Comput. Biol. 14, e1005944 (2018).
Guimaraes-Dias, F. et al. Expression analysis in response to drought stress in soybean: shedding light on the regulation of metabolic pathway genes. Genet. Mol. Biol. 35, 222–U259 (2012).
This work was supported by Hong Kong Research Grants Council Area of Excellence Scheme (AoE/M‐403/16), Lo Kwee‐Seong Biomedical Research Fund; Lee Hysan Foundation, National Natural Science Foundation of China Major Projects (91125027), and CUHK VC Discretionary Fund (VCF2014004). We also thank the Worldwide University Network (WUN) for support. Professor Silin Zhong and Dr. Yun-Ru Chen of CUHK provided help in the preparation of 250 bp sequencing library. Shigemi Sasamoto of KDRI assisted in preparing DNA samples from Japanese soybean cultivars. We also thank Professor Scott Jackson of University of Georgia and Dr. Carolina A. Contador of CUHK for their constructive suggestions to this manuscript.
These authors contributed equally: Min Xie, Claire Yik-Lok Chung, Man-Wah Li.
H.-M.L. and T.-F.C. coordinated and designed the experiments. F.-L.W., C.Y.-L.C., M.-W.L, S.-W.T., Z.X., K.F., A.F., Q.D., M.-S.N., X.Q. and T.T. contributed to sample preparation and data generation. M.X., C.Y.-L.C., L.Y., T.D., L.H. and L.C. completed the de novo assembly of the W05 genomes. M.X. performed annotation for W05 genome. X.W. constructed the binmap and performed QTL mapping. M.-W.L. and M.X. analyzed the QTLs, gene, and alleles. A.L. and Z.W. performed PCR validation for the I locus analysis. M.X. performed comparative genomic analysis. C.Y.-L.C., A.K.-Y.L., M.-W.L., and M.X. performed comparative OM analysis. T.-H.W. built and maintained the data dissemination website. M.X., C.Y.-L.C., M.-W.L., J.H., B.V., H.N., S.I., G.C., S.C., C.F., T.-F.C., and H.-M.L. analyzed and refined the scientific contents. M.X., C.Y.-L.C., M.-W.L., C.F., T.-F.C., and H.-M.L. wrote the first draft of the this manuscript. M.X., M.-W.L., C.F., and H.-M.L. wrote the final draft.
Correspondence to Ting-Fung Chan or Hon-Ming Lam.


% Free Dating Site, Free Online Dating Service for Singles at uralremstroy.com open minded with a heart of forgiving, loving and dqting with sense of humor. If you want to find out who likes you, start using the dating page. Create Free single parent dating site humor Profile For Free and find a friend or the possible love of your life. I have a lot of love to give, do you. Single Dahing And Dads is your % FREE Parent Singles Online Dating Site. Create Your Profile For Free and find a friend or the possible love of your life. I have a free single parent dating site humor of love to give, do you.
Parent Single Meet - Visit the most popular and simplest online dating site to flirt, chart, or date with interesting people online, sign up for free. As I said, there is ddating real requirement here.
So paernt here to meet single parents. Lees Summit Aite Parents Trensomnia. Ich bin sehr kommunikativ My inspiration for meeting new people hummor we can't meet new people if we don't look and I am a people's person and get along with anyone I don't have my picks it's not what the eye sees it's what's on the pagent that counts.
Free single parent dating site humor your free single parent dating site humor profile on Mingle2 today and meet other free single parent dating site humor parents in Missouri looking for love, romance, friendship, and more.
Imagination encircles the world. Even still, for those who are unsure about the types of single people you'd like to meet, as in, you don't have a 'type' then you may opt to hunt down your match via our powerful zip code dating feature.
Parent Single Meet - Visit the most popular and simplest online dating site to flirt, chart, or date with interesting people online, sign up for free. Parent Single Meet - Visit the most popular and simplest online dating site to flirt, chart, or date with interesting people online, sign up for free.
I want someone that can stand by herself free single parent dating site humor make decision. Skip to main content. With this, you'll find options to search via age and location thus narrowing down your short list to a select few. Lees Summit Single Siingle Trensomnia. So you're here to meet single parents. Alessio 46 I looking for my half part. Single Moms and Dads is for single parents who are looking for an outlet online to find love. Lees Summit Single Parents Trensomnia.
Feature rich dating tools for better dating. Place your free single parent dating site humor profile on Mingle2 today and meet other single parents in Missouri parnet for love, romance, friendship, and more. Calling all charming, funny and witty single dads. Ich bin sehr kommunikativ My inspiration for meeting new people is we can't meet new people if we paret look and I am a people's person and get along with anyone I don't have my picks it's not what the eye sees it's what's on the inside that counts.
Please enable it in sits browser to access site features. With this, you'll find options to search via age and location thus narrowing down your short list to humorr select few. I love woman who are thick and I find that there are son many amazing people out there to learn from. Sorry in advance Sorry for being so adorable.
Alessio 46 I looking for my half part. Sorry in advance Sorry for being so adorable. I want someone that can stand by herself and make decision. Hit me up if you wonna know more.If you want to find out who likes you, start parebt the dating page. Looking for a Man Woman. LunaC84 has 1 photo. I do generally prefer taller men though.
Hit me up if you wonna know more.If you want to find out who likes you, start using the dating page. Parent Single Meet - Visit the most popular and simplest online dating site to flirt, parentt, or date with interesting people online, sign up for free.
Looking for a Man Woman. Quick search tools and humog rooms. Lees Summit Single Parents Trensomnia.
Feature rich dating tools for better dating. Registration only takes minutes, so go datinh, we'd love to have you aboard and look forward to seeing you dating right here at America's free dating site and single parent dating resource.
As I said, there is no real requirement here.
To make a daring lasting relationship wih the person and have a wonderful family with. No fake profiles, no spam, just real single moms and dads looking for dates.
I did child care hard per person However, I am very lonely I want you to marry if I make friends I long for the emigration to the foreign countries. Feature rich dating tools for better dating. I'm an open book so just introduce yourself to me.
latin dating site single parent dating free single parent dating site humor dry sense of humor. So you're here to meet single parents.
No fake profiles, no spam, just real single moms and dads looking for dates.


Engineers Without Borders (EWB-HK) complies with the Personal Data (Privacy) Ordinance in Hong Kong.
EWB-HK collects only the personal information that is knowingly and specifically supplied to us by the user.
EWB-HK is committed to ensure the privacy of your personal information.
EWB-HK will not sell or pass on your information to any other organization, without your prior permission.
Personal information including name, address, telephone number, e-mail address, nationality, date of birth, etc. are collected only in connection with specific activities, such as membership registration (EWB-HK, and Register of Humanitarian Engineers & Professionals), promotion campaigns, feedback collection, surveys, and digital newsletter subscriptions, etc. You have the right not to disclose any of this information.
At any time, you can ask us for a copy of the personal information that we hold about you or request a correction or deletion of such information.
All reasonable efforts are made to ensure that any personal data held by EWB-HK is stored in a safe place.


text:
Russian Blogger Faces Jail – Kommersant Translation
By Robert Amsterdam | Published: August 14, 2007
[Note – yesterday we did a blog post about the case of a Russian blogger who is being criminally charged for something he wrote on LiveJournal – now we are pleased to bring you an exclusive (for now) translation of the original Kommersant article. UPDATE: Russophobe posts the exact text of Terentyev's comment]
Filter the Magazine – Procuracy Threatens Blogger with Two Years of Jail NEWSPAPER "KOMMERSANT" № 143(3719) OF 13.08.2007 By Alexey Lazarev, Syktyvkar; Alexander Voronov The procuracy of Syktyvkar (Republic of Komi) has filed charges under the article "Arousing hatred or enmity" against Savva Terentyev because he had commented unflatteringly about the local police on one of the internet blogs. This is the first criminal case in Russia opened for a commentary in a web journal, for which mister Terentiev may get two years of deprivation of liberty. The procuracy of Komi has filed charges against blogger Savva Terentyev under Art. 282.1 of the CC RF ("Arousing hatred or enmity, and equally denigration of human dignity"). This article envisions punishment in the form of a fine of up to 300 thsd. rubles or deprivation of liberty for a term of up to two years. The case in relation to Savva Terentyev was initiated by the procuracy of Syktyvkar in the spring of this year (Kommersant reported about this on 13 April). As is said in the decree of the procuracy on the initiation of the case, serving as the basis was "browsing of freely circulating information in the internet network". In the decree it is reported that on 15 February on the blog under the name suranov of the site livejournal.com ("LiveJournal"), policemen uncovered "news under the heading 'Cyberpolice being used at elections'", in which "a user using the pseudonym terentyev left a commentary containing a call directly aimed at arousing hatred or enmity". What specifically terentyev had written and who he is exactly is not indicated in the decree. During the course of a discussion of the role of the police in the activity of local opposition newspapers, terentyev left an extremely unflattering comment about the law-enforcement organs, in part calling for "a purging of society from a purging of society from filthy cop scum." Soon after the initiation of the case, policemen paid a visit to the apartment of Savva Terentyev and confiscated a computer from him. The blogger was interrogated by an investigator for particularly important cases of the procuracy of Syktyvkar, who took from him a signed pledge not to leave town. At the present time, the case is already being conducted by a third investigator, who is the one who filed the official charges against mister Terentyev. Senior assistant of the procurator of Komi for relations with the mass media Yuri Knyazev elucidated the necessity for punishing the blogger to Kommersant thus: "In the internet they have shut off all the brakes, anything you want they can write. And this is a crime." By the way, mister Knyazev declared then that his agency still had to clarify whether it was Savva Terentyev specifically who had left the criminally punishable message on the blog. As was clarified subsequently, the Syktyvkaran procurators did not really have a clear picture of how this is done. At any rate, in the capacity of experts they brought in not internet providers (capable of determining from what specific computer the message on the blog had been left), but philologists of Syktyvkar state university, who never were able to figure out who specifically had left the message the procuracy did not like on the blog. We will note that this is the first criminal case in Russia opened for commentaries in a web journal. Anton Nosik, head of the blogs service of the company SUP Fabrik, which manages the Russian segment of LiveJournal, considers the charges against the blogger "absurd". "The ignorance of district judges often plays a role in the outcome of cases associated with the internet, so this will be a test of the internet literacy of the courts of the Republic of Komi. This trial will become the most reverberatory in Komi in the past thousand years, and I hope that in the presence of a multitude of journalists, the judge will examine the case on the merits, and will not simply rubber-stamp a guilty verdict". Mister Terentyev himself has declared that he "hadn't planned to foment anything at all even in my wildest dreams", and has reported that as of Monday he is embarking upon familiarization with the case materials.

redpajama_set_name: RedPajamaCommonCrawl


In 2017, Jolie Brise took part in the International Rendez-vous 2017 Tall Ships Regatta – a 10,000 nautical mile transatlantic race visiting Portugal, Bermuda, America, Canada and France.
Pupils have crossed the Atlantic on Jolie Brise previously in 2000 and 2009. In 2017, the Captain, first mate and bosun on the leg from Portugal to Bermuda were all former pupils whose love of sailing was sparked by their experiences on Jolie Brise whilst at Dauntsey's.
In 2018, Jolie Brise competed in the 2018 Tall Ships Races. Four fantastic cities and two challenging races across the North Sea made these races a spectacular event.
The Tall Ships fleet embarked on an invigorating voyage across the North Sea, from Sunderland, UK to Esbjerg, Denmark. The international fleet then undertook a breath-taking cruise-in-company between Denmark and Norway, before racing down to The Netherlands.
Highlights for our pupils were the maritime celebrations at each port. Throughout their arrivals, crowds lined the harbour sides, giving the Tall Ships fleet a much deserved and warm welcome.
Jolie Brise's first off-shore cruise of the year. A five-day cruise, hopefully going to France and the Channel Islands (weather permitting) but mainly focussing on the students achieving their RYA Competent Crew Certificate during this cruise.
Joining the Tall Ships fleet in Fredrikstad, to enjoy the festivities with the anticipated quarter of a million spectators, before cruising with the entire Tall Ships fleet, through the Skagerrak, possibly visiting Oslo, Kristiansand, Stavanger and lots of spectacular Norwegian Fjords with amazing wildlife. There will be a lot of socialising with crews from other Tall Ships and local Norwegians.
As this is a Tall Ships Event you MUST have had your 15th birthday by 13 July.
Taking part in the end of the Tall Ships Festival in Bergen, then cruising through the astounding Norwegian Fjords, before heading across the North Sea to the remote but beautiful Shetland Islands. Then sailing on to the Faroe Islands, which are famous for their abundance of sub-arctic wildlife, incredible geography and their own unique culture. We will spend some time cruising through the islands and meeting local youth.
Cruising around the remarkable Faroe Islands, before heading North to South East Iceland, cruising the East Icelandic Fjords. Meeting up with pupils on Summer Cruise 4 at Seydisfjodur, swapping over for a four-day camping and four-wheel drive expedition across Iceland to Reykjavik to fly home. This will be a truly remarkable trip, going to places you won't easily ever be able to again.
Flying to Reykjavik, West Iceland, then a four-day camping and four-wheel drive expedition across Iceland before joining Jolie Brise in Seydisfjodur on the East Coast of Iceland. Cruising the East Icelandic Fjords before sailing to the fascinating Faroe Islands sailing round the archipelago meeting local Faroe Island youth. This will be a truly remarkable trip, going to places you won't easily ever be able to again.
Flying to Torshavn, capital of the Faroe Islands, to join Jolie Brise to cruise around this remarkable group of islands, before heading South to the Outer Hebrides in Scotland. Cruising the Outer Hebrides and North West Coast of Scotland, before finishing in Oban. Another remarkable trip, visiting some of the most remote parts of the United Kingdom.
I have sailed on Jolie Brise in Norway, and across the Bay of Biscay to Portugal, but the highlight for me was sailing across the North Atlantic for 19 days. I have seen incredible things that I could not have experienced anywhere else. It has been life-changing.
This film shows Jolie Brise setting sail for Canada from Torbay in April 2017.
Want to see where Jolie Brise has been and where she is now? Please use our Yellow Brick tracker to view.


text:
Generating fault-tolerant cluster states from crystal structures
© 2020 Verein zur Forderung des Open Access Publizierens in den Quantenwissenschaften. All rights reserved. Measurement-based quantum computing (MBQC) is a promising alternative to traditional circuit-based quantum computing predicated on the construction and measurement of cluster states. Recent work has demonstrated that MBQC provides a more general framework for fault-tolerance that extends beyond foliated quantum error-correcting codes. We systematically expand on that paradigm, and use combinatorial tiling theory to study and construct new examples of fault-tolerant cluster states derived from crystal structures. Included among these is a robust self-dual cluster state requiring only degree-3 connectivity. We benchmark several of these cluster states in the presence of circuit-level noise, and find a variety of promising candidates whose performance depends on the specifics of the noise model. By eschewing the distinction between data and ancilla, this malleable framework lays a foundation for the development of creative and competitive fault-tolerance schemes beyond conventional error-correcting codes.
Newman, M; de Castro, LA; Brown, KR
10.22331/q-2020-07-13-295


This post is a bit special for me. Anybody that knows me, knows that I like to give, and helping others is something I enjoy. When I first came across My Gift Stop, I was browsing for a new watch. Before I order from any company I tend to learn about it, owning one myself, how a business conducts itself is important. To my surprise, My Gift Stop is a truly stand up company. On every sale they donate 5% of the profits to Direct Relief.
The site offers shoppers a place to get last minute gifts for everyone. It doesn't matter if you are a male or female, it contains luxury and designer products to fit all needs. Since the works directly with the manufacturers they are able to offer incredible prices to their customers.
I have a bit of a watch problem and own way too many. When I was browsing the site for a new one, I came across this Orient Ray Raven II Watch and it caught my eye. I don't do Black watches often, but the simplistic look of that drew me in. I've also heard really good things about the brand. It's currently about 50% off, which is a crazy deal.


Born in Mobile, Alabama on September 17, 1951.
Deborah Moseley Belvin, 67 of Palm Bay passed away on Sunday, November 11, 2018. She was the Retired Director of Hibiscus Day Care and a member of The Cornerstone Church of God, a graduate of Melbourne High School Class of 1969 and an Avon Representative for many years.
Survivors include her sister Melanie Moseley (Fred) Karstens, nephew Tyler, and surrogate Mother Betty Coulter.
A funeral service will be Friday November 16th at 1pm in the Cornerstone Church of God. Donations in her memory may be made to the Cornerstone Church of God.


text:
Balachandran NATESAN
balachandran.n AT ifpindia DOT org
Coastal Transformations and Fisher wellbeing. Synthetized perspectives from India and Europe (FISHERCOAST)
Dynamics of Forest Diversity
G. Muthusankar, Christophe Proisy, Shuvankar Ghosh, Raphael Mathevet, Balachandran Natesan, et al.. On the urgent vulnerability assessment of the Coromandel coast of India threatened by increasing and combined environmental and human pressures. Paper presented at the International Symposium on Advances in Coastal Research with Special Reference to Indo Pacific (AdCoRe IP-2019), Dec 2019, Chennai, India. pp.92. (hal-02902376)
N. Balachandran, M Anbarashan, P. Uma Maheswari. Conservation significance of endemic vascular plants of Tropical Dry Evergreen Forest (TDEF) of Southern India. Paper presented in the Association For Tropical Biology and Conservation Asia Pacific Conference from 10-13 September 2019, MAS Athena, Thulhiriya, Sri Lanka, Sep 2019, Paper presented in the Association For Tropical Biology and Conservation Asia Pacific Conference from 10-13 September 2019, MAS Athena, Thulhiriya, Sri Lanka, Sri Lanka. (hal-02700842)
Raphael Mathevet, Elea Desmots, Anbarashan M, Balachandran N, G. Muthusankar, et al.. Looking for Integrated Conservation and Development of the Coramandel Coastal Wetlands: The case Study of Kaluveli (Tamil Nadu, South India). Paper presented at the International Symposium on Advances in Coastal Research with Special Reference to Indo Pacific (AdCoRe IP-2019) jointly organized by the National Centre for Coastal Research (NCCR), National Institute of Ocean Technology (NIOT) & Research and Information System for Developing Countries (RIS) in Chennai from 17th to 19th December 2019, Dec 2019, Chennai, India. (hal-02902229)
Anbarashan M, N. Balachandran, Raphael Mathevet, G Muthusankar, Ghosh S, et al.. Mapping and Assessing the Land Cover and Land Use of a fragile Coastal Landscape, Cuddalore District, Tamil Nadu, South India. International Symposium on Advances in Coastal Research with Special Reference to Indo Pacific (AdCoRe IP-2019), National Centre for Coastal Research (NCCR); National Institute of Ocean Technology (NIOT); Research and Information System for Developing Countries (RIS), Dec 2019, Chennai, India. (hal-02733648)
Ayyappan N, B.R. Ramesh, Soupramanien Aravajy, S. R. Ramalingam, N. Barathan, et al.. Dynamics of tree diversity in undisturbed Evergreen forest at Uppangala, Central Western Ghats. Paper presented at the Fourth Indian Biodiversity Congress (IBC 2017), Pondicherry University, Mar 2017, Pondicherry, India. pp.293. (hal-02889658)
Munoz François, Sylvain Schmitt, Valérie Raevel, M. Réjou-Méchain, Ayyappan N, et al.. Wood and leaf strategies in a low-elevation rainforest of Western Ghats. Paper presented at the Fourth Indian Biodiversity Congress (IBC 2017), 10-12 March 2017, Pondicherry University, Puducherry, India, Mar 2017, Puducherry, India. p. 38. (hal-02734452)
Valérie Raevel, Ayyappan N, N. Balachandran, S. Aravajy, B.R. Ramesh, et al.. Towards a comprehensive taxonomic name resolution tool of Westerh Ghats trees. Paper presented at the Fourth Indian Biodiversity Congress (IBC 2017), 10-12 March 2017, Pondicherry University, Puducherry, India, Mar 2017, Puducherry, India. p. 81. (hal-02734617)
Jeyaprakash K, Balachandran Natesan, Rathinavel S.. Traditional uses of medicinal plants by Adi tribe from Daying Ering Memorial Wild Life Sanctuary, Passighat, Arunachal Pradesh, India. National Conference on Emerging Trends in Plant Science, Mar 2016, Tiruchirappalli, Tamil Nadu, India. (hal-02910958)
Jens Kattge, Gerhard Bonisch, Sandra Díaz, Sandra Lavorel, Iain Colin Prentice, et al.. TRY plant trait database – enhanced coverage and open access. Global Change Biology, Wiley, 2020, 26 (1), pp.119-188. (10.1111/gcb.14904). (hal-02434220)
Kothandapani Raman, Sivangnanaboopathidoss Vimalraj, Bawa Mothilal, Natesan Balachandran, Abhishek Tomar. Range extension of the Gooty Tarantula Poecilotheria metallica (Araneae: Theraphosidae) in the Eastern Ghats of Tamil Nadu, India. Journal of Threatened Taxa, www.threatenedtaxa.org, 2019, 11 (10), pp.14373-14376. (hal-02271561)
Natesan Balachandran, Krishnamurthy Rajendiran, Walter Gastmans. Endemic plants of tropical dry evergreen forest, Southern India. Biodiversity Research and Conservation, BRC, www.brc.amu.edu.pl, 2018, 52, pp.11-23. (hal-02102399)
Jeyaprakash K, Balachandran N, Kathiresan D. Champereia manillana (Blume) Merr. (Opiliaceae) and Stemodia verticillata (Mill.) Hassl. (Plantaginaceae): New distributional records to North East India. Bio Bulletin, Research Trend, 2018, 4 (2), pp.17-20. (hal-02100879)
Pierre Ploton, Nicolas Barbier, Pierre Couteron, Cécile Antin, N. Ayyappan, et al.. Toward a general tropical forest biomass prediction model from very high resolution optical satellite images. Remote Sensing of Environment, Elsevier, 2017, 200, pp.140 - 153. (10.1016/j.rse.2017.08.001). (hal-01824235)
N. Balachandran, K. Ravikumar, K. Rajendiran, W. F. Gastmans. A new species of Tetrastigma (Vitaceae) from Tamil Nadu, southern India. Webbia, 2017, 72 (1), pp.113 - 116. (10.1080/00837792.2017.1308110). (hal-01555316)
N. Dhatchanamoorthy, N. Balachandran, K. Ravikumar. Rediscovery and lectotypification of Lepidagathis diffusa (Acanthaceae), an endemic species from southern India. Rheedea, http://www.iaat.org.in/journal.html, 2017, 27 (2), pp.96-98. (hal-01823655)
Valérie Raevel, Ayyappan N, Balachandran N, S. Aravajy, N. Barathan, et al.. Taxonomic and ecological database of trees of Western Ghats - TreeGhatsData (Version 1.1) [Data set]. Zenodo, https://zenodo.org, 2017. (hal-02123503)
Navya, R, Krishnakumar, C, N. Balachandran, S. Aravajy, Krishnamurthy Anupama. Resilience of the tropical dry evergreen forest in the human-impacted region of Coromandel Coast, India. Journal of Economic and Taxonomic Botany, Scientific Publishers, 2017, 41 (1-2), pp.31-46. (hal-02279128)
Sourabh Pargal, Rakesh Fararoda, Gopalakrishnan Rajashekar, Natesan Balachandran, Maxime Réjou-Méchain, et al.. Inverting Aboveground Biomass?Canopy Texture Relationships in a Landscape of Forest Mosaic in the Western Ghats of India Using Very High Resolution Cartosat Imagery. Remote Sensing, MDPI, 2017, 9 (3), pp.228. (10.3390/rs9030228). (hal-01556328)
N. Balachandran, K. Ravikumar, N. Dhatchanamoorthy. Two new plant records to Southern India. The Indian Forester, http://www.indianforester.co.in, 2016, 142 (9), pp.849-851. (hal-01374337)
N. Balachandran, K. Rajendiran. The taxonomic identity of Jasminum matthewii (Oleaceae). Rheedea, http://www.iaat.org.in/journal.html, 2016, 26 (1), pp.54-56. (hal-01374345)
N. Balachandran, K. Rajendiran. Cordia ramanujamii (Cordiaceae): new species from Tamil Nadu, India. Taiwania, College of Life Science, National Taiwan University, 2016, 61 (2), pp.74-77. (10.6165/tai.2016.61.74). (hal-01352524)
Balachandran Natesan, N. Dhatchanamorthy, K. Rajendiran. Interesting Plant Records from Tamil Nadu. Indian Journal of Forestry, M/s Bishen Singh Mahendra Pal Singh, India, 2015, 38 (4), pp.339-342. (hal-02915411)
N. Dhatchanamorthy, Raja P., Soosairaj S., Balachandran Natesan. Notes on the distribution of Crotalaria globosa Wight & Arn. - a rare and endemic legume in Tamil Nadu. Phytotaxonomy, Association for Plant Taxonomy, 2015, 15, pp.104-106. (hal-02916118)
Suvathimani S., Vinothini K., Balachandran N. Defense mechanism between the parasites: Dendrophthoe falcata and their host in and around Pondicherry, India. The National Symposium on Current Trends in Plant Sciences, Feb 2018, Tambaram, Chennai, India. 2018. (hal-02130657)
Vinothini K., Suvathimani S., N. Balachandran. Morphological relationship between host and parasite: Dendrophthoe falcata. The National Symposium on Current Trends in Plant Sciences, Tambaram, Chennai, Feb 2018, Tambaram, Chennai, India. (hal-02130650)
Valérie Raevel, N. Ayyappan, N. Balachandran, S. Aravajy, N. Barathan, et al.. Taxonomic and ecological database of trees of Western Ghats - TreeGhatsData. 2018. (hal-01824264)
Powered by HAL


At 06.35 am we untied from the dock we have headed out into a bright clear dawn for a while. There was little time to feel nostalgic, as we still had numerous tasks to perform and the morning fog soon formed to envelope us in its cloak of white ambiguity. When I started sailing Fog was a frightening experience. Compass and dead reckoning, estimating speed, strong tidal and river estuary cross currents. GPS, AIS and Radar take the bowel laxative part away, so no worries as long as the other guys are paying attention!
Frost in the morning at the dock and very cold. May and Marty knocked on our deck to make sure we are awake, up and ready. After making coffee we start the motor of Beautiful Swimmer and leave from Prince Rupert. At 10.30 a.m. we passed Hammer Island At 12.00 a.m. Passing Oona River 5.5 K no wind ….Foggy….. At 13.15 p.m. 6.2 K 13.45 p.m. White House 6.5 k 15.00 p.m.
At 20.30 p.m. We tided up and got free shore power. Hartley Bay is a generous dock that you can plug power line to the boat without charge. There is small fuel station, Water. People are friendly. Unfortunately our friends the Hills where out of the village. It would have been hard to say goodbye as Steve has known them for 30 years.
It was a long day and we had dinner at 21.00 p.m.
Luksana said I could add captions, but I think the photos speak for themselves. The North Coast is still one of the almost pristine wonderland's the planet. Could not but feel sad and concerned that this beauty is under real threat from the Tar Sands pipeline that is being fast tracked by the present Canadian Government is threatening this beauty for ever.
Funny they never build pipelines through the centre of cities or next door to the Houses of Governments. But these natural Cathedrals, Art Galleries and National Treasures that are all unique and can never be replaced are being put at risk so that a few can make money producing more of what is destroying the planet. I must admit I did feel selfish in leaving this special place as it faces its biggest threat since the glaciers retreated.
2nd night stay at Hartley Bay Check engine and so on. Beautiful Full moon tonight.


text:
Vinnytsia Bolshevik Uprising was an armed uprising of the Bolsheviks in Vinnytsia from November 10 (October 28) to November 12 (October 30) 1917 during the October Revolution.

The uprising preceded by a protest of revolutionary soldiers from quartered in Vinnytsia the 15 Reserve Infantry Regiment. On 5 November (23 October) they came out on demonstration of protest against the order of command of the Southwestern Front to dissolve the regiment.

The same day on the initiative of Bolsheviks of the Vinnytsia Soviet workers and soldiers deputies there was created a revolutionary committee (revkom) headed by a leader of local Bolsheviks Nikolai Tarnogorodsky. The revkom announced all orders of local authorities invalid without agreed upon by it. The committee also agreed not to allow departing to the frontlines the 15th regiment and other revolutionary minded formations that were located in Vinnytsia as well as prohibited to issue arms from the military warehouses. Punitive formations that arrived to the city issued an ultimatum to the Vinnytsia Soviet: to deport the 15th regiment to frontlines, issue arms out of warehouses for the front and arrest all Bolsheviks. The soviet rejected those requests and offered for punishers to leave the city.

Following the events in Petrograd and Kiev on 10 November (28 October) there started the uprising. Against the punishers came out Red Guards workers and various military formations (~6,000 soldiers). On 12 November (30 October) after a fierce fighting and by concentrating great military force, the counter-revolutionary command of the Southwestern Front forced the rebels to retreat out of the city. However to their help from Zhmerynka arrived two regiments of the 2nd Guard Corps that drove the punishers out of Vinnytsia. On 17 November (4 November) in the city was established the Soviet regime. According to Soviet encyclopedias, the consolidation of Soviet regime in Vinnytsia prevented the Central Council of Ukraine that cunningly disarmed revolutionary formations and temporary occupied the city.

References

External links

 Vinnytsia October Armed Uprising 1917 at the Ukrainian Soviet Encyclopedia

Russian Revolution in Ukraine
History of Vinnytsia
1917 in Ukraine
Conflicts in 1917
Bolshevik uprisings
Communism in Ukraine
Military history of Ukraine
November 1917 events

redpajama_set_name: RedPajamaWikipedia


text:
You are here: Parliament home page > Parliamentary business > Publications and Records > House of Commons publications > Parliamentary Commissioner for Standards Annual Report 2005-2006
Select Committee on Parliamentary Commissioner for Standards Annual Report 2003-04
4 Other registers
Brief history of the registers
4.1 Ten years after the introduction of the Register of Members' Interests in 1974, the Select Committee on Members' Interests (a predecessor of the Committee on Standards and Privileges) conducted an inquiry into concerns about parliamentary lobbying and recommended that:
"when they are approached, Members of the House must be able readily to identify the source and true nature of the approach. Equally the full purposes of those with access to Parliament should be known. We are convinced that greater openness would now be beneficial in certain areas."[31]
4.2 To meet those concerns the Committee concluded that registers should be set up for Members' staff, journalists and all-party groups. The Committee's recommendations were subsequently agreed by the House on 17 December 1985 and the first registers were published in 1986. The form and substance of each register are detailed in the sections below.
Members' Staff Register
4.3 Those holding a parliamentary pass as a Member's secretary or research assistant are required to register any other occupation or employment from which they receive income exceeding half of one per cent of a Member's salary from the same source in the course of a calendar year, if that occupation or employment is in any way advantaged by the privileged access to Parliament afforded by their pass.[32] They also have to register any tangible gift (e.g. glassware) and any other benefit (e.g. hospitality, service or facilities provided) which they receive, if the value of the gift or benefit exceeds that sum and relates in any way to their work in Parliament.
4.4 The number of staff on the register has increased significantly, from 1456 on 31 March 2003 to 1576 on 31 March 2004. The number of these registering an interest rose less markedly, from 361 to 388.
Journalists' Register
4.5 Those holding a pass as a lobby journalist accredited to the Parliamentary Press Gallery or for parliamentary broadcasting are required to register any occupation or employment from which they receive income exceeding one percent of a Member's salary from the same source in the course of a calendar year, if that occupation or employment is in any way advantaged by the privileged access to Parliament afforded by their pass.[33]
4.6 Turnover on the register is low. Of the 408 journalists on the register as at 31 March 2004, 62 had registered interests. This compares with 398 and 60 respectively at 31 March 2003.
All-Party Groups' Register
4.7 The membership of all-party groups consists mainly of backbench Members of the House of Commons and Lords but may also include ministers and non-parliamentarians. There are two types of groups: subject groups (relating to a particular topic, e.g. forestry) and country groups (relating to a particular country or region).
4.8 Inclusion on the Register of All-Party Groups is compulsory for any group which includes Members of the Commons from more than one party and has at least one officer who is from the Commons. Such groups are required to register the group's title and the names of its officers. Financial and material benefits received by the group as a whole must also be registered, where the group receives during a calendar year one or more benefits whose total value is £500 or more from the same source. Lastly, the group must register the name and paid employment or occupation outside Parliament of any staff servicing the group who hold a parliamentary pass, if that occupation or employment is advantaged by the passholder's privileged access to Parliament.
4.9 Groups that qualify for inclusion on the Register of All-Party Groups may also apply for inclusion on the Approved List. Both are compiled by my office. By being on the Approved List, a group qualifies for certain entitlements, largely to do with use of the House's facilities. Additional rules apply to groups on the List. For example, they must hold annual elections for their officers and must also register the names of a minimum of ten Members from the Government party and ten from the Opposition parties. All but 6 groups are on both the Register and the Approved List.
4.10 The number of registered groups as at 31 March 2004 showed a significant increase over that in 2003, being 401 as against 356. Of these 113 (101) were country groups and 288 (255) were subject groups. The number registering financial or material benefits fell slightly, however, from 211 to 209.
Overlap between the registers
4.11 Overlap between the Registers kept by my office is an increasingly common occurrence. Subject to the different financial thresholds that apply to each of the registers, examples arise when:
a) Hospitality (eg relating to overseas visits) is received by an all-party group. This should be registered on both the Groups' and Members' register (and may also need to be registered with the Electoral Commission, depending on its value to each individual Member).
b) An external organisation or individual subsidises a staff member's salary. This should be registered on both the Staff and Members' register, and if the staff member acts as part of the staff to an all-party group, on the Groups' register.
4.12 It is important that Members keep in mind the potential need to make entries in more than one of the Registers arising from the same circumstances. As mentioned earlier, in some cases Members will also need to address the Electoral Commission's registration requirements.
Access to the registers
4.13 The three registers are not published in hard copy form. However, a copy of the most recent edition can be viewed on the standards section of the parliamentary website at: www.parliament.uk/about_commons.pcfs.cfm and a paper copy is held at the House of Commons where anyone may inspect it by arrangement.[34] An updated edition of all three registers is usually issued each month when the House is sitting.
4.14 In the past year I received no formal complaints in connection with these registers.
31 Select Committee on Members' Interests, First Report, Session 1984-85, HC 261 Back
32 Over £275 during the period of this report Back
34 An arrangement may be made with the Secretary to the Committee on Standards and Privileges Back
Prepared 6 July 2004