Development of Genic and Genomic SSR Markers of Robusta Coffee (Coffea canephora Pierre Ex A. Froehner)


Coffee breeding and improvement efforts can be greatly facilitated by availability of a large repository of simple sequence repeats (SSRs) based microsatellite markers, which provides efficiency and high-resolution in genetic analyses. This study was aimed to improve SSR availability in coffee by developing new genic2/ genomic-SSR markers using in-silico bioinformatics and streptavidin-biotin based enrichment approach, respectively. The expressed sequence tag (EST) based genic microsatellite markers (EST-SSRs) were developed using the publicly available dataset of 13,175 unigene ESTs, which showed a distribution of 1 SSR/ 3.4 kb of coffee transcriptome. Genomic SSRs, on the other hand, were developed from an SSR-enriched small-insert partial genomic library of robusta coffee. In total, 69 new SSRs (44 EST-SSRs and 25 genomic SSRs) were developed and validated as suitable genetic markers. Diversity analysis of selected coffee genotypes revealed these to be highly informative in terms of allelic diversity and PIC values, and eighteen of these markers (,27%) could be mapped on a robusta linkage map. Notably, the markers described here also revealed a very high cross-species transferability. In addition to the validated markers, we have also designed primer pairs for 270 putative EST-SSRs, which are expected to provide another ca. 200 useful genetic markers considering the high success rate (88%) of marker conversion of similar pairs tested/validated in this study. 

Introduction

Coffee tree belongs to the genus Coffea, comprising two main cultivated species C. arabica L. (2n54x544) and C. canephora Pierre ex A. Froehner (diploid, 2n52x522), yielding arabica and robusta type of coffees, respectively. Arabica coffee is known for excellent cup quality but suffers from a narrow genetic base due to its domestication history and susceptibility to diseases and pests. In contrast, robusta coffee though poor in quality has better adaptability to various stresses. To keep pace with the environment and also of the sensibilities of market, there is a continuous need for genetic improvement of coffee, which unfortunately is severely constrained owing to inherently slow pace of tree breeding using conventional methods, and variety of other reasons [1,2]. The situation demands development of new, easy, practical technologies that can provide acceleration, reliability and directionality to the breeding efforts, as well as characterization of cultivated/secondary gene pool for proper utilization of the available germplasm in coffee genetic improvement programs. In this context, DNA polymorphism based genetic markers becomes important that have proven to be of immense value in characterization and genetic improvement of plant germplasm resources. 
Among different types of DNA markers, microsatellites or SSR markers are the most ideal for studying genetic diversity, population structure, phylogenetic relationships, construction of frame-work linkage maps, QTL interval mapping, marker-assisted selection (MAS), etc., thereby aiding in genetic improvement of crop plants [1]. In the last few years a number of efforts have lead to development of a few hundred SSR markers in coffee [2][3][4][5][6][7][8][9][10][11][12], but these are insufficient to realize the full potential of markers for mapping/linkage studies in coffee, more so in arabicas which have an extremely narrow genetic base. Moreover, most of the described markers are poorly validated, especially for their utility in cultivated genepool comprising arabicas and robusta coffee. The situation thus calls for newer efforts to generate additional validated markers for them being of any gainful utility in marker-based genetic studies/coffee breeding. 
With advancements in genomic studies, there has been an huge burst in the EST sequences in the public domain that provide an easy and economic/costeffective opportunity to identify and develop EST based SSR markers, which have the additional advantage of assessing the functionally effective genetic diversity [13,14], and also have very high cross-species transferability [8,11]. In this study, we have used the coffee EST database containing 13,175 unigene [15] to identify SSRs in the expressed part of coffee genome, and use the same to develop novel coffee-specific EST-SSRs for use as efficient genetic markers. Thus we describe here 44 new validated genic-SSRs, and another set of 270 putative similar markers that need further validation. In addition, we also describe 25 new genomic SSR markers that were developed using an affinity capture approach based SSRenriched partial, small-fragment genomic library. 

Materials and Methods


Plant material and DNA extraction

The plant material used for the validation of SSR markers comprised a set of 16 elite coffee genotypes belonging to C. arabica (tetraploid arabicas) and 

Construction of an SSR-enriched small-insert genomic library/ development of genomic SSRs

A partial genomic DNA library enriched for microsatellite repeats was constructed using the methods described earlier [17]. Briefly, the method involved: one-step restriction digestion of genomic DNA with Hae III enzyme (NEB) and ligation of resulting fragments with ds Mlu-I adaptor (Mlu-F: CTC TTG CTT ACG CGT GGA CTA and Mlu-R: pTAG TCC ACG CGT AAG CAA GAG CAC) [18]; amplification of the restricted-ligated DNA pool using Mlu-F primer; SSR enrichment of the amplified DNA pool using liquid phase hybridisation (in 6X SSC) with streptavidin coated paramagnetic beads (Dynal) attached with biotinylated equimolar pool of four oligos (CA) 15 , (GA) 15 , (GAA) 15 and (CAA) 15 . This was followed by amplification of the hybridized/trapped genomic DNA fragments by PCR and construction of partial genomic library in TA vector (Invitrogen) as per the manufacturer's instructions. A number of positive (white) recombinant clones were randomly picked up from the library, amplified and sequenced for both the strands using M13 universal primers on ABI 3730 DNA Analyzer (Applied Biosystems, USA). The sequences were aligned and edited using Autoassembler (Applied Biosystems, USA). The SSR-positive sequences were identified and used for development of new genomic SSRs as described earlier [2]. 
The amplified PCR products generated using all the new SSRs were resolved using capillary-based ABI 3730 DNA Analyzer and were precisely sized for major, comparable and conspicuous peaks using GeneMapper 3.7 (Applied Biosystems), using default parameters. 

Statistical, genetic and diversity analysis

The data for EST-SSRs and genomic SSRs were analyzed separately for various genetic parameters, viz., mean, standard deviation, expected heterozygosity (H e ), Hardy-Weinberg equilibrium (HWE) and linkage disequilibrium, (LD), using Arlequin ver 3.1 [19], polymorphism information content (PIC) and private alleles (Pas) using Convert ver. 1.3.1 [20]. Cross-taxa transferability (T mark ) was calculated over 15 species (except C. canephora) as proportion of primers showing successful amplification vis-a `-vis all the tested primers whereas primer conservance (C taxa ) was calculated as proportion of the species displaying successful amplification vis-a `-vis all the tested markers. 
Genetic diversity analysis to infer generic relatedness/affinities was performed over informative Pms (polymorphic markers) for cultivated genotypes/related species using MicroSatellite Analyzer [21] with Nei's genetic distance [22]. The genetic distance matrices were used to construct Neighbour Joining (NJ) consensus tree using Phylip ver 3.6 [23], which was viewed using Treeview ver 1.6.6 [24]. 
We also attempted mapping of the new markers on a robusta linkage map using JoinMap ver 4.0 [25] as described earlier [2,26] using group LOD score of 5.0. 

Results

In this study, we undertook in-silico analysis of a robusta coffee transcriptome to identify and develope coffee-specific EST-SSR markers. Simultaneously, we also attempted development of genomic-SSRs by constructing a small-insert SSR enriched partial genomic DNA library. The new markers (44 EST-SSRs and 25 genomic-SSRs) were validated for their utility in genetic studies using panels of elite coffee genotypes, and related taxa of coffee for cross-species transferability. The details of these new markers viz., locus designation, primer sequences, repeat motifs, amplification temperature, amplicon size, and SGN ID or Genbank accession numbers are given in Tables 1 and2. Details of additional primer pairs for 270 putative EST-SSRs, which were designed but need to be validated have been provided in Table 3. 

Types, Frequency and Distribution of SSRs in the coffee transcriptome

The coffee EST unigene database analyzed here, comprised 13,175 unigenes having a total length of 8923 kb and an average lengthof ca. 677 bases/unigene [15]. These ESTs were found to contain a total of 2,589 SSRs (having a minimum S1 &S2). The mean length of repeat iterations (RI) for all the SSRs was 5.2, whereas average length of DNRs was maximum (9.6 RI) followed by TNRs (4.6 RI). Among the individual SSRs, AC had the maximum average repeat length of 10.5 RI, followed by AG (8.6 RI), AT (8.3 RI), CG (6.3 RI); all the TNRs had RI in the range of 4.3 to 4.8, whereas all other larger SSRs had an average RI of less the three. The identified SSRs having a repeat core of 18 bp or more were selected as candidate 'usable' SSRs for further primer designing/marker conversion. Overall, in-silico analysis of the EST unigenes revealed one EST-SSR (having a minimum repeat core of 12 bp) per 3.4 kb and one usable SSR (having a minimum repeat core of 18 bp) per 15.9 kb of robusta transcriptome (Table S2). Among the individual SSRs, the most abundant EST-SSR motif was AG, followed by AAG. 

Development of microsatellite markers from usable EST-SSRs

Only 483 (18.7%) of the total 2589 identified SSRs had a repeat core.18 bp, which were used for marker conversion. Primer pairs could be designed for 320 of these SSRs, of which randomly chosen 50 pairs were further tested for validation studies. These included SSRs with DNRs (30%), TNRs (64%), PNRs, HNRs (2% each) and complex SSRs (see Table 1 for marker ID, primer sequences, repeat motifs, amplicon size, sequence ID and functional identity). Of the selected 50 primer pairs, 44 could be successfully amplified as single locus SSR marker, indicating 88% primer to marker conversion ratio. Considering this high conversion ratio, another ca. 200 useful genetic markers are expected from the remaining 270 putative EST-SSRs (Primer IDs: CCESSR51 to CCESSR320) that are lsited in Table 3. S3). 
From the 56 SSR+ive sequences, a total of 41 primer pairs could be designed successfully (with five pairs containing two SSRs each). Of these, 25 pairs (encompassing 28 SSRs) resulted in robust PCR amplifications (Table 2), and all of them could further be validated as single locus markers indicating ,61% primer to marker conversion ratio. 

Validation of EST-SSRs for use in genetic studies

All the new 44 EST-SSRs resulted in good amplicons exhibiting low to medium allelic diversity when tested on a panel of 16 elite robusta and arabica genotypes ( Figure 1). Overall, a maximum of six and seven alleles (N A ) with an average of 2.1 and 3 alleles/SSR were obtained for the tested markers of which 65.9% and 81.8% were polymorphic/informative for tetraploids and robusta genotypes, respectively (Table 4). Fifteen markers in the case of tetraploids and eight for robustas were found to be monomorphic. Moreover, 14 markers resulted in double alleles (i.e. consistent presence of two allelic amplicons across the tested samples) indicative of duplicated loci in case of all the tested tetraploid arabicas. In general, no private alleles were evident except in one robusta genotype (Sln274) for marker CCESSR14. 
The PIC values were comparable (0.19-0.67 and 0.11-0.77), and no significant differences were seen in the observed/expected heterozygosity (H o /H e : t-value50.70; P50.49; and t-value50.68; P50.40) for the new markers across the tested tetraploids and diploid robustas, respectively. However, significant differences were observed in the total number of amplified alleles (N A : t53.74, P,0.005), as well as, the behaviour of the polymorphic markers (Pms) when tested for HWE and LD in the tested tetraploids and the robusta genotypes ( Table 4). In general, more markers were in HWE and only a relatively small proportion of markers exhibited LD and heterozygote excess and/or deficiency in case of robustas, in comparision to tetraploid arabicas. 

Validation of genomic SSRs for use in genetic studies

A total of 25 putative genomic SSRs were also validated as genetic markers ( Table 5). When tested on the panel of 16 elite robusta and arabica (tetraploid) genotypes, five of these markers in arabicas and one in robustas were found to be monomorphic. Twelve ofthe polymorphic markers in arabicas resulted in double alleles (putative duplicated loci). In total, a maximum of seven and eight alleles (N A ) with an average of 2.7 and 4.3 alleles/marker were obtained for the tested polymorphic markers of which 32% and 96% were informative in arabicas and robustas, respectively (Figure 1). The PIC values varied considerably, with mean PIC value being 0.47 (range 0.12-0.78) for tetraploids, which was significantly less than 0.60 (0.12-0.85) observed for robusta (Table 5). Further, the Student's t-test revealed significant differences in N A (t54.09, P50.00) but non-significant differences in PIC estimates (t51.26, P50.13), as well as, for the observed/ expected heterozygosity estimates (H o /H e ) for the comparable markers of arabica and robusta genotypes. Further, it was notable that while.83% of the Pms were in HWE and only few markers showedsignificant heterozygote deficiency to varying extent in both arabicas and robustas, the number of marker-pairs that exhibited LD was significantly more in arabicas (28.0%; 8 of 28 pairs) that that seen in robustas (14.2%; 36 of 254 marker pairs). 

Mapping of new EST-and genomic SSRs

The 69 new SSR markers were also tested for their suitability in linkage mapping. In total, 11 of the 44 EST-SSRs (,39%) and seven of the 25 genomic SSRs (28%) could be mapped onto an existing first-generation framework linkage map of robusta coffee [2,26]. This map comprised a total of 374 mapped markers (71 SSRs, 185 RAPDs and 118 AFLPs) on 11 major and 5 minor linkage groups. The new markers developed in the present study were mapped using the existing SSRs on the map as anchor markers. The 18 new markers that could be mapped, occupied positions on eight distinct linkage groups, with eight markers on CLG03; two markers each on CLG06, CLG11, CLG15; one marker on CLG02, CLG04, CLG05, CLG08 (Tables 1 &2). The position of these 18 markers on robusta linkage groups alongwith positions of SSRs used as anchores (CM62, CM115, CM12, CM100, Cof_EST01_150, CaM46, CaM44, CM39_302, CM39_273) is shown in figure 2. 

Cross-species/2genera transferability and marker conservation

New SSR-markers when tested on 13 related Coffea and two Psilanthus species, exhibited robust cross-species amplifications with alleles of comparable sizes in the tested taxa (Figure 1, Tables 4 &5, Table S4). The EST-SSRs showed 100% transferability accross the tested Coffea and Psilanthus spp., whereas the genomic-SSRs indicated 96% amplification and transferability for Coffea spp. and 92% for the related Psilanthus spp. The analysis also indicated some private alleles (PAs), which possibly could be species-specific (Tables 4 &5). 

Generic affinities within/between cultivated and wild coffee germplasm by new SSRs

The SSR allelic data were examined for their utility in ascertaining the genetic diversity and generic inter-relationships between the cultivated, as well as, the wild coffee genepool. The average genetic distance values calculated using the EST-SSR allelic data were in general, significantly less but comparable to that obtained using the genomic SSRs for the tested arabicas, robustas, as well as, for different Coffea and Psilanthus species. 
The NJ phenetic tree generated using the genetic distance estimates of EST-SSRs allelic data clearly resolved the tested germplasm in two distinct clusters, one representing all the tetraploid arabicas, while the other comprised all the diploid robusta genotypes (Figure 3a) with significant branch support. The selections formed a single cluster within the tetraploids cluster, while pure arabicas and hybrid-selections appeared in distict sub-clusters. Similarly, in clustering analysis of 14 related species (12 Coffea and two Psilanthus spp.; Figure 3b) along with two genotypes each from C. arabica and C. canephora, tetraploid Erythrocoffeas (C. arabica) and diploid Erythrocoffea (C. canephora) formed coherent clusters. Moreover, the grouping of the related taxa, in general, was as per their botanical type with few changes. Though all the entries from Erythrocoffeas fell into one ) appeared as independent strong groups. Single Melanocoffea species (tested in this study), C. stenophylla was not grouped with any of the above species cluster but was found close to the Coffea species than the Psilanthus spp. 
Similar results were obtained using the data from genomic SSRs (CCRMs, data not shown). 

Discussion


SSR motifs in coffee transcriptome, and development of new EST-SSR markers

In the present study, 15.4% of the coffee ESTs were found to contain SSRs, which is comparable with our earlier study [11], but much higher than 2.7-10.8% that was reported for 18 representative dicotyledonous species [27], and 7 210% reported for monocot species [28]. Notwithstanding this apparent enrichment/ higher abundance, the SSRs in coffee transcriptome were very comparable to other plant species in observations like: 1. Abundance of TNRs than DNRs; 2. Abundance of AG among the DNRs followed by AT; 3. CG as the least abundant among the DNRs; 4. Abundance of AAG among TNRs (among the dicots); 5. Predominance of GC-rich TNRs (but not CCG/GGC) than the non-GC-rich TNRs. 
A total of 18.7% of the detected EST-SSRs were found to be suitable candidates for primer design, a comparatively lower proportion (,50%) then we reported earlier [11]. The main attributes that rendered majority of the identified SSRs unsuitable for marker development were: a shorter repeat core (,18 bp) and/or flanking sequences of low complexity (AT/GC-rich and/or regions prone to secondary structure formation) or shorter lengths seriously constraining designing of optimal primer pairs. However, in this study, primer-to-marker conversion ratio (ca. 88%), was higher than many earlier similar studies in other crops. Such differences in marker conversion ratios are expected due to differences-in the quality of primers designed), GC content of the genome, the genome complexity, and/or genome size [13]. 

SSR enrichment and development of genomic SSRs

The genomic DNA library constructed in this study, resulted in very high proportion of SSR+ive sequences, with very low degree of redundancy (9.1%; 6 out of 66 identified SSRs positive sequences). This was notable, as in earlier similar studies the apparent high success rates were generally confounded by high degree of redundancy [29]. Similarly, the proportion of SSR positive sequences found suitable for primer designing was also higher (87%) in our study than the average of 54¡3% recorded in other species [30]. These obsrevationssuggest that the enrichment approach used in this study may be a desirable strategy for efficiently entrapping and targeting the SSRs even in genome(s) like coffee that are relatively poor in SSR motifs [2]. 

Utility of new EST-and genomic SSRs as genetic markers

The SSRs provide desirable markers for studying genetic diversity, germplasm characterization, constructing reference panels/bar codes, for individualization of genotypes, linkage mapping, population biology, and taxonomic relationships of related taxa [2]. Therefore, it becomes desirable to validate the new markers for their utility in genetic studies, which unfortunately has been lacking in majority of published studies describing development of coffee-specific SSR markers. 
Various genetic parameters viz., allelic diversity, PIC, H o , H e , HWE, LD calculated for all the new EST and genomic SSRs and mapability on linkage map, amply suggested their possible utility as genetic markers (see Table 4 & 5). In general, the extent and pattern of allelic/genetic diversity revealed by the new markers conform to that reported earlier for the coffee genomic SSRs [5,6,31], and the EST-SSRs [8,11]. 
Different genetic parameters/tests such as H o , H e , LD, HWE are important indicators of origin, evolution and distribution of diversity in the available genepool. The heterozygosity measures (H o , H e ) for the new SSR markers indicated heterozygote decay (deficiency) in the tested germplasm. The HWE and LD analysis of the polymorphic markers were in general agreement with our earlier observations with genomic as well as EST-SSRs [5,8,11]. Overall, these studies indicated that the tested robusta germplasm comprised allogamous, relatively unrelated genotypes, while autogamous tetraploids comprised mostly of hybrid varieties/selections with overlapping/shared pedigrees. The results thus suggest the suitability of the new markers for reliably ascertaining genetic diversity in the coffee gene pool. 

Cross-species/2generic transferability

All the new EST-and genomic SSR markers revealed very high and robust cross species/2generic amplifications with alleles of comparable sizes when tested on 12 other Coffea and two Psilanthus taxa. The data revealed that the markers described here show much higher taxa transferability than earlier published genomic2/EST-SSR markers [2,5,7,11]. This is significant as successful cross-species amplification is generally restricted to related species within a genus and reduces when tested for different genera [32]. Further, it was interesting to note that the new SSRs that were monomorphic/uninformative for the tested arabica/robusta germplasm, exhibited considerable polymorphism across the tested related taxa (the only exceptions were the marker CCESSR16 and 18 that showed a very low conservation even across the Coffea spp.). Thus the new SSR markers described here strengthen the possibility of their use as Conserved Orthologous Sets (COS) for genetic characterization of different related wild coffee taxa, and also for coffee taxonomic/synteny studies. 

Diversity analysis and genetic relatedness within/between Coffea and Psilanthus species

The EST2/genomic-SSRs described in this study were able to group all the 16 genotypes (representing the cultivated genepool) in phenetic clustering that was indicative of their species status and known pedigrees (Figure 1a). Similarly, the analysis 14 Coffea and two Psilanthus species, revealed generic affinities that were largely in agreement with their known taxonomic relationships (Figure 1b), based on their geographical distribution as well as Chevalier's botanical classification [33]. Importantly, the analysis distinctly separated the two Paracoffea species (P. bengalensis and P. wightiana) from all the other Coffea spp. These results are similar to the earlier published studies undertaken to ascertain species relationships using SSRs [2,7,8,11], as well as other marker approaches [34][35][36]. These results, thus, amply demonstrate that the new SSR markers developed in the present study can be considerably informative in exploring the taxonomic relationship of coffee species complex. 

Conclusions

The present study describes a total of 69 new validated SSRs; 44 EST-SSRs developed from coffee transcriptome using in-silico methodology, and 25 genomic SSRs developed using SSR enrichment approach. In addition, it provides primer pairs for additional 270 putative EST-SSRs. Analysis of the identified SSR-positive ESTs also provided insights into the relative abundance and distribution pattern of different SSR motifs in the coffee transcriptome, which was found to be relatively rich in its SSR abundance. Among the identified EST-SSRs, TNRs followed by DNRs were more abundant than other SSRs, and among different types of SSR motifs, AG was the most abundant. All the 69 markers were found to be polymorphic in the tested coffee/related germplasm and their utility as efficient genetic markers could be demonstrated for diversity analysis, germplasm individualization, linkage mapping, cross-species transferability and taxonomic studies. As many of these SSRs showed a very high cross-species transferability, they can aid in conservation, management and resolving taxonomic relationships, as Conserved Orthologous Sets (COS) for Coffea and Psilanthus species and more importantly as efficient, and informative genetic landmarks on molecular linkage maps. 

Figure 1 .

 1 Figure 1. Representated Gene Scan profiles showing the SSR alleles obtained using the new SSR markers for some of the coffee genotypes tested in the study for marker validation. The right side set of 3 panels is for the genomic-SSR CCRM-33, and the similar set on the left is for genic-SSR CCESSR05. The 3 panels in each set represent 8 genotypes each of arabica and robusta coffee, and 8 of the related coffee species, respectively, from right to left end. doi:10.1371/journal.pone.0113661.g001 


11 -Figure 2 .Figure 3 .

 1123 Figure 2. Map positions of 18 new SSR markers developed in this study (11 CCESSR and 7 CCRM markers) on robusta linkage map; mapping population was derived from a cross between CxR (a commercial robusta hybrid) and a local selection Kagganahalla [26]. The SSR markers of the existing map used as anchor markers are shown in italic and bold face. doi:10.1371/journal.pone.0113661.g002 




  cluster, it contained two entries from Pachycoffeas (C. dewevrei with C. canephora and C. liberica with C. congensis). The remaining four of the Pachycoffeas (C. excelsa, C. arnoldiana, C. aruwemiensis, C. abeokutae) grouped with each other with good bootstrap support. The C. salvatrix a Mozambicoffea was also grouped with these Pachycoffeas, while the other three Mozambicoffeas (C. racemosa, C. eugenioides, C. kapakata) and two Paracoffeas (Psilanthus spp. 




  




  


Table 1 .

 1 Details of the new EST-SSR markers developed using EST database and validated using diverse coffee genotypes in the present study. 


	Sl. No. Marker Id	Primer sequence	Repeat motif	Allele size (bp)* Source EST ID** Linkage group
	1	CCESSR01	F: TGGTAGCACTGTCGGAAGCATAT	(AGC)7	239	-
			R: GACCCATCTAACTTGCTGCATTTT			
	2	CCESSR02	F: AAGATATGTTTTAGCCCAAGTAGTGAC	(AT)11	168	-
			R: ATTGGTTGGTACTGTTTAGCTGTTCAT			
	3	CCESSR03	F: CAGCCGTATCAGCACCAGCAT	(TC)10	217	CLG02
			R: TTCCCAACCCGTCAAAGTCCT			
	4	CCESSR04	F: GTCGACAACAGCGCTCAAGATC	(TCT)6	259	-
			R: CAAAAAAGACTGGAAAGAGGGTATTAG			
	5	CCESSR05	F: AGGGGCTGGTTATTTTTTGGG	(GCA)6	141	-
			R: GGGGGTAAATACGGGAAAGCAGA			
	6	CCESSR06	F: AACCCTCCCTCCTCCACCTTTTC	(AC)23	224	CLG09
			R: GGAGGTGGTGGTGGTGTAAAAAAAG			
	7	CCESSR07	F: CCCCCTTCCCATTTTTTCCCCT	(AGC)6	194	-
			R: CCGGGTGGTAGAGAGATTGCTGCA			
	8	CCESSR08	F: AAACCACAACATCCCCCAAGAGT	(GCA)8	149	-
			R: GGCAGTAGAATTGGAGCGGTGAAA			
	9	CCESSR09	F: CCCCCACCCACTTCTCTTTG	(TTC)8	184	-
			R: ACAACAAACGAACGCTCTCTGATAA			
	10	CCESSR10	F: GCAGAAGAAGCACCAGTAGCAGAA-	(GCT)7	207	-
			GAAG			
			R: TGCCTTCTACTCTTCACTCTTCTCCACT			
	11	CCESSR11	F: AGGGAAAAAGAAGAGTGAGAAGA-	(GAA)6	140	-
			ATATT			
			R: GGGACACTCACATATACTGCAAAC-			
			TTAG			
	12	CCESSR12	F: CCGCCATCCCTTTTGCCTTTC	(CCA)6	200	CLG02
			R: ACGGCAGCAGAAGTGGAGGTGTT			
	13	CCESSR13	F: GCGGGGTAGTTTTGGGAATATGG	(TTA)6-tt-(TTC)6 124	CLG02
			R: TTTGGGGTCCTTTTTCTTTCACACAT			
	14	CCESSR14	F: CTTGCCCCCTTCCCTCCCACTC	(CT)10	232	-
			R: TTCGGCTCCTTGTGTTTGGGTA			
	15	CCESSR16	F: AGAGCAATGAGAAACAAACGAAACT	(CAT)6	173	-
			R: AGGTGCCCAACTATCCCAGAAT			
	16	CCESSR17	F: CTCCACACCAACAAAATCCCACTT	(CAG)6	169	-
			R: CCCACATCCTGAGTCTGCTGCTAA			
	17	CCESSR18	F: GGGGAGGATGCTTATGATATGAGG	(CAC)6	152	-
			R: TCCGGTTCACCTGCTTTTCCTT			
	18	CCESSR19	F: CATCGTATCTCGCCCTCTCTCTTTC	(CA)13	219	-
			R: CCACAACAAGTACAACCAACCGAAAC			
	19	CCESSR20	F: TTCTGGCCGATTGATTGTGAT	(AAG)7	143	-
			R: GCGACAAGGCTGACAAACTACTAC			
	20	CCESSR21	F: CGAGCTAGTGCAGACAGATTGAGAT	(AG)17	164	CLG13
			R: GTCCTTGGCGAAATCCCTCAG			
	21	CCESSR22	F: CCCTCAATCTCGTCCCCCTCT	(TC)9	153	-
			R: CCCTCCATAAATCTTCTTCACGTACTC			

Table 1 .

 1 Cont. 


	Sl. No. Marker Id	Primer sequence	Repeat motif	Allele size (bp)* Source EST ID** Linkage group
	22	CCESSR23	F: GGCCTCTCTTTAATTTTCTTGTCTTT-	(TTC)8	160	-
			TTTC			
			R: ATGGAGGGTAGGGTTTCGAGAGTGA			
	23	CCESSR26	F: AACCGGCCTTCTTGTATGATTCTCTA	(CAT)8	206	CLG07
			R: TTGGCTAACCCTCACTCTCTCCCTACTA			
	24	CCESSR27	F: GCCCACTCCATTCGTACTTGTTTCC	(CT)12	120	-
			R: GCGGTGCTGCTCAATGCTCAT			
	25	CCESSR28	F: AAAATGAGTGACGATGGGAAAGACA	(CCA)7	193	-
			R: GAGGGAAGCCGATCACTGGTTG			
	26	CCESSR29	F: GGCGCTAGAGTTGGTTGTTTGC	(CTTCT)5	95	-
			R: CAGGCATTGGAACCAGCGAAC			
	27	CCESSR31	F: AGAAGAGTACTGAAGGCCTGGAAGA	(GTG)6	220	-
			R: AGCATCTGCAGCCTCCATAGC			
	28	CCESSR32	F: CTTGGCGTTTAGCGTTCTCACATT	(TA)9	163	-
			R: GCTCAACCAAACCAATACATACCTCTT			
	29	CCESSR33	F: GCCCGCATGGACGACTTGGA	(AGG)7	227	CLG13
			R: CGCTTGACGTATCCTTTGGCCTCT			
	30	CCESSR34	F: GCATTGCTCCCCCCACTTCA	(CTC)6	168	-
			R: GAGCATGGGGACGAGGAGGA			
	31	CCESSR35	F: CTGCTAATGCTGCTGAAAAAGAGATACC	(AG)10	107	CLG13
			R: GGCTGTGAATTCTTGTGACTTGTGACT			
	32	CCESSR36	F: AGCCTTCTGCAATTCCCTCGTACA	(AG)9	100	-
			R: GGCGTCGTAGAGGGCATTCAGA			
	33	CCESSR38	F: GCCCGAGGGTTAGATTGATCA	(AG)12	162	-
			R: CTTGTCTTCTGTTTGATTTTGTGTTCTA			
	34	CCESSR39	F: GCGACCGGACGACCAAAAATAAT	(GAA)7	131	-
			R: CGCCGTCGTCAGAGTCATAATAATCA			
	35	CCESSR40	F: CGTGGGGGTTTGTTTTTCTCG	(AGG)7	205	-
			R: GTCCCCCCTCAGCCGTTTTTG			
	36	CCESSR41	F: GGGCTGCAGGCTTGTCACCAC	(GA)10	205	-
			R: AATCGGTTTAGTTTTTTGTTTCCTCAC			
	37	CCESSR42	F: CGGGCGGAAACGGTCAGATC	(GCA)7	117	-
			R: TGCCGTTGTTGTTGTCCAGGTG			
	38	CCESSR43	F: CCCAGCAAGAACTCAACCCCATCA	(TTC)8	172	CLG02
			R: TGGCCTAATGAAGATGACGTTGCTGATG			
	39	CCESSR44	F: AGGAATAATGGAGGAGACGTTGTTG	(CTT)7	236	CLG02
			R: GCACAAATCCCAGTACTTCCTCATAGA			
	40	CCESSR45	F: AAATGGCCGAGATAGAGAAGGAGAAG	(AGG)7	135	-
			R: CCCACTCCTCCGCGGTACTGATC			
	41	CCESSR47	F: GCAGCAACAATCACTTCCACAGC	(GCA)6	198	-
			R: TGCTGTTGTAACTGCGGGATTTG			
	42	CCESSR48	F: GCAACCTTATCTAGATTCAACTTCAACTT (AAATCA)5	188	-
			R: CGGGAAGAAATGGCAGCCTATAC			
	43	CCESSR49	F: GCGGCCATCCTTGTCTTCG	(ATC)8	186	CLG02

Table 1 .

 1 Cont. 


	Sl. No. Marker Id	Primer sequence	Repeat motif	Allele size (bp)* Source EST ID** Linkage group
			R: TAGCCGCTGACGTAATCTTCCTT				
	44	CCESSR50	F: GGGATGATGTGGATTCTATGGTCTACTA	(CAG)7	108	123181	CLG10
			R: ATGCCATTTTAACACTTCCTCCTCA				
	CCESSR: CCMB Coffee EST SSR marker;				
	F: forward primer;					
	R: reverse primer;					
	-: Unmapped;					
	CLG: Combined Linkage Group [1];				
	*: Predicted amplicon size based on source EST sequence;				
	**: Source EST ID as per the downloaded SGN database (ftp://ftp.sgn.cornell.edu/coffee/).			
	doi:10.1371/journal.pone.0113661.t001				

Table 2 .

 2 Details of the new genomic SSR markers developed using streptavidin-avidin affinity capture SSR-enriched library in the present study. 


						Allele size	GeneBank acc.	Linkage
	Sl. No. Primer Id	Primer sequence	Repeat motif T a ( ˚C)	(bp)*	No.	group
	1	CCRM02	F: AATGGTGGCAGTCCTGAAAGATC	(GA)12	57	268	KM874369	-
			R: AACATCAACTTTCCTGGTCTTC					
	2	CCRM06	F: TTCTTATCACCTTGGGCTACCTTTCTTC	(AG)8	57	146	KM874370	-
			R: AAGCGGTTTAGTTTTTTGTTTCCTCAC					
	3	CCRM07	F: TAAAGGATGGTATATGTGGCTGGAGTA	(AT)8	57	126	KM874371	CLG01
			R: CCACAGCCTCGGCATTTACTATATAT					
	4	CCRM10	F: AAAAAGACAAGATTCAACCTGCAGTAGT	(GT)9	57	104	KM874372	-
			R: TTCCCACCCCCCAAAAAAAA					
	5	CCRM14	F: ATTTGATTTCTTCTTTCTCTGTTGTC	(CT)22	55	130	KM874373	-
			R: ACAAAAGCCCTGAAAATAATAGATCTA					
	6	CCRM15	F: CGAAATTGACGAAGCTCTTGTT	(CTT)6	57	243	KM874374	-
			R: TTGCTAGTTTCGAAATCGTGTAAGGAC					
	7	CCRM16	F: TCCTATAGCAGAAACACAAAATGACACAG	(TC)26	55	223	KM874375	-
			R: GGTTTTTGGGTTCTTTTTAGCATATACA					
	8	CCRM17	F: TAAGCGTTGGAATTCCTCACTCTATCT	(CT)17	55	228	KM874376	CLG01
			R: ACAGCTAAAGAAACAATGAACCAGT					
	9	CCRM19	F: GTTTTTTTTTTCTTTTTTCTTTTTGAGCT	(GA)26	57	252	KM874377	CLG06
			R: AAGGCAATGTTGGTCAGCAGTGG					
	10	CCRM21	F: CACCCCTCCCATCCGTTGAAACAT	(GA)16	57	258	KM874378	CLG06
			R: AATGATGCTCCCAGTGTTTGATGA					
	11	CCRM22	F: CTTGCAGTTTACTTCCCTTTGGTTG	(GA)29	57	241	KM874379	-
			R: TTTTCTTCTGTATATTGTTGGAGTTCTTC					
	12	CCRM23	F: CGGCAGTGTGGTCCCCTTTGAAT	(GTT)6	57	141	KM874380	-
			R: AAAAAAAAACTCACACTCTATCAAAACTAAGG					
	13	CCRM24	F: GAGTGTGAGTTTTTTTTTGTGACCTTAA	(GA)9.(GA)9	57	213	KM874381	-
			R: ACCCCACATTCCTCTCATCCATTC					
	14	CCRM28	F: GGGGCAACAAGTGGTAGGATATGAAGAC	(CA)10	57	209	KM874382	-
			R: CGCCTTCACTATGGTTTTGCCTTCTAA					
	15	CCRM31	F: CTTTTATGTCTATCTGTCTCTGCTTTTC	(CA)10	57	114	KM874383	-
			R: CCTGCAGTAGTTTCACCCTTTATCC					
	16	CCRM33	F: ACAGCCGTTGAACTTATGGGATTACA	(CT)12	57	118	KM874384	-
			R: ACAAAGGGATGGAGAGGATGGAATATAC					
	17	CCRM34	F: CCCCAGAACGAAAGGCAATCAT	(GA)10	57	165	KM874385	CLG05
			R: TTGGGACTATTTATACTGGGGAAGAA					
	18	CCRM35	F: GGGGTTAAATCAGGGGAAAAGTGG	(TG)12	57	144	KM874386	-
			R: AAGCGAGGGAGAGAGCAGCAGATC					
	19	CCRM36	F: CCATGGGGCAAAAGGCAAATTCTAT	(CT)18	57	171	KM874387	-
			R: TCCAGACCGCCGTTCACGAAGTATA					
	20	CCRM37	F: TGCTTCCCTTCTCATTCTGGTACTTT	(GT)10	57	146	KM874388	-
			R: AATCCATCAACAACTTCAGCATACCA					
	21	CCRM38	F: TGAGAATTAAAGCAGCAGGGGTATG	(CA)10	57	204	KM874389	CLG05
			R: GCAAAAAAAGGCAAAAGCATTACATC					
	22	CCRM40	F: ATTCACGCTTTCATTACTTTTCTC	(CT)29	57	176	KM874390	CLG05

Table 2 .

 2 Cont. 


						Allele size	GeneBank acc.	Linkage
	Sl. No. Primer Id	Primer sequence	Repeat motif T a ( ˚C)	(bp)*	No.	group
			R: TTTGTATTTCCTTTCCATTTCTTTTGTA					
	23	CCRM41	F: AGCAGAAACACAAAATGACACAGAGCA	(CT)24	57	161	KM874391	-
			R: AATGGTCCAAGGAAAATGAAAAATGTT					
	24	CCRM42	F: CGGAGAAGAGCAATATACAAGCAAGG	(GA)13	55	143	KM874392	CLG11
			R: GCCACCCCAGAACTTTTGCAA					
	25	CCRM45	F: CTTCAAGCAAAATTTTCAACAGCACAG	(GA)10	57	187	KM874393	-
			R: GGCCCTTTTTTAGTCTCACCACATT					
	CCRM: CCMB CXR Microsatellite marker;					
	T a : annealing temperature;					
	F: forward primer;						
	R: reverse primer;						
	-: Unmapped;						
	CLG: Combined Linkage Group [1];					

*: Expected amplicon size in the robusta variety CxR. doi:10.1371/journal.pone.0113661.t002 
Table 3 .

 3 Details of primer pairs for additional new EST-SSR markers designed in the present study. 


						Predicted		Predicted
						Tm ( ˚C)		Tm ( ˚C)	Predicted
	Sl		Unigene	SSR repeat		forward		reverse	amplicon
	No. Marker ID	ID*	unit	Forward Primer	primer	Reverse Primer	primer	size (bp)
	1	CCESSR51	119463 (AGAGCA)5 AGCAGAGATCGAGACAGAG-	59	ACATATCTGAAACC-	59
					AGA		CTCGGC	
	2	CCESSR52	119606 (CT)9	AATCGGAGGATTTGTGCTTC 59	GCGCCTAAATCACC-	59
							CATATT	
	3	CCESSR53	119736 (GA)11	CCGCGGTCAGTCTTACTACA 59	ACACAAATCAACAC-	58
							CCATCC	
	4	CCESSR54	119787 (GGA)6	CGCAATCTTGAATGAGGAGA 59	TGGGAGGTTGATCA-	59
							TCTGAA	
	5	CCESSR55	119820 (CT)11	AGGACAGGAGTGTGATCCCT 59	GCCATGTCCTCCTTT-	59
							CGTAT	
	6	CCESSR56	119897 (TC)11	AGGTTTTTGTGTCCCTTTGG 59	CATCGATGAAAAGCA-	59
							GCAGT	
	7	CCESSR57	119919 (AT)15	GAATTTTGTCAGCCAGAGCA 59	GACGGAAAGATTCTG-	58
							GCTTT	
	8	CCESSR58	119987 (TCC)7	AGCTACGCTAGGCAATTGGT 59	GACAACAACAACAGC-	58
							CAACA	
	9	CCESSR59	120008 (TC)12	GGAACAAGACTCTCTTGCCA 58	TCATCACACAAGGAG-	59
							GCAAT	
	10	CCESSR60	120008 (TG)9	ATTGCCTCCTTGTGTGATGA 59	CCGGTCGATCAACAA-	59
							TCTTA	
	11	CCESSR61	120045 (TCT)6	GGTGAAGGGTCCTTACCTGA 59	GAGATGTGCTACTGG-	59
							CTACTGC	
	12	CCESSR62	120064 (GATT)5	CTCTTGTTTCCAACCCAACC 59	CGGACACTGTGAGGA-	59
							GAGAA	
	13	CCESSR63	120107 (TCT)6	AGTCCAGTCCAGTCCTGTCC 59	CCGATATGATTTTGGT-	59
							GCTG	
	14	CCESSR64	120121 (GCT)7	CGAAGTTGTGCAGGATGAAC 59	GGAGCTGCTTGCTCT-	59
							TCTTT	
	15	CCESSR65	120179 (AT)10	GTGATGCTCGGTGTATCTGG 59	ACTAGAGGCCGAGAA-	59
							TTGGA	
	16	CCESSR66	120206 (AGA)10	GATGAGCTCCAAAACAAGCA 59	AAAACTTCCCAGGCTT-	59
							CAGA	
	17	CCESSR67	120260 (CTCC)5	GTCGTCTGTTCCTCCTCGAC 60	CCACTAATCCCGAGCA-	59
							AAAT	
	18	CCESSR68	120291 (AAG)8	CCACGCGAATAATCATCAAC 59	AAGCACCTTATCCCCA-	59
							ACAG	
	19	CCESSR69	120316 (TCA)6	TTGGAAAACCATAGAAGGGC 59	ACCCTCATCAATCTCT-	59
							TGCC	
	20	CCESSR70	120320 (GCCACC)4 TTAAATCAGCCCTCAAGCCT 59	CTTGAAATCTTGCGCA-	59
							CTGT	
	21	CCESSR71	120322 (TTC)6	TTAGAAAAGCTGCGAGACGA 59	TTGACCATTTCCCCTT-	59
							C-	
							TTC	
	22	CCESSR72	120543 (AT)9	GATTGCTCTCTTTCTTCGGG 59	TCCGCCCCAGTTAGAT-	59
							TTAT	
	23	CCESSR73	120545 (AT)9	GTTTTCCGGCTAGCTTGTTC 59	GTGCATGAGGTGAAAA-	60
							TGGA	
	24	CCESSR74	120579 (CCT)7	TGTAATATTGCTTCGCTCGG	59	AAGGGATGATGCCTAG-	
							TTGG	

Table 3 .

 3 Cont. 


						Predicted		Predicted
						Tm ( ˚C)		Tm ( ˚C)	Predicted
	Sl		Unigene	SSR repeat		forward		reverse	amplicon
	No. Marker ID	ID*	unit	Forward Primer	primer	Reverse Primer	primer	size (bp)
	25	CCESSR75	120596 (TCC)7	GGCTCAATCGATAAGCAACA 59	CATGAACATATCCTGG-	59
							AGCG	
	26	CCESSR76	120656 (AAC)6	AAGCGAAGATGGAGAGCAAT 59	CCGCCTTTTGTAGAA-	59
							GAACC	
	27	CCESSR77	120656 (TCA)6	TTTTAGGCCAATCCTTCACC	59	GTATGTGAGGGCGTA-	59
							ACGTG	
	28	CCESSR78	120720 (GAA)6	GAGCCCTCTTTTTCCTCCTT 59	CCGTAATGATACAAAC-	59
							CCCC	
	29	CCESSR79	120764 (CATATA)	AGCAGCACCAGACTAGCTCA 59	GTCCCAGAAACGAAG-	59
				4-13 bp-			ATGGT	
				(AT)9				
	30	CCESSR80	120821 (ACAGG)4	CAGAATCCCATATTCCCCAC	59	TTGCTGTCTGATTTCC-	59
							AAGC	
	31	CCESSR81	120883 (TCAAT)4	CAGATTCCGGTGTCACAAAG 59	TTTGCTGCAGTGTCAC-	58
							TTGT	
	32	CCESSR82	120965 (TCGAAA)4 GACTATGGATGGCTTTGCCT 59	GGCGGATTGGTTGAT-	59
							AGAAT	
	33	CCESSR83	121010 (CCTGCA)4 TGAATCAACTCCTGCTCCTG 59	GGTAGTTCCAGCCA-	59
							CCAGAT	
	34	CCESSR84	121086 (TC)11	GGATCAAACGTGGCTAAGGT 59	AAGAAAAGGGCTAC-	59
							AACGGA	
	35	CCESSR85	121131 (AAG)6	CCCCGGGCTGCAGAAACAA-	60	AGCTCCGGTAAGCC-	59
					GT		TCAATA	
	36	CCESSR86	121240 (GCA)6	AACCCTCCACTCCATTTCAG 59	ACTATTGTTGCTGCTG-	59
							CTGG	
	37	CCESSR87	121289 (TTC)6	GAAGCAACCCCTCAACTGAT 59	AGCACCCTCTGCTTC-	59
							AATCT	
	38	CCESSR88	121377 (GT)9	CACGCGGATAATACACCG	59	AGTTGCTCCTGCTT-	59
							CTCCAT	
	39	CCESSR89	121392 (GCT)6	GCACTGTTTTGAATGGTTGG 59	TCCGCACTACAAGT-	59
							ACCGTC	
	40	CCESSR90	121439 (GATTA)4	TACTTGAGCGATCAGAACCG 59	TAATCCTGCGTGCTA-	59
							TTTGG	
	41	CCESSR91	121548 (CTTCT)5	AGGCGCTAGAGTTGGTTGTT 59	CAAAGTAAGCAGCA-	58
							GGCATT	
	42	CCESSR92	121580 (TC)11	CCTACATCCCACGACCTTCT 59	GTGGTAGTGCTTTG-	59
							GTGGTG	
	43	CCESSR93	121610 (AT)9	CTGCCCTATGATGAATGGTG 59	TCATCTCAAGCATCG-	59
							TCTCC	
	44	CCESSR94	121752 (TGCTCC)4 CCTCTCATGCCAGCAACTAA 59	AAAGCACTGGGAAC-	59
							TGAAGC	
	45	CCESSR95	121841 (CT)10	TGGAGCAGAGATTGTCAAGG 59	CCAGCTGGAACTTC-	59
							CCTG	
	46	CCESSR96	122004 (AT)19	ACATTCGAAAACTTGGGGAG 59	CAAGGTCTTGGGTT-	59
							TGTCCT	
	47	CCESSR97	122006 (AG)14	CTCGTGCTGTAACCCTCTCA 59	AGTGTGATGGAAC-	59
							GCGAATA	
	48	CCESSR98	122012 (AGC)6	GACGCGCAGTCTTTCAAGTA 59	GCTTCACAGTCGTCT-	
							TCCAA	

Table 3 .

 3 Cont. 


						Predicted		Predicted
						Tm ( ˚C)		Tm ( ˚C)	Predicted
	Sl		Unigene	SSR repeat		forward		reverse	amplicon
	No. Marker ID	ID*	unit	Forward Primer	primer	Reverse Primer	primer	size (bp)
	49	CCESSR99	122077 (CGC)6	TCGGACTGCTATAGTGGACG 59	TCCTTCGAGACGTTA-	59
							GCCTT	
	50	CCESSR100 122089 (AG)9	TTCTGCAATTCCCTCGTACA	59	AGGGGTCAGAAGAA-	59
							GGGATT	
	51	CCESSR101 122184 (AT)10	AGGGGTGGATTCAGTAGACG 59	ATGCAATTGAACCGA-	59
							CTCAG	
	52	CCESSR102 122195 (AGGCTC)6 AGTCAGCTTCTTGCTGCTCA 59	CCCTCCAGAGTACCC-	59
							ATTGT	
	53	CCESSR103 122254 (TCGT)5	GGAATGAGCTAAAAGCCCAG 59	TTACCGTGTAGGTTG-	59
							GTGGA	
	54	CCESSR104 122257 (TTCA)5	GCTCAGTGAGGTTCCAAACA 59	ACCCTGTAGGTTGGT-	59
							GGAGA	
	55	CCESSR105 122324 (AGA)6	AGGCCATAGTGGTGAACACA 59	ACAAAGCACAGAACC-	59
							AACCA	
	56	CCESSR106 122340 (TC)10	AGCTGATTGAGGATGGCTCT 59	CATTGGCTTTCCCCA-	59
							ATACT	
	57	CCESSR107 122383 (CTT)8	CCTTCTCCCTCTTCCCTTCT	59	TGAGTGACAGCGTC-	59
							AACAAA	
	58	CCESSR108 122387 (CTT)7	GTGGGAAATCTCAAATGGCT 59	CTCTTTCTGCTGGG-	59
							TTGTGA	
	59	CCESSR109 122514 (AGG)6	GAAAGACCCCAAACCAAGAA 59	GGAGTCATCAACAA-	59
							TGGCAC	
	60	CCESSR110 122533 (TA)10	GTGCCGTTGTTTGTTTCATC	59	GTGCATGAGTGGAT-	59
							TCAAGG	
	61	CCESSR111 122550 (TCA)6	CTTCATGGACCATTCTCACG 59	TAGCCGAAACAGAG-	59
							CATTTG	
	62	CCESSR112 122619 (GAA)6	TCCCCTGAATTGGACTCTTC 59	ACAAAGGCCAACGTT-	59
							TTACC	
	63	CCESSR113 122646 (AGC)6	GCATCCTTGTATTCGGAGGT 59	ACCACTCGCTTCAAC-	59
							CTCTT	
	64	CCESSR114 122681 (AAAG)5	CCAGGCTAAGTGCTCATCAA 59	GGCAGCGCAGTAAA-	59
							GTCATA	
	65	CCESSR115 122758 (TC)13	CTCCCATTTCCTCTTTCTCT	55	TAGGGTTTTGAAGG-	59
							GCAATC	
	66	CCESSR116 122793 (TC)18	GAGAAGTTTGCCAGAACCCT 58	ATCAATCTTCAAAG-	59
							GGCCAC	
	67	CCESSR117 122797 (CT)12	CTCGTCCCTCTCCCTGTACT 58	ACCACAACAGCGAA-	59
							CATCAT	
	68	CCESSR118 122811 (AAG)7	CTTTCCTCAGCTTCACCACA 59	CCCCTGTTTCTGGT-	59
							CTTGTT	
	69	CCESSR119 122842 (ATA)6	TGCAAGTGTGATGAATGTGG 59	CTAAGGCGAGAAAA-	59
							CAAGGG	
	70	CCESSR120 122880 (GAAGCA)4 TAGGGGGACCAATAGCAAAG 59	ACACGTCTTGCGAC-	59
							AAAGTC	
	71	CCESSR121 122996 (TTA)6	CCGGACAGGAACAAGAAAAT 59	CATTCAATTGCCTG-	59
							ACCATC	
	72	CCESSR122 123010 (TCC)	GTCTGCATCTGCTTGCTCAT 59	AAACCATCTATCCC-	
				6-30 bp-			AGCCAG	
				(TCA)7				

Table 3 .

 3 Cont. 


						Predicted		Predicted
						Tm ( ˚C)		Tm ( ˚C)	Predicted
	Sl		Unigene	SSR repeat		forward		reverse	amplicon
	No. Marker ID	ID*	unit	Forward Primer	primer	Reverse Primer	primer	size (bp)
	73	CCESSR123 123023 (CTGCCT)4 GACAGAGGAATCCTTGCCAT 59	TCCCAGAAAAATCACC-	59
							TTCC	
	74	CCESSR124 123110 (AACTA)4	GAGAAGCGAACCACACTTGA 59	ACTTGAGCAGCAACCC-	59
							TTTT	
	75	CCESSR125 123132 (CTATGA)4	GAGCGCTATGGATGAGCATA 59	GCTGTACAGGATTGGG-	59
							AGGT	
	76	CCESSR126 123134 (CTG)6	ACCACCACAAGCACAACAAT 59	TGACCCCCAACAAAAC-	61
							AAAG	
	77	CCESSR127 123160 (TA)11	CTGATGGGCGTCATCAATAC 59	GGGAAAGGGAGTCTC-	59
							AACAA	
	78	CCESSR128 123185 (ACG)6	GAGGAAATTCGGGAAGTTGA 59	CATCGTCATCATCATC-	59
							GTCA	
	79	CCESSR129 123240 (TTC)6	CCAGAACCAACTAGGGTTTT 56	TCACTTGAATTGCAGA-	59
							AGGC	
	80	CCESSR130 123248 (GCA)6	TCTTCTACAGCTGCCACCAC 59	ATTGCGAAGAATGAAG-	59
							GGTC	
	81	CCESSR131 123291 (CT)10	CTTCTCCAAACGGACCAAAC 60	CTCAGCCTCCATTGCA-	59
							CTAA	
	82	CCESSR132 123309 (AT)20	CGTAAATTAGGGGCGTTGTT 59	TTGGCACAAACCTGAT-	60
							GGTA	
	83	CCESSR133 123323 (ACAT)5	TCTCCCCATGGTACTTCACA 59	CCCATGCATTTCCAGA-	59
							CATA	
	84	CCESSR134 123329 (TC)13	TCACTGCCTGAGGCTTTATG 59	GAGCATGGTCCCGATA-	59
							AGAT	
	85	CCESSR135 123332 (AG)10	TTGTTCTCTCCAGGGGAGTC 59	CGTAGAACCAATCCAG-	59
							CAGA	
	86	CCESSR136 123342 (AC)9	AATATGAATGGAGGCGAAGG 59	ACAATGTCCCGAACTG-	59
							TTGA	
	87	CCESSR137 123362 (AAT)6	GGTCTGAAAGTGGCCAAAAT 59	GGGAGATACGCTCAAG-	59
							GTGT	
	88	CCESSR138 123381 (CGG)6	AAACCTAGTGGTGGAGGTGG 59	CAGCTTGGTTCCAGTC-	59
							TCAA	
	89	CCESSR139 123445 (TC)9	AATCTTGTGGAGGAAATGCC 59	GCTTTTTGCTTCGGTA-	59
							CCTC	
	90	CCESSR140 123562 (CT)9	CGAGGCTCTATCCTCCTCTC 58	CTGACGCATCTGACCT-	59
							CACT	
	91	CCESSR141 123682 (CTC)	AGTACCTCGACAACCCCAAC 59	AGGCTCTGCCAAATCA-	59
				7-10 bp-			AAGT	
				(TTC)7				
	92	CCESSR142 123741 (CAGCAA)4 GAAGTTTGAAGCCTGAAGCC 59	TTCCGCGTATCTGAAT-	59
							CTTG	
	93	CCESSR143 123742 (TTGTA)4	AACCGATTTGATTCAGAGCC 59	CTCCACTCCACTGGGT-	59
							TTTT	
	94	CCESSR144 123743 (TTGTA)4	AACCGATTTGATTCAGAGCC 59	AAAAAGGTCAAACGTC-	59
							AGCC	
	95	CCESSR145 123795 (GCG)8	AGGCTTGAAGACGAAATGCT 59	GCTCTTTCCAGTCGAT-	59
							CTCC	
	96	CCESSR146 123812 (ATCTTT)4	ACACTCGACCCTCAAGTTCC 59	AGTGATCATCCATCC-	
							ATCCA	

Table 3 .

 3 Cont. 


						Predicted		Predicted
						Tm ( ˚C)		Tm ( ˚C)	Predicted
	Sl		Unigene	SSR repeat		forward		reverse	amplicon
	No. Marker ID	ID*	unit	Forward Primer	primer	Reverse Primer	primer	size (bp)
	97	CCESSR147 123819 (CTTC)5	ACGAGGGCAAAACAAAGAAG 59	TCAAGACGAAGAAATG-	59
							CAGG	
	98	CCESSR148 123822 (GCT)6	ATCCGCGAAGCTACTTGTTT 59	ACGAATTTCTGGTCCA-	59
							CTCC	
	99	CCESSR149 123903 (CCG)6	GGCTCTGGAGGCTTTGAATA 59	GGCGAGCATGATTAG-	59
							ACAGA	
	100 CCESSR150 123909 (CT)14	CTTCCAGGTTCCTTGTCCTC 59	CCATCAGCACTTCCAT-	59
							CATC	
	101 CCESSR151 123967 (TTTC)5	CTCTTCCCTACTTTGCTGCC 59	AGGGTTTCTGTCGCTG-	59
							TTCT	
	102 CCESSR152 123993 (TG)10	GGCCTAGTACTCCCAATCCA 59	ATGATGCAGTTGATCC-	59
							TCCA	
	103 CCESSR153 124161 (CT)12	CTCGGCTCCCAAATATTCAT	59	ACATGCGGTTATGCAC-	59
							TCAT	
	104 CCESSR154 124171 (CCA)6	ACCACCACCTCCAGAGAATC 59	CGTAGAAATTGCTATC-	59
							CGCA	
	105 CCESSR155 124195 (AGC)6	ATCCCCATCAGAAGACCTCA 59	GAAGCTCGACAAACA-	59
							GGACA	
	106 CCESSR156 124198 (GAACT)4	CCCAGAAGAAATCCCAGTGT 59	GACCAATCGAGGCC-	59
							AATAAC	
	107 CCESSR157 124268 (TGC)6	TTTCTCTCACACGCCAAAAG 59	GCTGATGATGCGAT-	59
							CAAAGT	
	108 CCESSR158 124269 (CCT)7	AGTCGTCTTCTGGTGGCTTT 59	GAGGAGGAGGTGG-	59
							TGGTG	
	109 CCESSR159 124309 (AAAGG)4	TTCGAGTTCTATGGCTCGTG 59	TGGTGGTATGGAAAAC-	59
							AGGA	
	110 CCESSR160 124355 (CAAAA)4	TCGCCTTTTCTCTCACTTCA	59	GCTCGTTTCCTCTTGA-	59
							CTCC	
	111 CCESSR161 124358 (ACA)6	ATGAGGATGAGGAGGGATTG 59	TCCCTCAAGCTGTCTT-	59
							TCCT	
	112 CCESSR162 124358 (AAG)6	AGCTTCCTCCAGAACCTCAA 59	ATCATCATCAGGGCCT-	59
							CCT	
	113 CCESSR163 124365 (AAC)6	TTGCTCTTCCACTTTCATCG	59	CTTCACGAAATGCCTC-	59
							AGAA	
	114 CCESSR164 124426 (TTTTTA)5	GACGTGGAGGGATCAGTTTT 59	CAATTCCGACGTTTC-	59
							ATCAG	
	115 CCESSR165 124428 (TTTTTA)4	GACGTGGAGGGATCAGTTTT 59	TGAATATGACCCGCAT-	59
							GAGT	
	116 CCESSR166 124436 (GAG)6	CGAGTACCATTGGGATGATG 59	ACTGTCATTTGAGTGC-	59
							TCCG	
	117 CCESSR167 124457 (AC)9	CAAAGAAAAACCGCTCTCG	59	AGTGGAGGATTTGG-	59
							AAAACG	
	118 CCESSR168 124461 (AATA)6	CTCTGCCCACTTTCTCCTTC 59	CACTTGGGCATCTGA-	59
							GAAGA	
	119 CCESSR169 124465 (TTCA)6	TCCCCCATTCATTTGGTAGT	59	GCAAGGAGACCTTCA-	59
							AGAGG	
	120 CCESSR170 124468 (TTCCTC)	TCTCCCATTCATTCCAGGAT	59	GTAAGTGCGGATGAT-	
				4-24 bp-			GATGG	
				(ATC)6				

Table 3 .

 3 Cont. 


					Predicted		Predicted	
					Tm ( ˚C)		Tm ( ˚C)	Predicted
	Sl	Unigene	SSR repeat		forward		reverse	amplicon
	No. Marker ID	ID*	unit	Forward Primer	primer	Reverse Primer	primer	size (bp)
	121 CCESSR171 124565 (ATT)6	GCTCTGTTCAGGAAGGGAAG 59	GTATCAGAATCCCCAC-	60	
						CCAA		
	122 CCESSR172 124577 (AAG)6	TGGCTTTTCTCCGTTATCCT	59	TTCTTCTTGGTGCTTG-	59	
						ATCG		
	123 CCESSR173 124612 (CGG)6	TCCTTTCCTAGCCTTCTGGA 59	AGAGAAAAAGAGCGC-	59	
						GAGAG		
	124 CCESSR174 124697 (GAC)6	GAAAATCGAGAAAGGGTGGA 59	GAGTGGTCCTCAAGT-	59	
						CCCAT		
	125 CCESSR175 124700 (GCT)6	ACACCAACGACGGGTAAGAT 59	CAGCCAGATTCGAATA-	59	
						CCCT		
	126 CCESSR176 124759 (AG)11	GGGATTGCTGCATAGGAAAT 59	CATCTCAATCAAATGC-	59	
						CCAC		
	127 CCESSR177 124765 (CCA)7a(AA-	CTGCGACTCAGACATCCACT 59	ACGCTGTCTCCTCCG-	59	
			C)6			TAACT		
	128 CCESSR178 124767 (TCAT)5	GCCGGCTAGTCTATACGGAG 59	ACCTTGAAGTTTGGT-	59	
						GGAGC		
	129 CCESSR179 124817 (TC)9	TCATTGCTGCTTCAGTCTGTC 59	TCTCTTCAAAGCGGAT-	59	
						TCCT		
	130 CCESSR180 124851 (TTC)11	GACTTCTCTCTCCCCCTTCC 59	TTGGAGCGTAATGTCG-	59	
						TAGG		
	131 CCESSR181 124871 (AGC)6	ACCAATTTCAAACCTCCAGC 59	CGAACACCACATGCAT-	59	
						TACA		
	132 CCESSR182 124949 (CCT)7	CCCCTTAAAATCGATTCCCT	59	CGGAGAGGAATTTTC-	59	
						GACAT		
	133 CCESSR183 124969 (AATTG)4	TCTAGCTTGAGGGGATCGTT 59	TAAGCCTAAGCAAAGC-	59	
						AGCA		
	134 CCESSR184 124995 (GAGAAG)4 CACCAAAGCCATGAAAGCTA 59	TCCCAAATCTCCTCTC-	59	
						CATC		
	135 CCESSR185 125025 (TC)11	CCTCCTTAAAACCCTAAACCG 59	TCTAGTATTTTGGTGG-	59	
						GGGC		
	136 CCESSR186 125054 (TA)9	TCTTGTCTTGCACATTTCCC	59	GGTCTCCGTTGTTGA-	59	
						GGATT		
	137 CCESSR187 125099 (ACC)6	TTCCCGCTTTACCAGATACC	59	TTCCTCCAACAGACA-	59	
						ACAGC		
	138 CCESSR188 125102 (ACC)6	TTCCCGCTTTACCAGATACC	59	AAACATAAGGTTGGG-	59	
						GTGGA		
	139 CCESSR189 125104 (AT)11	CTGGCTCTGACATGCAATTT 59	TTCAGAAGCGCGTAG-	59	
						TCTGT		
	140 CCESSR190 125128 (TC)12	TGCCACTTTCTTCTCCTCCT	59	CTGGAAAAGGTGAAA-	59	
						AGGGA		
	141 CCESSR191 125139 (CAGCGG)4 AGCACATCCAACACCACAGT 59	AACCCAACGTTAAGG-	59	
						GACAC		
	142 CCESSR192 125151 (ACA)8	ATTCCGAATCGGGTACAGAG 59	AATGTACATCCCTTCC-	59	
						CCAC		
	143 CCESSR193 125186 (AAG)6	GCCTCAACCACTTGCCTATT 59	CGTGATCATGATGCCC-	59	
						TAAC		
	144 CCESSR194 125232 (AT)13	ATTCAGTTGCAGCTGTGGAG 59	ATGATCTGGGAAAGG-		
						ACAGG		

Table 3 .

 3 Cont. 


					Predicted		Predicted	
					Tm ( ˚C)		Tm ( ˚C)	Predicted
	Sl	Unigene	SSR repeat		forward		reverse	amplicon
	No. Marker ID	ID*	unit	Forward Primer	primer	Reverse Primer	primer	size (bp)
	145 CCESSR195 125265 (GT)14	TGATGATAGCTCCGCTTTTG	59	AGCCAATCACCAGCA-	60	
						AGATT		
	146 CCESSR196 125286 (CTC)8	GGGTCCATGTTCGTCTTTTT	59	GAGACCAAGCATTGAA-	59	
						GCAA		
	147 CCESSR197 125289 (ATTTT)5	GAGGATCACTTTTGCCCCT	59	GAAAAAGGTCAAGACA-	56	
						TCCC		
	148 CCESSR198 125382 (AC)16	CTCTCCCCATCCCTCAACT	59	GTGAAGGAGGTGGTG-	59	
						GTCTT		
	149 CCESSR199 125416 (AT)16	ATCATCCCCTCGATAGCAAC 59	TCCCGAAAGGAAAGAA-	59	
						TGAG		
	150 CCESSR200 125444 (TG)11	TGGTTGTAGGTGGTCGTCAT 59	GCTCGGGGATATCGAT-	59	
						CTTA		
	151 CCESSR201 125484 (CTCCGA)4 AGGGTTTTTCGGAGATGACA 60	CCGGGAAAACTAAGA-	59	
						GCAAG		
	152 CCESSR202 125529 (TC)9	GTGCTCGTGCAATTGAAACT 59	ATGCCGAGTGGATGT-	59	
						CTATG		
	153 CCESSR203 125676 (AAACAA)4	TGATCTTGATCCCCTCATCA	59	GATGCATTCACAAAAC-	59	
						CGTC		
	154 CCESSR204 125734 (GAG)6	TCCTTGGCTCGAGATCTTTT	59	TTTGTGTCCTCTTCTC-	59	
						GCAC		
	155 CCESSR205 125776 (CT)14	AGGATTGCCTTCCGTTTGTA 60	GATTCAACTCGTGGGA-	59	
						CCTT		
	156 CCESSR206 125893 (CAT)7	CCCCTTGTTTCTCTTTGTGG 60	TGCATCATCCCTTGTA-	59	
						TCGT		
	157 CCESSR207 126139 (GGC)6	TCCGTTCCTCTTTCTCGTTT	59	CACAGCACCCATGG-	60	
						TAACAA		
	158 CCESSR208 126219 (GAG)6	TGGTCTTCAATCACCAAGGA 59	GACACATTGCACGT-	59	
						AGTCCC		
	159 CCESSR209 126243 (CAC)8	GGTGGACGAGGCTTTTATGT 59	CTGTGAGCAAACTTG-	59	
						ACGGT		
	160 CCESSR210 126250 (CAC)9	GCCAAAATCCCTGTTCTCAT	59	TGTGGATGCACCAGA-	59	
						TTCTT		
	161 CCESSR211 126255 (GAA)6	ACGAAAGGGTTGAAGGTGAC 59	GACAGAGACGACGAA-	59	
						GACCA		
	162 CCESSR212 126427 (ATC)7	TCCAATGGTTTACAGGAGCA 59	TCTCCGGTGTAAAACT-	59	
						GCTG		
	163 CCESSR213 126427 (CAGGAG)6 GGCGGCTTCGGTATTATTTA	59	GCACTAGTTTCTTGGA-	59	
						CTGGG		
	164 CCESSR214 126506 (TTTC)6	TTCATTGTCTTTGAGCCAGG 59	CCCCATCACTCATTCC-	59	
						TTCT		
	165 CCESSR215 126540 (AAG)9	AAATCGAATGGCTTGGAGAC 59	GTGCAAGTAAATCCGA-	59	
						GCAA		
	166 CCESSR216 126661 (AGT)6	TTTGTCATTTCTCTCGCTGC	59	AGTTGGGGAGAATTG-	59	
						ACCTG		
	167 CCESSR217 126701 (TTCA)5	AAAGGATACGGCTCACAAGG 59	GAATGAATGAACGAG-	59	
						GGGTT		
	168 CCESSR218 126701 (TTCA)6	AAGGAGCCAATCGTTCATTC 59	TTACCCTGTAGGTTG-		
						GTGGAG		

Table 3 .

 3 Cont. 


					Predicted		Predicted	
					Tm ( ˚C)		Tm ( ˚C)	Predicted
	Sl	Unigene	SSR repeat		forward		reverse	amplicon
	No. Marker ID	ID*	unit	Forward Primer	primer	Reverse Primer	primer	size (bp)
	169 CCESSR219 126730 (CT)10	GAGTTGGCTTGAAAGCATGA 59	CCATCCCCAAGGAAA-	60	
						GTGTA		
	170 CCESSR220 126767 (GA)9	GCACAAGACCTTCCCTTTGT 59	CTCTTGCACGTCGTTT-	59	
						TGTT		
	171 CCESSR221 126881 (GCA)6	CGGTGCTGCAGTTTCACTAC 60	CAAGGCAGCAAGTATA-	59	
						CCGA		
	172 CCESSR222 126910 (AAT)8	CCAACAGAAAAGTTGGGGAC 59	TTAGCCGAATCGTTATT-	59	
						CCC		
	173 CCESSR223 127055 (TTC)6	GAAATCCCACTCGGTGAAGT 59	CCCACCCCGAAGATAT-	59	
						AAAA		
	174 CCESSR224 127171 (AAGG)5	ATTTCAGTCTGGGTCTTCCC 58	TGAGGCAGACCTTGTT-	59	
						CTTG		
	175 CCESSR225 127376 (GT)10	TCCCAGAACAAAACGAGACA 59	CTTGTCGTAAACGGCT-	59	
						TCAA		
	176 CCESSR226 127478 (CT)23	CAGCCTCCGGTTAGCAAG	60	TGTCCAATACCCACTC-	59	
						TTCG		
	177 CCESSR227 127479 (ACC)10	GTACCCCCACAACACTCACA 59	GATGCTTCTCATCGTC-	59	
						TCCA		
	178 CCESSR228 127712 (TTTA)5	GATTTTGTGCCAAAGGGAAG 60	GCTTTGACCGGAGAA-	59	
						GTCAT		
	179 CCESSR229 127774 (CCT)6	CCGCCGTTAAATCAAACTCT 59	CAAAGAAAGCTGCTC-	59	
						ACTGC		
	180 CCESSR230 127785 (AT)13	GCTGCTGCTTTGCTTGTTCT 61	AGCTTCAGAAATGGT-	60	
						GGCAT		
	181 CCESSR231 127800 (GAA)9	GCTACAGCGTGCAGAAAAAG 59	TACTCCTCAGGCCTC-	59	
						CATTT		
	182 CCESSR232 127828 (TCA)6	ACCTGGGAGGACTGATAACG 59	CCAGATTCCATACTTG-	59	
						GGCT		
	183 CCESSR233 127910 (AAAAGG)4 GAAGACTCTGTAAAGGAACG-	58	GTGTTATGGCTCAAC-	59	
				ACC		CATGC		
	184 CCESSR234 127938 (CTG)8	GCGGCTTAGTTCTATCCTCG 59	ATGGCTGACAGCTTC-	59	
						CTCTT		
	185 CCESSR235 127992 (TAA)6	AGTGGGAAAAGTGAAGGTGG 59	ATTAATCCCCTCCTCC-	59	
						GAGT		
	186 CCESSR236 128005 (CTC)8	AGTCAGGTATGCTGCCATTG 59	GGAAATCTTTGGGGT-	59	
						CTGAA		
	187 CCESSR237 128130 (CCT)6	CGTCAATAAATTGGTGTGGC 59	GGGAGAATGGGAGAT-	59	
						GAAGA		
	188 CCESSR238 128268 (TA)11	CCGTCGTTACTGCAAAAGAA 59	ACAGAGGGGTTTCAA-	59	
						ACAGG		
	189 CCESSR239 128301 (AAGAAC)4 TCCCAAGTCCCTATCTCTGC 59	CAGCTGATGGTTTAG-	59	
						GAGCA		
	190 CCESSR240 128306 (TCT)7	TCTGTGTCCCTTTTCAGCAG 59	GGATGGTCAATGCA-	59	
						GATGAG		
	191 CCESSR241 128309 (CGG)8	ATATCATGGATGGTGCTGCC 61	TCCAGATTGTGCTGC-	59	
						TTTTC		
	192 CCESSR242 128315 (TTC)6	GCACGAGGCTTTCTCTCTTT 59	CAGCAGGAAACTGG-		
						AGAAGTC		

Table 3 .

 3 Cont. 


					Predicted		Predicted	
					Tm ( ˚C)		Tm ( ˚C)	Predicted
	Sl	Unigene	SSR repeat		forward		reverse	amplicon
	No. Marker ID	ID*	unit	Forward Primer	primer	Reverse Primer	primer	size (bp)
	193 CCESSR243 128377 (AG)12	GGTTCTTCTGTGTGGGTGTG 59	GTAGAAGCCAGCTAAT-	59	
						GCCC		
	194 CCESSR244 128432 (TG)15	CACACACCCAACACACAGAA 59	GCGAGAGGAATTTTGA-	60	
						CCAG		
	195 CCESSR245 128504 (GAG)6	TCCATCCCATATTCCCATCT	59	GTCGGGTCTCTTCTTC-	59	
						TTGC		
	196 CCESSR246 128527 (GA)10	CACCAACAATTCCCTATCCC 59	TCCATCTCCAACACTA-	59	
						CCCA		
	197 CCESSR247 128527 (CTG)6	CACCAACAATTCCCTATCCC 59	TCCATCTCCAACACTA-	59	
						CCCA		
	198 CCESSR248 128527 (CTG)6	TGGGTAGTGTTGGAGATGGA 59	CAATGACCGCCTGTAT-	59	
						TCTG		
	199 CCESSR249 128550 (ATC)6	CTAGTAGCCATGGTCACCCA 59	TTCTTCGTAGGCAGCT-	59	
						CTGA		
	200 CCESSR250 128587 (TC)11	GAGGCAAACCAAATGGAGAT 59	ATTGAAAAGCCTGCTG-	59	
						AACC		
	201 CCESSR251 128673 (CT)9	TTCTCTGCTGCTGCTCATTT	59	GGCACACATTTATCCA-	59	
						CTCG		
	202 CCESSR252 128894 (AAAAG)4	GCCCAAAGCTGTTAGAGACC 59	TCCTTGTCATACGCTT-	59	
						GCTC		
	203 CCESSR253 129060 (TCT)7	CAATTTGCAGTCAGTCCGTT 59	ACATCTGCCACTGTCT-	59	
						GAGC		
	204 CCESSR254 129129 (GAG)7	ACAGATGCTTTATGGGAGGG 59	GGAAAGGTATTGCTG-	59	
						GGGTA		
	205 CCESSR255 129269 (GCA)9	TGAAGGTTCACTGATTGGGA 59	TGCTGTTGTAACTGCT-	59	
						GCTG		
	206 CCESSR256 129431 (AAG)9	CGCCACCAGTACAGATCAAG 59	TCCGCATATCGAACAC-	59	
						TCAT		
	207 CCESSR257 129439 (CA)10	AGTCACCACACAGATGCTGC 60	GGCCATTCAAGGTTTC-	59	
						TTGT		
	208 CCESSR258 129524 (GAA)8	CTAATCTTCCACGGCTCTCC 59	TTTTGTTCAGCAGCAA-	59	
						GGTC		
	209 CCESSR259 129581 (AAAAG)4	CCAACCTCACGTCTCACCTA 59	GCCAGTGACACAGAC-	59	
						GACTT		
	210 CCESSR260 129646 (TC)11	CTTCAGCCACAACAGATGCT 59	AAAAGAGCACACAGT-	59	
						GCAGC		
	211 CCESSR261 129668 (CT)12	CATGATTATGCGTTGGCTTC	59	GGTTGTCATGTTGAAT-	56	
						GAGG		
	212 CCESSR262 129745 (CAC)10	GCCTCCAGTCAACAAACAAA 59	TGGTGCTTCTCTTCCT-	59	
						TGTG		
	213 CCESSR263 129793 (CA)10	TAGCGGGGAAAATTGATAGG 59	AGGGGCTTTTTCTCCA-	59	
						TCTT		
	214 CCESSR264 129797 (TCC)6	TCGATGATGGCTACAGCTTC 59	TTGCTCCTGAGAAACA-	59	
						CTGG		
	215 CCESSR265 129906 (TA)11	GACCAGAGAGAGACACCTA-	57	CTCTTCCTAGCAGCAG-	59	
				CTTTT		CAGA		
	216 CCESSR266 129913 (CAG)6	TTCCTTTAAAACCGTCAGGG 59	CTGCATGCTCTGTTGT-		
						TCCT		

Table 3 .

 3 Cont. 


					Predicted		Predicted	
					Tm ( ˚C)		Tm ( ˚C)	Predicted
	Sl	Unigene	SSR repeat		forward		reverse	amplicon
	No. Marker ID	ID*	unit	Forward Primer	primer	Reverse Primer	primer	size (bp)
	217 CCESSR267 129917 (AAGAGC)5 GAGATGGAGAGACCATCATC-	58	AACTAAGACTTTTGCC-	59	
				C		GCGT		
	218 CCESSR268 129950 (TCCACT)4	TTCCCCATCTAAGGCAAAGT 59	CTGATTCAGGGCTCTC-	59	
						CATT		
	219 CCESSR269 129957 (GAGCAG)4 AACCCACCTTCCTCTCCTTT 59	CATGCATATCGGACAC-	59	
						AACA		
	220 CCESSR270 129964 (GAT)7	AAGGCTCGACATGTTTTTCC 59	CCTACGCAACATCATC-	59	
						AACC		
	221 CCESSR271 129972 (AG)9	CTTTCCGCCTTTTCGTACTC	59	AACACACGCACAACA-	59	
						CACAG		
	222 CCESSR272 130030 (AG)19	GGCTGCAAGAACATAGAGCA 59	GAAATCCTTGTTGGCT-	59	
						GGTT		
	223 CCESSR273 130066 (GAG)6	AAAGAAAGAGCCCTCCACAA 59	GCTCCAAAACCACCA-	59	
						TTACC		
	224 CCESSR274 130235 (TGC)6	GGTTTATGACGTTGTGGACG 59	GGCCTATGATGCGGT-	59	
						AGAAT		
	225 CCESSR275 130276 (GGC)7	GGCGGCTACTCAGAGACTTC 59	TGGAACTCCAATTCCT-	59	
						TTCC		
	226 CCESSR276 130329 (AT)13	AGGCTGCGTTCCATATAAGA 57	CCCAGAAACTCCATTT-	59	
						CGAT		
	227 CCESSR277 130342 (ATC)6	CTCTGCAGAACAACCCTTCA 59	CCAACAGCTAAAAGT-	59	
						GCCAA		
	228 CCESSR278 130353 (CA)11	TCTCTCTCTCTCCATTCGCA	59	GGTACGAGGTTGATC-	59	
						GGAGT		
	229 CCESSR279 130353 (CAC)6	ATGGCTCCATCATCTCATCA	59	ATTGGTGGGTCCTTC-	59	
						AATTC		
	230 CCESSR280 130368 (AG)17	GGCTGTCTTAAGGCCCTAGT-	59	ATCAAGTGATGCTGT-	59	
				T		CGAGC		
	231 CCESSR281 130475 (CTC)7	CGCCAACACCCCTTATCTAT	59	CATTGAGGAATTGGA-	59	
						ACGTG		
	232 CCESSR282 130516 (CCT)6	ACCCTGCTCCACCTTATCAC 59	GACAGAAACCTTCAT-	59	
						CCCGT		
	233 CCESSR283 130575 (AGA)7	GTGGTCAGTATGGTTGCTGG 59	AGCCACAAAAGAACC-	59	
						ATTCC		
	234 CCESSR284 130635 (AG)13	AGCACCGTCAGACTCTCCAT 60	TAAGCCAAACGTCGT-	59	
						CGTAG		
	235 CCESSR285 130647 (CAC)6	GAGGCCGAGTTACGAGAGTC 59	AGCATGCCAACCCTT-	59	
						TATTC		
	236 CCESSR286 130785 (TAAAA)4	TGCTCGGACCCTCTAAGTTT 59	TGGAGTCGTTTAAAA-	59	
						AGGGG		
	237 CCESSR287 130804 (AG)10	CGTTACAGAATTTGCGGATG 59	CAACTCTAAGCCGTC-	59	
						GATGA		
	238 CCESSR288 130947 (GCAGAG)4 TTTTGAACACGTCAAGGCTC 59	CGAAGAGGGGACTG-	59	
						AGAAAG		
	239 CCESSR289 130972 (TCA)9	GCACAACCCATATGACGAAG 59	GGAGGAACAGTTGG-	59	
						AGAATGA		
	240 CCESSR290 130978 (AG)16	TTATTATTGCACAGCCCCG	60	AACAATCCATTCTTGC-		
						TCCC		

Table 3 .

 3 Cont. 


					Predicted		Predicted	
					Tm ( ˚C)		Tm ( ˚C)	Predicted
	Sl	Unigene	SSR repeat		forward		reverse	amplicon
	No. Marker ID	ID*	unit	Forward Primer	primer	Reverse Primer	primer	size (bp)
	241 CCESSR291 130993 (TC)14	CGGTGGGGTTCTGATAATTT 59	CGGTTTCAAGCTAACG-	59	
						AACA		
	242 CCESSR292 131127 (TC)11	ATCGTGTTTAGCAGTGACGC 59	GCAGATATCATATACTC-	55	
						CCGTC		
	243 CCESSR293 131130 (AAG)6	CACTCACTCAACACTTCGCA 59	CCATCTTCCACCACCT-	59	
						TTCT		
	244 CCESSR294 131306 (TCGCA)4	CTTTGGGGGCTGAGAGTAAG 59	GCTCCATATTGCAACG-	58	
						TCTT		
	245 CCESSR295 131334 (CCA)7a(AA-	TAATACATCCACTCCGCCAA	59	TACTCATCAACAACCT-	59	
			C)6			CGCC		
	246 CCESSR296 131479 (TG)9	GCAATTGTGGATGGTCTGAG 59	AAACACACTGGGAAA-	58	
						GAGCA		
	247 CCESSR297 131560 (ACC)6	CAATCTGTTCCCACGTATGC 59	ACAAGTGCTCCAACAA-	59	
						ACCA		
	248 CCESSR298 131583 (AT)12	CCACACACTGCAGATTGGTT 60	AAGATCTCTGGGGCAT-	59	
						CAAC		
	249 CCESSR299 131633 (CTT)6	AGGTTCGAGCCAGGAAGATA 59	TTGCCTTGGTTGATGA-	59	
						CTTC		
	250 CCESSR300 131669 (AG)12	TCCGGGAAGGATAGTGGTAG 59	TTGCTGAAACAGTCTC-	58	
						CCTC		
	251 CCESSR301 131714 (GA)22	ATTAGTGCGGAATTCCCTTG 59	TGATAATCTCGAGTGA-	59	
						CCGC		
	252 CCESSR302 131742 (TA)18	TAATGCACGCACAAACACAC 59	ATCATTGCAAATCTCC-	59	
						TCCC		
	253 CCESSR303 131761 (CA)10	CATGGATTGGAATTCTGCTG 59	CAGATTAGCTCCGCA-	59	
						ATCAA		
	254 CCESSR304 131806 (ATG)8	GCACCTAAGAGAGGAGTGCC 59	ATGAGTTTGGCTGGAT-	59	
						GTGA		
	255 CCESSR305 131831 (GCCTGC)4 CCGATTTCGCTCGTAGTGTA 59	TATCAATACGGACTG-	59	
						CTGGG		
	256 CCESSR306 131899 (GAT)9	TCCGCTGGTCACAAGAAAT	59	ATCAAACTTCGACAC-	59	
						CCCTC		
	257 CCESSR307 131901 (AAC)6	AAACTTTGATATCCCGGCTG 59	GCCGCCTTTTGTAGA-	59	
						AGAAC		
	258 CCESSR308 131958 (TCA)9	GATTCCAGTATTTCGGCTCC 59	TTGCTCAGATTAGGCT-	59	
						GTCG		
	259 CCESSR309 131966 (AC)10	TTTGAACTTGCTGGAGCATC 59	TGGTGAACTTCTGTTT-	58	
						TCCC		
	260 CCESSR310 132036 (AAG)6	CTCCTCAACACTCCTGACCA 59	CCCTCCTGAAGCTGC-	59	
						TTATC		
	261 CCESSR311 132205 (GT)10	CCTTGATTGTCACGTGTATGC 59	AAGATATGCCCAGGT-	60	
						CATGC		
	262 CCESSR312 132207 (TCGT)7	AAAGGATACGGCTCACAAGG 59	ACCCTGTAGGTTGGT-	59	
						GGAGA		
	263 CCESSR313 132325 (AAG)8	TGAGCACTCAAGGGAAATTG 59	TTCGTCCAGGGAGTA-	59	
						GCTTT		
	264 CCESSR314 132326 (AAG)6	GCTTTTGTCCAAGGGATGAT 59	GGAGCTCCCAGGAG-		
						GTAGTA		

Table 3 .

 3 Cont. 


					Predicted		Predicted	
					Tm ( ˚C)		Tm ( ˚C)	Predicted
	Sl	Unigene	SSR repeat		forward		reverse	amplicon
	No. Marker ID	ID*	unit	Forward Primer	primer	Reverse Primer	primer	size (bp)
	265 CCESSR315 132398 (TA)11	ACGTTTATTACGGCGGATTC	59	ATCGCCGTCCCATAAC-	58	174
						TATT		
	266 CCESSR316 132466 (AGA)6	TCTGTCTGGGCAACACTATAC 55	AGAAAGGGAGGTGGA-	59	338
						GGAAT		
	267 CCESSR317 132484 (CT)9	TTTGCAGTTATGACTTGGGC 59	TCCTCACTGGAAAGG-	59	449
						GATTC		
	268 CCESSR318 132502 (GTCTT)4	GGGGATCAGCGTAAGAATGT 59	TTTCCACCCACGTAAT-	59	346
						AGCA		
	269 CCESSR319 132516 (TCA)6	CCGTCCACGTTCACATCTAC 59	AGTAGCGCGTAGTGA-	59	130
						CCTGA		
	270 CCESSR320 132606 (AGC)6	GACCAGAAGAACAGCGATGA 59	CTCCTTCTTCTTCTCG-	59	285
						GCAG		

*: Unigene ID as per downloaded from the SGN ftp site ftp://ftp.sgn.cornell.edu/coffee/. doi:10.1371/journal.pone.0113661.t003 
Table 5 .

 5 Highly significant HW dis-equilibrium at P,0.01; The putative DL (duplicated loci) markers were not considered for calculation of various estimates as these appear to be fixed exhibiting no segregation. doi:10.1371/journal.pone.0113661.t004 Allelic diversity attributes of the newly developed 25 genomic SSRs when tested over cultivated and wild related coffee genera. 


	1-4 0-4		2.11 0.34	0.89 0.81	0.14 0.12	
	0-5		1.57	1.59	0.24	
	1-17		7.52	3.62	0.55	
	0.11-	0.77	0.44	0.19	0.03	
	0.13-	0.85	0.53	0.22	0.04	
	0.00-	1.00	0.54	0.26	0.04	
	00-0.23-0.19-1-7 0-1	0.88 0.78 0.67	Average 2.14 0.00 0.47 0.54 0.43 3.00 0.02	SD (¡) 1.21 0.00 0.36 0.13 0.16 1.57 0.15	SE (¡) 0.18 0.00 0.10 0.03 0.03 0.24 0.02	Note: N A : Number of amplified alleles; PA: Number of Private Alleles; H

o : Observed heterozygosity; H e : Expected heterozygosity; PIC: Polymorphism Information Content; PI: Probability of Identity; NA: Not amplified; *: Significant HW dis-equilibrium at P,0.05; **: 
PLOS ONE | DOI:10.1371/journal.pone.0113661 December 2, 2014 

Acknowledgments

RKA thanks the Director, CCMB, Hyderabad for the facilities to undertake the study; Director Research, Coffee Board, Bangalore for coffee materials, and R. Phanindranath for providing lab help as and when required. PSH acknowledges CSIRfor junior and senior research fellowship during his doctoral research and DBT for the Postdoctoral fellowship. 


The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files. New genomic sequences have been submitted to GenBank/ NCBI, and accession numbers are provided within the paper. 


This work was supported by: the FYP project, PLOMICS_BSC0301, to RKA, from the Council of Scientific & Industrial Research (Govt of India); and Project grant ''BT/PR/6292/Agr/16/575/ 2005, dated 4/12/2006,'' to RKA from the Department of Biotechnology (Govt of India), under which some of the DNA samples/sequences used in the present study were collected/carried out. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. 


Supporting Information

Table S1. Summary statistics of screening of the coffee unigene ESTs for SSRs. doi:10.1371/journal.pone.0113661.s001 (PDF) 
Table S2. Summary statistics of distribution and abundance of detected SSRs in the unigene ESTs and SSR frequency estimates for coffee transcriptome. doi:10.1371/journal.pone.0113661.s002 (PDF) 
Table S3. Characteristics and distribution of the detected SSR motifs (without MNRs) across the non-redundant 56 SSR+ive sequences generated using SSR enrichment approach. doi:10.1371/journal.pone.0113661.s003 (PDF)
Prasad S Hendre, Ramesh K Aggarwal
Development of Genic and Genomic SSR Markers of Robusta Coffee (Coffea canephora Pierre Ex A. Froehner)