Genetic analysis of quantitative trait loci for cocoon and silk production quantity in Bombyx mori (Lepidoptera: Bombycidae)

Keywords: Lepidoptera, Bombycidae, Bombyx mori, cocoon and silk production quantity, genetic linkage map, QTL mapping

Silk production quantity is the most economically important characteristic of the domesticated silkworm moth, Bombyx mori. It is controlled by multiple loci. The cocoon and silk production quantity of silkworm strains Jingsong and Lan10 have significantly diverged. A backcross population (BC1) was bred using Jingsong and Lan10 as parents to identify quantitative trait loci (QTLs) for silk quality. In this research, a genetic linkage map of the silkworm was constructed using the BC1 mapping population. The map contained 85 sequence-tagged site markers, 80 simple sequence repeat markers, and 16 single nucleotide polymorphisms. A linkage map was constructed from the data, which consisted of 181 markers distributed over 28 expected linkage groups and spans 2147.1 cM in total length. Fourteen QTLs were detected for cocoon filament length, whole cocoon weight, pupae weight, filament weight, and cocoon shell weight. The 14 QTLs were distributed in 5 linkage groups (linkage groups 1, 14, 18, 23 and 25) based on the constructed linkage map. In addition, five QTLs, which had the highest logarithm (base 10) of odds (LOD) values, were located on the first chromosome, three of which located at the same region in linkage group 1. These results represent an important foundation for the map-based cloning of QTLs and marker-assisted selection for improving the silk quality of economically important silkworm strains. 

INTRODUCTION

The silkworm moth, Bombyx mori L. has been domesticated over the past 5,000 years from the wild progenitor, Bombyx mandarina (Moore) (Xiang, 1991). The silkworm is important for genetic research as well as an economically important insect. More than 400 mutations have been identified in silkworms, and more than 1,000 silkworm strains are maintained as genetic resources (Ito et al., 2009;Meng et al., 2009). It is also a key model species of the order Lepidoptera, the second largest group of holometabolous insects, which includes many beneficial species and the most destructive agricultural pests. 
Cocoon quality is very important because it influences sericulture yield and the selection of silkworm lines for silk production. Currently, modern techniques such as transgenesis and marker-assisted selection are the most effective ways of improving silk properties and they are applied widely in silk production (Lu et al., 2004). Molecular markers are powerful tools for genome analysis; they are applied comprehensively in the mapping functional genes and the molecular breeding of B. mori (Li et al., 2005b;Sreekumar et al., 2011). The construction of linkage maps is a fundamental aspect of gene analysis. It provides guidelines for marker-assisted selection and map-based cloning. An accurate linkage map is also necessary for further genetic analysis. In recent years, several linkage maps for B. mori have been con-structed based on various molecular marker techniques, including restriction fragment length polymorphism (RFLP) linkage mapping (Shi et al., 1995), random amplification of polymorphic DNA (RAPD) linkage grouping (Promboon et al., 1995;Yasukochi, 1998), amplified fragment length polymorphism (AFLP) frame mapping (Tan et al., 2001;Lu et al., 2004), simple sequence repeat (SSR = microsatellite) linkage mapping (Miao et al., 2005), and single nucleotide polymorphism (SNP) (Yamamoto et al., 2006;Sreekumar et al., 2011). In many cases, maps from different parental populations and species have been integrated (Yasukochi et al., 2005;Zhan et al., 2009). However, some maps have relatively low density and could not be used effectively as guides for breeding B. mori. 
In the present study, STS (sequence-tagged sites), SSRs, and SNPs were adopted to construct a linkage map of silkworm to map quantitative trait loci (QTLs) for silk quality because of their good transferability, high reproducibility, and co-dominant inheritance. These markers are particularly suitable for high-throughput genotyping, which allows the rapid analysis of hereditary monogenetic traits and QTLs (Maddox et al., 2001;Ihara et al., 2004;Lee et al., 2005;Huang et al., 2006). A linkage group was constructed and the QTLs were localized based on the cocoon filament length, cocoon shell weight, whole cocoon weight, pupae weight, cocoon filament weight, and cocoon shell ratio. 

MATERIAL AND METHODS


Silkworm strains

Significant hereditary and ecological divergence occurred in the genes responsible for the silk quality of silkworm strains Jingsong (JS) and Lan10 (L10), which are preserved in the Sericultural Research Institute, Chinese Academy of Agricultural Sciences. The JS strain is used widely in various applications and its silk properties are advantageous for silk production. In contrast, L10 has high stress resistance, but produces smaller cocoons and has poor silk-producing properties. Additional crosses between strains from different origins may increase the mapping efficiency of markers because of the increased potential for genetic diversity (Zhan et al., 2009). A backcross population (BC1) was bred from JS and L10 parents. The 93 male individuals were selected randomly from the resulting BC1 population. 

Silk quality collection

The fresh cocoons were weighed before dipping in 0.6% sodium silicate solution (Na2SiO3) for 60 min at 24°C. The cocoons were transferred in pure water and then the silk fibers were reeled. The length of the cocoon filament was measured during reeling. The silk fibers were washed and dried after measurement. The cocoon filament weight and pupal weight of each cocoon were also determined. The cocoon shell weight was calculated using the following formula: 
Cocoon shell weight = whole cocoon weight -pupal weight -weight of castoff skin inside the cocoon. 

DNA preparation and PCR amplification

DNA was extracted from the parent moths, and pupae of the F1 progeny, and the 93 BC1 progeny as described by Xia et al. (1998) The purity and concentration of the prepared DNA were determined with a BioPhotometer Plus (Eppendorf, Hamburg, Germany) and diluted with 1 × TE buffer (10 mM Tris-HCl, 1 mM EDTA ), pH 8.0. 
A total of 1120 pairs of STS/SNP primers were designed based on the silkworm genome database (The International Silkworm Genome Consortium, 2008), and 428 pairs of SSR primers were selected from the established SSR linkage groups 206 Fig. 1. The distribution of the cocoon and silk quality in male individuals in BC1 population. (Miao et al., 2005;Zhan et al., 2009). The 93 DNA samples were diluted to a final volume of 10 ng/µl with sterilized double-distilled water and stored at 4°C prior to amplification using the polymerase chain reaction (PCR). The PCRs and the amplification using the SSR markers were performed according to Miao et al. (2005). The PCRs were performed via STS markers with an initial 3 min period at 94°C (denaturation), followed by 35 cycles of denaturation at 94°C in 30 s, 30 s of annealing at 56°C, 70 s of extension at 72°C; and a final extension time of 5 min at 72°C. The PCR products were electrophoresed using 2% agarose gel to determine their lengths. PCR products of the SNP markers were purified and then sequenced using an ABI 3130xl sequencer to detect SNPs. 

Linkage map construction and data analysis

A genetic linkage map was constructed using MapMaker/Exp V3.0 (Lincoln et al., 1992). The QTLs for silk length, whole cocoon weight, pupal weight, silk weight, and cocoon shell weight were scanned using MapMaker/QTL v1.1b.Whole chro-mosomes were scanned every 2 cM for the presence of QTLs. Positions with LOD  2 were considered QTLs (Basten et al., 1994). 

RESULTS


Phenotypic variations of five characteristics in the BC1 population

Whole cocoon weight, cocoon filament length, cocoon shell weight, cocoon filament weight, and pupal weight showed patterns of continuous and normal distributions, which indicate the quantitative inheritance of these characteristics (Fig. 1). 

Genetic relationship among different characters of cocoon and silk production quantity

The genetic relationships among cocoon and silk qualities were analyzed. All characteristics were positively 207 Fig. 2. Linkage map of Bombyx mori. Marker names were labeled at the right side of the groups, and the distance between markers are indicated at the left side. correlated (Table 1). The genetic correlations between silk weight and cocoon shell weight, as well as between whole cocoon weight and pupal weight, were extremely high (0.999 and 0.996, respectively). The lowest correlation was between cocoon filament length and pupal weight (0.699). These results suggest that the QTLs for some characteristics are linked. 

Construction of molecular linkage maps

A total of 182 markers were polymorphic between the two parents JS and L10. The DNA polymorphisms from the 93 individuals of the BC1 population were amplified. 
The genotyping results of the 181 polymorphic primers (Supplement 1), including 85 STS markers, 80 SSR markers, and 16 SNPs accorded with the 1 : 1 ratio of the population. These markers were analyzed using Mapmaker/Exp (Version 3.0) and assigned into 28 linkage groups (Fig. 2). The total genetic distance was 2147.1 cM. The mean distance between adjacent markers was 8.5 cM. 

QTL analyses for cocoon and silk production quantity

A total of 14 QTLs were detected for cocoon filament length, whole cocoon weight, pupal weight, cocoon fila- ment weight, and cocoon shell weight using Mapmaker/QTL (Version 1.1b) (Table 2). The QTLs were distributed in 5 linkage groups (linkage groups 1, 14, 18, 23 and 25). Interestingly, the 5 QTLs located on the first chromosome had the greatest logarithm (base 10) of odds (LOD) values, which meant they were highly reliable, 3 of which located at the same region in linkage group 1 (232S1 and S0106). These findings indicate that some silk traits are significantly correlated with each other. 
The clustered distribution of the QTLs is in accordance with the genetic relationship among these traits, which in turn is consistent with the relationships among whole cocoon weight, cocoon shell weight, and pupal weight. These findings highlight the significant relationship between the traits for cocoon production quantity, and explain why the highest QTLs were mainly detected in the same linkage group. 

DISCUSSION

The two parent silkworm strains used to construct the linkage map have numerous genetic differences, which facilitated the expression of numerous extensive polymorphism markers leading to the construction of a molecular linkage map. The silkworm is one of the lepidopteran species in which no genetic exchange occurs in females (the heterogametic sex) during the formation of female gametes. Using the BC1 population as the mapping population circumvented this problem and allowed analysis using mapping software. In our research, the silk fibers were reeled from the cocoons while the pupae were still alive. Data, including the cocoon filment length data and cocoon filament weight, were recorded to utilize live pupae to extract DNA for linkage and QTL analysis. In addition, JS is one of most economically important strains and it has been used in silk production for nearly 30 years. Therefore, the study has a more direct applied importance to sericulture and the production of silkrelated products. 
The silkworm has attracted increasing attention as an important genetic and biomaterial resource. China has a long history of sericultural research and an accumulation of genetic data on silkworms. Xia et al. ( 2004) reported whole-genome shotgun sequencing and provided public access to the assembled silkworm genome data. In 2008, The International Silkworm Genome Consortium published the fine scale genome sequence of the silkworm, which is necessary for molecular genetics research in this insect. Cocoon quality is an important characteristic of silkworms; however, the genes of this insect are more difficult to map than single Mendelian factors (Zhan et al., 2009). 
A total of 14 QTLs for the silk quality characteristics were identified. Cocoon filament length, cocoon shell weight, whole cocoon weight, pupal weight, and cocoon filament weight, which are controlled by multiple genes, are the economically significant characteristics of importance to the silk industry. The present investigation pro- vides an excellent foundation for the map-based cloning of major genes that control silk production. Compared with previous research on QTLs for cocoon quality (Lu et al., 2004;Li et al., 2005a;Sima et al., 2005), the locations of the QTLs in the chromosomes were not identified because of the difficulties involved in duplicating both the AFLP or RAPD results. In research, the exact location of the STS, SSR, and SNP markers were determined from the silkworm fine scale genome sequence, and these markers can be duplicated in other silkworm strains. Our results are thus consistent with those by Zhan et al. (2009). Firstly, the detected QTLs were distributed mainly in groups 1 and 23. Secondly, the LODs of the QTLs in group 1 are extremely high. These results indicate that the data are accurate and repeatable. Both results show that one or more QTLs on chromosome 1 affect silk and cocoon quality. Our results also show that all of the QTLs on chromosome 1 are located at a region of 290 kb, and there were 12 predicted genes in that region. We are now trying to clone all the genes and compare them between Jingsong and Lan10. The findings by Zhan et al. (2009) indicate two QTLs for cocoon shell ratio in groups 18 and 19; however, we only detected two QTLs at the same locus and which controlled whole cocoon weight and cocoon shell weight on chromosome 18, but did not contribute to cocoon shell ratio, and no other QTLs were detected for cocoon shell ratio either. This result could be attributed to the fewer markers available in those groups, which were insufficient for detecting QTLs. Although we constructed a linkage map that covers all 28 chromosomes of the silkworm, the marker density was still low, and many QTLs that control cocoon and silk production quantity characters were not detected. More SSR and STS/SNP markers will be designed in future studies to finely map the QTLs and allow map-based cloning thereby leading to a better understanding of the mechanism of silk production in silkworm). 
209 2.2 S2323-N2301 23 Q-PW-2 5.2 C214S4-C233S2 1 Q-PW-1 Pupae weight 2.2 T2501-T2505 25 Q-FW-3 2.6 S2304-T2320 23 Q-FW-2 11.2 C33S2-S0106 1 Q-FW-1 Cocoon filament weight 2.0 T2501-T2505 25 Q-FL-3 3.1 T1406-T1403 14 Q-FL-2 8.8 C214S5-C214S4 1 Q-FL-1 Cocoon filament length 2.3 S2304-T2320 23 Q-CSW-3 2.4 N1814-T1810 18 Q-CSW-2 6.2 C33S2-S0106 1 Q-CSW-1 Cocoon shell weight 2.1 S2323-N2301 23 Q-CW-3 2.3 N1814-T1810 18 Q-CW-2 11.0 C33S2-S0106 1 Q-CW-

Fig

  Fig. 2. continued. 


TABLE 2 .

 2 Information of QTLs detected from BC1 in Bombyx mori. 


	Trait	Locus	Linkage group	Mark interval	LOD value
		1			
	Whole cocoon weight				

TABLE 1 .

 1 Comparative genetic correlations of cocoon quality in Bombyx mori. 





  The Primer Sequences in the Linkage Groups. XIA Q., ZHOU Z.Y. & LU C. 1998: The molecular phylogeny study of the geographical races of silkworm Bombyx mori. - 


	S0705 S1915	TCCAATTTGTGTGTTTGTATGTGTG GCCGCTGCTAACCGAAAGA	GGTTGAGAAGCAAGTCTGCGATA TATGATCTTATCTTTTCAGAATTTGGG	nscaf2986 nscaf2204	3772kb 762kb	SSR STS
	T07215 T1904	CGCCACGACAACGAATAGAC ACTGTATGGTACTGTACGGTGTT	GAAGAACGATGAAGCCTTATACCA TTGACATTGATGGAGCCAAGTTC	nscaf2986 nscaf2204	4526kb 3312kb	STS STS
	S0702 T1905	AGTTCCTTTCACAATGTCTTATGCTA ACAAGCGATACTGCGACACA	CACCATACATGGGCTCCGAT ACCTAACAAGATGGCTGAGATACA	nscaf2912 nscaf2204	1428kb 1865kb	SSR STS
	T0820 S1904	AATGTATCTTCGACAACCTCCG AACTTAACATTTAAGCAAACCCA	CCTACTCACGCTCCCTCATCT CCGCACAGCCAACTGACAA	nscaf98 nscaf2204	14kb 4211kb	STS SSR
	S0820 S2019	GAGGTACCAGTGATTGCAGACGT AAATTGCAGACATTGGCATCATC	CCCAGGTGTACTCGGAGTCATTTA AATCTTATCAGTTTTAGAAGGTACCGG	nscaf463 nscaf2938	642kb 350kb	SSR SSR
	T08410 T2013	ACGGTGATGATGTGAATAGTGATG CGACAGAGATACAAGAGAACACAA	TCCATCGTAAGAATCCATCCAGAA CCAAGAGACCTAGAAGCCTGAA	nscaf2828 nscaf2937	3893kb 467kb	STS STS
	T08419 T2020	CCGTAACTGAAGGTGATTGAACTG GAAGATTGAAGGCGACGCATAA	AGCCTATTAACGCCACTACTGAAT GGATCAGAAACCAGGGAGTT	nscaf2970 nscaf2789	960kb 826kb	STS STS
	T0904 S2005	TTCCTAATCCGAGTGCTGTCTA GCCGCAAAACAATCAAGTGG	TGCTGGTCGCTTGCTTGA CGTATTTTAGTGTATGACTCGGATGA	nscaf2889 nscaf2795	3609kb 246kb	STS SSR
	S0902 T2002	CCTAAGACTACATAACTTGTCAATCTACGA GTAATACTTATCGGAGGACCACAA	AAAAGTTTGATACTAAATGAAGAGCACA GCACATTGGAAGCAGAGGTT	nscaf3045 nscaf2795	1405kb 2625kb	SSR STS
	T09ZJ S2002	CCATGACTTTTGTAGGGGTAC ACTCACCAACAAACCGCAAGA	CATCGTTTTCTTGGTCGTTA CACCGCAACATGCCTGCTATA	nscaf2511 nscaf3089	590kb 90kb	STS SSR
	T09217 T21305	CCGCAAGGAGCAGTACAATT GTGTGGCGTAAGAGCAAGCA	GGAAGAGCCACCGCATAGAT GGAAGGTTATGGCGACAAGTGA	nscaf2511 nscaf3041	5049kb 1569kb	STS STS
	T1004 S2114	CGTTCAAGTCCGAAGTCTGTC TCAAGGAAGAGTAGACGGTCAAAAC	CGAGGAAGGCTCACTCCATAA CGTCTTATTGTTTGATTGCGGCT	nscaf2855 nscaf3044	3444kb 657kb	STS SSR
	T1007 T21321	ACAGACTATGGCACTGAGATACC CCACGACGACTAAGGTATAGAGAA	GCATTACAACATTGGAAGACCTTC AACAGGACGGCGATGAGATG	nscaf2855 nscaf2868	6414kb 676kb	STS STS
	T1013 S2111	TTCTGAATCGGCAAGTCCAATC AGTCACAGATTTCGCCAATTAAGAT	CCATAGCAGCACGCAATATAGC ATTGTGCGGACGGAGGAGTA	nscaf2860 nscaf2136	409kb 1035kb	STS SSR
	S1019 T2112	TCAGACTGTTGAGCCAAAGCG TTGTGCGTTGTGCCTTAACTATC	GGCGTTTTGTAGTTGTTTTCAGTC ACTACTAGGACAGATGGAGGAGAC	nscaf2575 nscaf2136	137kb 5255kb	SSR STS
	T1018 T2115	ATTGAGAAGCCGACAAAGTAGGA AATCCAGGCAAGCAGCACAT	CGAATAAGTGAAGCGAAGCCATT GCAAGTGAAGTGTACCGATGG	nscaf2575 nscaf2136	2729kb 8279kb	STS STS
	T1101 T2211	ACGAAGTGGAGGTTCAGATACA GACTCATCTCCATTCACCTCCAT	GGATGTCTATGTTCTCTGGAGTC ATCAGCAAGTTGACGGTTCATAC	nscaf2176 nscaf1681	237kb 572kb	STS STS
	T1103 S2215	ACCTACCAGTTGATGATGTCACTT TGTCAGCCAAAGAATGTGTAAATGT	ACAGTTCCTCGTGCCAATCC ATACAAATGTAAAACTTTGCCGTTG	nscaf2176 nscaf1681	2360kb 4208kb	STS STS
	S1124 T22313	CGGACAGCTATAAATTGTACCATCG ACTCGCATAACCATAGTAAGAACC	CGATAAGACCGCCTATTGCAC CATACACATCGTCGCCAACAA	nscaf2176 nscaf2980	2818kb 49kb	SSR STS
	S1135 S2206	CGGCTTTGGCTATTGTTTACTTAC GGGTTAGAGGTCCCAAACGATG	CCAGTTTACATGACCCAAATTTAGAT CAGGTCACTTAGTCTTAACATTGCG	nscaf3032 nscaf3008	1344kb 1493kb	SSR SSR
	S1111 S2218	AACCTGTGAGCTGACCTAACCG TTACTGGCGTAGATAGTTGAATGC	AACAAGATAGTCCGCAACCCAG GGATACCTTCTCCGTGTTGCT	nscaf3031 nscaf3005	1088kb 1703kb	SSR SSR
	S1115 S2202	CCCTCAACCAGATGCAGCGA TTTAGTCATTCACAAAACAAACAAGG	ACCGTTATAGATGCTAAAGATATGATTGT TGAGATGACCTAAGGTAAGGGGAA	nscaf3031 nscaf3005	4832kb 819kb	SSR SSR
	T1216 T22210	CTGGCGTTACAAGAATAGTAGGAC CATCGTAGCACTTGTCGTTGTC	GCAGAACAGTGAGGTATTGACAG ACTTGGTAGTATGTGAAGCCTCTG	nscaf2842 nscaf3055	1266kb 15kb	STS STS
	STS STS SSR STS STS STS STS SSR SSR SSR STS STS SSR SSR SSR SSR SSR SSR SSR SSR SNP STS SSR SNP SSR STS STS STS SNP SSR SNP SSR SNP SNP SNP SNP SNP SNP SSR SSR SSR SSR SSR Marker type* STS SSR STS SNP SNP SSR SSR SSR SSR SSR SSR SSR SSR SSR STS STS STS SSR STS STS STS STS SSR STS STS STS STS SSR SNP SSR STS STS STS STS STS SSR SSR STS SSR STS STS SSR STS STS SSR * SSR -Simple Sequence Repeat, PCR products size polymorphism; STS -Sequence taq site, PCR products size polymorphism; SNP -Single 2089kb nscaf2986 TTGAACATATCGGTCGTATAGTCC GAAGCGTCGGCTCTAAGGTA T07207 286kb nscaf2983 CTTCAAAGGAGGGTAAGGGAC GGGCGTTACGAAACCAAA T0702 1862kb nscaf2853 GCACGGAAGCTCCAACGAC TGCCAGCCCTCGCCTAA S0609 1621kb nscaf2852 TTAGGCACACTACACATCACAAG CTCCAGGCAGTCCTCTCAAG T06210 1969kb nscaf2852 CCTACCGTAATCTACCGTATCTTC GCAACTGTCCATTCCTCCTAAT T06211 2152kb nscaf2852 CGTTGACATTCCTCGTTGG ATTGGATATTCTCGGCAGTTT T0616 472kb nscaf2556 CAGACAATGCCGAGAATATAATGC CTACGCCTTGACCTGTGACT T06216 452kb nscaf2556 GATGGTGAGTGGTTAGGGACGT CCTAAGCTAAAGGAGTCCTAATCACA S0601 3007kb nscaf2529 TGTCGTGCGTTTGAAAAGGTT CCAAATGTCGCGTATTAAGCAAA S0515 1867kb nscaf2529 GAGGATGTTGTGCGGACGC CAGGCTTTGGATGATGATAAATACA S0514 3460kb nscaf2674 TTCGCACCATTATCGTCAAA CAAACAGCGGAGGTCGTATA T0515 14kb nscaf2674 CACGCACTTTAATGTCTTCCTG ACGAGTTCATCTGCCGATCC T0512 1066kb nscaf2838 ACTTAGTTCGCCCACCTGACC CCAAGGGCATCGGTGACA S0502 463kb nscaf2681 TCATTAGAGTGGGATAGCGTGTTT TGGGTCATTTTGATTTGGGG S0421 1639kb nscaf2589 CAATCATCAATTAGGTCGTCTATCCA TTAGCGATAAGACCGCCTATTGTA S0417 4851kb nscaf2847 CGGTTGTCTGGAAGAGGTCGC TCGTACTAGGGTGACAGTCCAAAG S0411 4625kb nscaf2847 AACCCCGGTTCTATGCAATCT GCGAGAAATTCGTTTCCAAATG S0408 4546kb nscaf2847 TCGTAGTTATCACTTGCGAATGC CGAAGGCAGAGCAAAGTGGAC S0409 4243kb nscaf2847 GATTCCCACATCGGGCAAA AGCAAACAAACCGCCTGATG S0401 654kb nscaf2925 GTGGTATGGACATAATATGATGCGTA CGGCGTGACCGATTTCCA S0302 4894kb nscaf2930 CAAATTCATGCCAAACGAGA CGACTGAGTTGAGGCTGGAG C0311 3634kb nscaf2930 CTGGCTGTCGTGCTTACTTT GTCTGCATTCCTGAACTCGT T0310 713kb nscaf2886 GCTACGGTATTCGCACTGGAG CGACGCACTCCACCAAGC S0315 1114kb nscaf2886 TCTAATGAAAGCATTCCATCTT AGTTTGGCCTACAAATACAGC C0305 2671kb nscaf2883 CGCCTACTGTCACAAGTGTATCTGC CTTTAACCCTTTGACTGCCATGTA S0314 1104kb nscaf2623 CGATAGATGTCACTACTGCGATTC ATCTCTTCTGGTGTTCTTAGGTCT T02203 1386kb nscaf2623 GATGAATGTGAGGACAGAGATGAC AGCCGAGGTGAGTTGGAATC T02202 1596kb nscaf2964 GACCAAGAAACTGCTATGTAAGAAC GTATCATGTGAGCCAACGACC T0206 4896kb nscaf2964 GCTTATAGTGATTATGGAGTGCTT TTCAGTATGTCCGATGGGTT C0208 4944kb nscaf2964 GAGTTGTTGACCCTTGCGATTAT TGTTCAGAAATGAGGTTGCCCA S0203 AATTACCGCATCCACGTGTT CATTTTGCAGCGGATAAGGT C232S2 3758kb nscaf3040 TGGCACAGTCTTTATGTTTGGCT GCATTCAAACCTAAGTCAAATCAAA S0106 3755kb nscaf3040 AATTACCGCATCCACGTGTT CATTTTGCAGCGGATAAGGT C233S2 3468kb nscaf3040 TATTCCATGGGCGCTTTTTA TTTCGTGCGTCTTTCATTTTT C214S4 3472kb nscaf3040 TAAAAGAGCGGACCGTTAGC CCAATTTTGCTTCGCTTCTC C214S5 3362kb nscaf3040 CGAAACAGCAAAAGAAGAAACA AGGTCATTTTCACAGTCATGTTC C208S1 3272kb nscaf3040 GGAATCGGTGTTTTTCATGC GAGATGCCGAATGGATCTGT C200S8 2862kb nscaf3040 GGGGTTGGTTTTAGCTGTCA GGAACCAAATGAAATGCTCTTT C164S5 1535kb nscaf3040 TTAGGAGCGTCTATTTTAATAATCTGTTC ATAACAGTGACGCAAAGAACTAAACA S0104 1385kb nscaf3040 GAGAAGTGCAGAGTGCCCATATT CAGATTTCGCCAGGACTACACTTT S0105 4529kb nscaf1690 CATTATCGCTCACATCGCTCCT TTTATTTGGAATTAGTGGGGTTA S0108 243kb nscaf1690 CGATGCTCGATCTGATTTACCA CCCTATCCCTAATGATTCTTGTCCA S0107 4153kb nscaf2210 AATAGGAAGTTTTGAAGTATCTCGACC TTGAAACCAGCTCCACATAATAGG S0102 Locus Scaffold R primer (5'-3') F primer (5'-3') Loci name 526kb nscaf2770 ACGGTCGCATAGCAGGATAGA AGACTCCGAGCGGCTGTTAT T1913 1582kb nscaf2903 GTAAAGTTGCATACTCAGGTGTCA AATAGAAGGGAGAAGGAAGGTCT T1817 8871kb nscaf2902 CAAAAGCACAGCTACTTCCGC TTTATTTGTTAGGCTCGTTACTGTCA S1807 8692kb nscaf2902 TAACGGACGCTGTTGGCATT AACTCGGACATACTTGACTGGTT T1810 4517kb nscaf2902 TGAAGCGTGTCGTATCGT AGTATCTATCGTGCCCTGAA N1814 3456kb nscaf2902 ATGTGAACAACCCAAACT CTCAGATGAATCGCAAGA N1807 252kb nscaf2902 TGGCTTGGCTCTGCTCCTG TTCCCCTTTTGAATTTTAACGAG S1816 947kb nscaf2901 AGCGATAAGACCGCCTTTTGT GGCTACGTAAAATACATGGCAGTT S1817 1582kb nscaf2901 AAGATGACAGCCAAGTTCCAC GCGACCCTCACAACCAGA ZR22 964kb nscaf2901 TCCGACGGCTATCAAACAC AGGGAGGTAGTCAGGACAAGG ZR53 460kb nscaf2901 TTCTTATCATTTATAGGGTGAGGC AAGTCTAGTCGTGAAACTAGGGAG ZR33 633kb nscaf2901 GCCGAGCCTTCTCAAACG CCACCAACCTTGCGACAC ZR09 3278kb nscaf2829 CTGACAGTGGTGAGTTAATAAAACAAAA CGGCACTTAAAAGTTTTCATATCAATC S1711 3202kb nscaf2829 TGAGACCCAATAATGTCCCGAG GGGATAAGTGGGTCGTTTTGATT S1710 3178kb nscaf2829 ACGGGCTTTCTTTGCTAGATGT GTCATTGGGAGTTTGAAGTTTCG S1701 45kb nscaf2873 GAAGAGGCGGTGTTGGCTAA TCAGGTGGCATACAGAGAAGAC T1709 4938kb nscaf2865 AACAATAAGCCACTGGACGC AAACTCTGTAGAAGTAGGGGAACC T17215 900kb nscaf2766 TACTTACCCTCCGCTGTTCG ACTGTTATTCGGGGCTTGTG T17225 331kb nscaf3063 CCCTTGGGCTTAAGTCGGTTT CACCTAGTTTTACTTCACGGACCAT S1615 1417kb nscaf3063 CCGAGGAAGAGATAGTTGGACTC AAGCATCAGGCAGTAATGTGGTA T16322 246kb nscaf3062 TGACTTCCAGTTACACGCATCTA TCGCACACTCGCTCTCATTG T16312 67kb nscaf3058 GGCGATAACTTTGGGATACAC ATCAGCGAACGAAAATAGACG T1617 629kb nscaf3058 CGAATGTGCGTGATGTAACCAA GCGAGAGGCTATTATTCTGACTTC T16308 1638kb nscaf3058 ACAGATTGATTCCAGAAACGAGC AAATAGAAACTGACGATGAGGTAATAGC S1606 2642kb nscaf2655 TAACAACTCACAGTCATCGCTAAG TCACAGTCAGCACAGATGGTT T1515 3336kb nscaf2655 CCGCAAGGTAGCATAACACAG CACGAGTCCATCAGAAACACAAT T1516 9504kb nscaf2888 CGCTCCGCTATCTCATTACCTTAG CACAGACAATTCCTCCACGAGTA T1501 8349kb nscaf2888 TACTTGTTATTGGACCTCGTGGAT GGTGTTATGGAATATGCGGTAGG T1502 5086kb nscaf2888 CGTATTAGGGAAGAAAAGATGGCT GTGGTTATTGGGAACTTTTATGATGT S1509 811kb nscaf2888 GGAGCAGCCTCATCTTAACAT TGGACAGTGGCACCTTTCT C1501 473kb nscaf2888 GCGTAGCCACTGAAGGTTTACTT TGCCCCACCCTTCAAACC S1507 25kb nscaf2953 GTAGATAAATCCCACAAGCAAGA AACCATAGTCGGGGTCATTC T1417 1534kb nscaf2953 GTAGGCATACTCATAGGCATAAGC GGTTACCAGTGTTCATAGCAGAG T1403 335kb nscaf2943 CTGGCGTGTCACTTGTCTCT ACAGGTAGGTCGGTGTAGCA T1406 268kb nscaf2948 ATTGCTACTGGTTCGCTTGGA CTGGAGCCTGTAGTGGACTG T1413 1245kb nscaf2948 ACGGTGGTCTTACGAATGTCAT GGTCACAGACGAGGTCATCAC T1414 2568kb nscaf2948 AACTAATAACCGTCTCCGACAAATAA ACCTCGGGACAGATTCGTTTT S1424 5514kb nscaf1898 TACACAGAGACCTGAACCTAGAAC TTGCTTCGTGACCTTGAGATAAC S1313 6336kb nscaf1898 GGTGAAGGTGAACGGAGGAC CTAGTGTGATTGGATCGGTGAGTA T1312 7459kb nscaf1898 TTATCTAAAATGGCATCGCAAATC TCCCTGGCATTGCTGACG S1307 15213kb nscaf1898 CATTCTGCTTCTGCCGTTGATAA ATACCGACCACACCATACAATACT T1303 14462kb nscaf1898 GAGATTCCACAGGAGTGTCTTAGT GGCTATCCGAACGCTAATCAGA T1304 1130kb nscaf3093 CCTTCAAACCGAAACGCATTACT GAGACTTTGTGTCGTGGGTCCTAC S1320 106kb nscaf2993 TGGTTCAGCGGAAGGACTTG GCTCCAATCTCGTTAGGTGTTC T12203 7562kb nscaf2993 AAGGACCAACGACGATTCTGAA CTTATGCGGACTACATTGTGTCAA T1209 4498kb nscaf2993 TTGGAATAGAGTGGCACGGTTT GCACTTTATCTCCTCAGCTACCG S1215 nucleosis polymorphism. SSR 1101kb nscaf2797 GAAGCAGCAGCCCTCCGA CGGAATTAGATGTCTCACTGCCA S2803 STS 582kb nscaf2797 CCGAATCTTCACAATTCTCCACAA GGAACTGGCAATAGCGAACAA T28214 STS 386kb nscaf2801 AGGTGAGAATCTACTCCAGCAT TACCGATACACGACTCGCATA T28213 STS 575kb nscaf2836 GCAGTGAGGTGGCAATGATG GGTAAGGTGAGGTGGCGTAG T28208 SSR 86kb nscaf3071 CTGTAAATTAACACGGTTGTCTGGA TGTTTGCTGTGGCGGTAGTG S2816 SSR 1862kb nscaf3098 ATATTCAGCACAGCGTTTCCTTAA GGCAAAGACTGGATGTGAGATGTAA S2721 SSR 442kb nscaf3098 CCAGAAATAGCGGGTGAATCC TGAAGTGTCCTTAGGAGTCGTCG S2702 SSR 568kb nscaf3097 CCAGGCAGTTGAGGTCGTGA GGCACCCGACCATTTGTTGT S2710 STS 4032kb nscaf3099 GCATCTATTCCGTTCGCTTATATG GTCGTAGCCAGGAACACTATTAG T27207 SSR 2927kb nscaf3099 CGTTGTAGATGTCTATGGGCTCC ACAGACTTAACTTAAAACGGATTGAAA S2714 STS 1867kb nscaf3099 AAGACCTATCACCACCGACTG GGAACCTCTACCGCATCATCA T27204 STS 3204kb nscaf3003 GCATTGGACGCAGTACCTCATA TGCCTCTGTAGTTGTTACTTGTGA T2604 SSR 1776kb nscaf2330 GGATGGACGGAAAATCGCAG GCCAAATGATTATTGTCAGCCTG S2621 STS 2324kb nscaf2330 CGATGTTCCTCTCCGAAGACTT ATGCTCTGCTGAAGAAGGTGAT T26308 SSR 2521kb nscaf2330 GGCGCAATTTTGTCTGCACTTA TGAATACTGAAATAGCGGTCGTTG S2620 STS 3868kb nscaf2330 TCACCTTGGAGCAGCACCTA CTCGCCAGTGTTATGAAGAGAAC T26305 STS 935kb nscaf1071 TGTCTCGCTCATCCTCATACG CACAGCAGATTATACACGCATTAC T2613 SSR 1056kb nscaf1071 AAACTTATCTCAAGGTAGGTGGCG AATAGCGGAGGAACTGGGGA S2624 SSR 1829kb nscaf2815 TAGCGATAAGACCGCCTATTGTAC CCAGCGAACGTAGGGGACA S2518 STS 1155kb nscaf2815 TGCTTCCAACCGATTAGTAGTTAC TTCTCTGGTGAAGGCAAGTCTA T2515 SSR 4234kb nscaf2823 GGTTTTACGGGATTCCCTCAGT CTTCGCATATCATAATAGGCTTTTG S2508 STS 1711kb nscaf2823 TATTTGCTTCGGAGTGTCGT GGTTTCGGTGATTCTGATGT T2505 STS 1176kb nscaf2823 TTGGAATTGATGACCCGTTT CATTGCTAGGGCTAGTGTTGG T2501 SSR 1152kb nscaf2823 ACTGGTTTCGCATTGCTTGTG AAACATCAAACAGTTAGGTGGGC S2514 SSR 930kb nscaf2823 ACCCTTAGCAGTCGAGTACAATTTT AATGAGCTGTCTTGCTTAGAAAACTTAT S2525 STS 1666kb nscaf2822 CGTCAGGTGCGTCACATAGA TCTCTGGTATTCACTCCGACAAC T2506 SSR 963kb nscaf2822 CCAAGGAAAATCCCTGGAATCT GGGTGAAAAGTGATGACCAAAAC S2529 SNP 1172kb nscaf1705 GCTGACGACTATGCCTCAACA TGATGACACTGCTGCTTGGAA C2502 SNP 461kb nscaf1705 ATCCGAGAACGCTAATGTATCAGA GTTCAAGCAAGGTCTTCAGTGT C2501 STS 199kb nscaf3035 GTTACAGGCTTCCAGAGTGAGAG TCCTTCAGACCACAGCATTCATT T2419 STS 31kb scaffold606 AGGACTAACTCACACTGTCATCAT TCTTCAGCACTCGTCATCTATACT T2415 STS 213kb nscaf2962 TTGATCGTGCGAATTGTTGCTA CGTAGTCCTCGGTGTGATTGA T2411 STS 1200kb nscaf1108 CAGGAATCGGTGAGTTCTTCTTAA TGTTAGGCTGTTGCGGAGTC T2407 SSR 1295kb nscaf3066 GGTAGAGGGGCAAGAGGTCG TTTCTTTGTCCCCGCCTTCT S2410 SSR 436kb nscaf2891 GAATGTCGGTAGTTTGTACTTTGTTTG ATTAATATTTAGTATACAAAGCGTCCCAT S2404 SSR 1420kb nscaf3022 TGCCCACGGTCTCCTCG CGGGGACGGTGCTCCTTAC S2326 STS 143kb nscaf3022 TGAGAGCCAACGGATACTAGGT CAGGACATACGCACGGAGTC T2320 SSR 5670kb nscaf3026 ATGGGTTAGTTCGCTCGTCG ATGACAATACGCTTTATCACCAGG S2304 SSR 5155kb nscaf3026 TGGAAATTGTAGTGCAAGGTAGAGG GTGCTCACGAACGACTTGTACG S2308 STS 5133kb nscaf3026 CTTGTCGCATAAGAACCACATCTA GTAGCCACTCACAGCCAGAC T2318 SSR 4843kb nscaf3026 TTTAATGTATCGCACTGTTTATTTGTTT GGAACGCTGAATCCACGGA S2327 STS 305kb nscaf3026 TAGAAGGTGATGACAGGCTCAATT TGAAGGTCTTGCCGAAGTTATGA T2313 SNP 2334kb nscaf3027 CCTTTCAGATGACCTGCCGTTGG TTCGGATTACTTCTGTATTTGGTC N2301 SSR 209kb nscaf3027 GGTTTTCTGATTTATTGTTCTATGGTATTA GCCTCTGTTTTATTCAACTCACGC S2323 SUPPLEMENT 1. STS SSR 585kb nscaf2825 TCCGTGGAATTTTATTTCAGTTGTA CTCAGTTGGTCGCATTTCCG S1207 STS 294kb nscaf1962 ACCTACCAGGACGGACTTACC CACGGTCACGGCATTCTCTT T2301

Received August 17, 2012; revised and accepted November 28, 2012 

ACKNOWLEDGEMENTS. This project was supported by National Natural Science Foundation of China (grant No. 30972145, 30825007), and Natural Science Foundation of Jiangsu Province (BK2009220). We thank H. Loxdale for his helpful editorial comments on the manuscript. Acta Entomol. Sin. 41: 32-40 [in Chinese]. XIA Q., ZHOU Z., LU C., CHENG D., DAI F., LI B., ZHAO P., ZHA X., CHENG T., CHAI C., PAN G., XU J., LIU C., LIN Y., QIAN J., HOU Y., WU Z., LI G., PAN M., LI C., SHEN Y., LAN X., YUAN L., LI T., XU H., YANG G., WAN Y., ZHU Y., YU M., SHEN W., WU D., XIANG Z., YU J., WANG J., LI R., SHI J., LI H., LI G., SU J., WANG X., LI G., ZHANG Z., WU Q., LI J., ZHANG Q., WEI N., XU J., SUN H., DONG L., LIU D., ZHAO S., ZHAO X., MENG Q., LAN F., HUANG X., LI Y., LIN F., LI C., LI D., SUN Y., ZHANG Z., YANG Z., HUANG Y., XI Y., QI Q., HE D., HUANG H., ZHANG X., WANG Z., LI W., CAO Y., YU Y., YU H., LI J., YE J., CHEN H., ZHOU Y., LIU B., WANG J., YE J., JI H., LI S., NI P., ZHANG J., ZHANG Y., ZHENG H., MAO B., WANG W., YE C., LI S., WANG J., WONG G.K. & YANG H. 2004: A draft sequence for the genome of the domesticated silkworm (Bombyx mori). -Science 306: 937-940. XIANG Z. 1991: Genetics of the silkworm. In Lv H.S. (ed.): The Sericultural Science in China. Shanghai Scientific and Technical Publishers, Shanghai, pp. 223-290. YAMAMOTO K., NARUKAWA J., KADONO-OKUDA K., NOHATA J., SASANUMA M., SUETSUGU Y., BANNO Y., FUJII H., GOLDSMITH M.R. & MITA K. 2006: Construction of a single nucleotide polymorphism linkage map for the silkworm, Bombyx mori, based on bacterial artificial chromosome end sequences. -Genetics 173: 151-161. YASUKOCHI Y. 1998: A dense genetic map of the silkworm Bombyx mori, covering all chromosomes based on 1018 molecular markers. -Genetics 150: 1513-1525. YASUKOCHI Y., BANNO Y., YAMAMOTO K., GOLDSMITH M.R. & FUJII H. 2005: Integration of molecular and classical linkage groups of the silkworm, Bombyx mori (n = 28). -Genome 48: 626-629. ZHAN S., HUANG J., GUO Q., ZHAO Y., LI W., MIAO X., GOLD-SMITH M.R., LI M. & HUANG Y. 2009: An integrated genetic linkage map for silkworms with three parental combinations and its application to the mapping of single genes and QTL. -BMC Genomics 10: 389-404.
Bing Li, Xiuye Wang, Chengxiang Hou, Anying Xu, Muwang Li
Genetic analysis of quantitative trait loci for cocoon and silk production quantity in Bombyx mori (Lepidoptera: Bombycidae)