Screening SSR And SNP Markers From BAC-end Sequences And Their Preliminary Application In Zhikong Scallop (Chlamys Farreri)

Zhikong Scallop (Chlamys farreri) is one of the traditional and most importantmolluscs that are being widely cultured in China. The marker-assisted selection (MAS)provides the fundamental theories and applied experiences for this task not only inbetter utilizing the available resources but also increasing production in aquaculture.This research is to develop and characterize BAC-based molecular markers such asSSR (Simple Sequence Repeats) and SNP (Single Nucleotide Polymorphisms) forZhikong Scallop.To further understand scallop genome, a total of10,237BAC clones wererandomly selected from two scallop BAC libraries and both ends were sequenced withSanger method,17,447BAC-end sequences (BESs) including7,314paired-ends wereobtained, with an average read length of446bp after trimming and quality filtering. Atotal of7,773,272bp were generated, representing0.63%of the scallop genome.Based on this survey, the scallop genome was found to be highly AT-rich, with63.45%AT and36.55%GC.17,785repetitive elements were found in8,550BESs.For the SSR (1-6bp tandem repeats,≥12bp), hexa-nucleotide motifs were the mostabundant, followed by penta-nucleotide and tetra-nuceotide motifs, tri-nuceotidemotifs were the least. After the blast with Nr/Nt and EST database, we found2,083、1,375and1,901hits specifically. Among of them,446(2.56%) BACs have hits to thescallop related genes on both ends. A few of scallop BESs were anchored to thegenome of some sequenced species respectively. These BESs were identifed as amajor genome resource for scallop and mollusc genomic research. Microsatellite markers were developed from the BAC-end sequences and used toanalyze the genetic structure and genetic differentiation in two C. farreri populations(Dalian ang Qingdao).14polymorphic SSRs were chosen to amplify and then analyzethe genetic diversity in the two populations. A total of395alleles were obtained at thefourteen microsatellite markers and the number of alleles in each locus ranged from8to38in the two populations. The average number of alleles (Na) was18.9286and26.2143respectively. The average effective number of alleles (Ne) was11.7505and17.0891respectively. The mean observed heterozygosity (Ho) was0.5100and0.4204.The mean expected heterozygosity (He) was0.9156and0.9450. The data suggestedboth the two populations have high genetic diversities. The mean polymorphicinformation content (PIC) was0.8940and0.9302, which were both greater than0.5,indicating the fourteen loci were highly polymorphic. The unbiased genetic identityindex was0.4879, and the genetic distance was0.7177. The coefficient of genedifferentiation (Fst) and gene flow (Nm) between two populations were0.0243and10.0179respectively. Low genetic differentiation was observed between the twopopulations, and the variance mainly came from individual difference. Significantdeviation was detected by Hardy-Weinberg equilibrium test. There was heterozygotedeficiency at all loci. The results showed BAC-end sequence was an effectiveresource for development of SSR markers for genetic and genomic researches.Two strategies were applied to develop SNP markers based on the resources ofBESs and next-generation sequences of the two mapping parents which conserved inour laboratory. Firstly, we designed370pairs of primers with the BatchPrimer3.0software.260pairs were successfully amplified, which occupied70.25%The PCRproducts were sequenced with Sanger method. Then we used the BioEdit andSequencer5.0Demo software to do blasting.342SNPs were detected in112BACclones, and among of them,154SNPs in76BAC clones met the "pseudo-testcross"mapping strategy. With this method,19Indels were also found in13BAC clones.These SNPs and Indels are good resources for Zhikong Scallop genetic mapping after genotyping and linkage analysis. Secondly, the SNP searching software ssahaSNPwas used to develop BAC-based SNPs. The genome of two mapping parents weresequenced with next-generation sequencing technology (Illumina HiSeq-2000)resulted in11.67Gb and11.78Gb clean data respectively. Then we performed thealignment process by ssahaSNP for SNP detection. The number of SNPs detected isdifferent due to different set of parameters. The more strict parameters are set, thefewer SNPs are detected. When we set the parameters “match”,“identity” and “map”to90,95and5respetively,222,182SNPs and41,250Indels were developed. Oncethe parameters were increased to90,95and5respectively, the number of SNPs andIndels is accordingly decreased to53,398and7,092. To validate these SNPs andestimate the false positive rate,32SNP sits were randomly selected for primer design,PCR amplification and sequence alignment. The results showed that84.38%pairs ofprimers were successfully amplified and the success rate of sequencing was77.78%.Among the SNPs available for alignment analysis,76.19%of SNPs were proved to betrue positive. The research demonstrated that it is practical to detect SNP usingssahaSNP with BESs as the reference sequence. It is vital for the large scale SNPdevelopment of Zhikong Scallop and the SNP genetic map construction.TP-M13fluorescent-labelled system and MALTI-LOF Mass Spectrometry wereused to genotype SSR and SNP markers. The genetic linkage map of C. farreri wasconstructed using12microsatellite markers,39SNP markers,373AFLP markers andone sex marker. Linkage mapping was performed using the F1outbred families,which contained149and201markers respectively in the female and male linkagepopulation. The number of linkage groups was18in female and19in male, whichwas consistent with the haploid chromosome number of C. farreri.33contigs on thephysical map were integrated on the linkage map of C. farreri, which is important forwhole genome sequencing, QTL analysis and Marker-Assisted Selection.