| The triangle sail mussel,Hyriopsis cumingii,is the most commercially important pearl mussel species in China.Colour is one of the most important criteria to judge the quality and value of freshwater non-nucleated pearl.It is well known that the shell nacre colour is related to pearl color in some pearl mussels.In this study,an full-sib family were used for a larger numbers of SNP markers development by SLAF-seq method.Then the developed SNP markers were used to construct the high-density genetic linkage map and QTL mapping for pearl quality-related traits in H.cumingii.Meanwhile,HcTyr and HcTyp-1 genes were used to detect shell nacre colour-related SNPs and genetic mapping.Then,SNPs selected from shell nacre colour-related QTLs were used to screen positive clones based on an established BAC library.Subsequently,high-throughput sequencing technology and bioinformatics tools were used to sequence the positive clones and predict candidate genes,respectively.The results were summarized as follows: 1)SLAF marker development and construction of the high-density linkage map for H.cumingiiIn this study,we first constructed the SLAF-seq library for H.cumingii with 157 individuals and two parents.Using high-throughput sequencing and bioinformatics,a total of 139,113,148 pair-end reads were obtained for the SLAF library.Among them,88.64% bases were of a high quality(Q score > 30),and GC content was 37.45% on average.After correcting or discarding low-depth SLAF tags,239,704 high-quality SLAFs were identified,of which 201,805 and 206,806 were detected in the male and female parents,respectively.The coverage of SLAFs for paternal and maternal parents was 19.25-fold and 20.70-fold,respectively.Among the 239,704 high-quality SLAFs,129,967 were polymorphic with a polymorphism percentage of 54.2%.A further screening was performed and 4,508 SLAF markers and 506 SSR markers from the framework map were used for the linkage map construction.After linkage analysis,4,920 markers were mapped to 19 linkage groups.The final map was 2,713 cM in length,with an average inter-marker distance of 0.55 cM.The number of markers mapped to each linkage group varied from 114 to 436,with an average of 258 markers per LG.The mean chromosome region length was 142.80 cM.The ‘Gap ? 5 cM' value(indicates the percentages of gaps in which the distance between adjacent markers is smaller than 5 cM)of the 19 linkage groups ranged from 80.28% to 100%(average,97.79%).2)QTL mapping for shell nacre colour-related traits and the validation of QTLs in H.cumingiiOn the basis of the high-density genetic map,a total of seven QTLs were detected for five shell nacre colour-related traits,including,one for AL,one for Aa,three for Ab,one for AdE and one for purple mantle scar length.The phenotypic variability explained by each QTL ranged from 17.9% to 22.8%.Of these seven QTLs,five significant QTLs related to shell nacre colour tended to colocalize,clustering in a special region 17.3-36.1 cM on LG17 and explained 19.7% to 22.8% of the trait variation;this suggests that some important genes or loci determine shell nacre colour in LG17.To examine whether the SNPs located on QTLs were associated with shell nacre colour,three SNPs were selected from shell nacre colour related QTLs and used for primer design.Then,the primers and markers were assessed in three families of H.cumingii,and each family contained one hundred individuals.Shell nacre colour was assessed with reflectance spectroscopy with transformation into color parameters based on the tristimulus values.Statistical analysis showed that different genotypes of these three markers were significantly associated with L,a,b and dE(P < 0.05).This suggested that the shell nacre colour related QTLs identified in the study had high accuracy and reliability.3)Detection of shell nacre colour-related SNP and gene mapping of HcTyr and HcTyp-1 genes in Hyriopsis cumingiiGenomic sequence fragments were amplified according to cDNA fragments of HcTyr and HcTyp-1 genes.Then 8 and 7 SNPs were identified from HcTyr and HcTyp-1gene respectively after sequencing the PCR products and blasting.Regarding these SNPs,the genotype and gene frequency of 144 Hyriopsis cumingii were further analyzed,which showed the polymorphic information(PIC)of these SNPs were low and medium genetic diversity.Associations between SNPs and shell nacre colour related traits were analyzed using Chi-square test.The results showed that there were 7 SNPs on HcTyr gene and 4 SNPs on HcTyp-1 gene were significantly associated with shell nacre colour(L,a and dE).Haplotype analysis revealed that the frequency of four major predominant haplotypes(?,?,? and T1)in the white strain was significantly higher than that in the purple strain.And the other three major predominant haplotypes(?,? and T4)showed significantly higher frequency in the purple strain.Gene mapping showed that HcTyr and HcTyp-1 genes were both located on LG16.4)Construction of BAC library and screening of shell nacre colour candidate genes for H.cumingiiA healthy H.cumingii was selected for construction of BAC library with HindIII.The BAC library was pooled in 4 x 96-well plates with a total of 163,200 BAC clones.Plate 1 and plate 2 containing about 350 independent primary clones with each well,and the average insert size of BAC was 135 kb.Plate 3 and plate 4 containing about 500 independent primary clones with each well,and the average insert size of BAC was 150 kb.The genome coverage of the library is about7.8 –fold assuming that the genome size is 3×109 bases.SNP and SSR markers selected from shell nacre colour-related QTLs were used to screen positive clones based on this BAC library.Then,6 BAC clones were detected by PCR and used for high-throughput sequencing.And bioinformatics tools were used to analyze the positive clones sequences and predict candidate genes.Finally,two genes that code for cytochrome P450-10 and ABHD6-B-like proteins were selected from BAC2G12 and BAC1D6 respectively,which were considered as candidate shell nacre colour-related genes. |
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