Transcriptome Analysis Of Argopecten Irradians Concentricus And Study On The Molecular Markers Of Its Progeny Crossed With Argopecten 'Bohaihong'

Argopecten irradians concentricus is an important economic species cultivated in the southern China sea area.Since its introduction in 1991,it has created good social and economic benefits.However,the continuous breeding of small groups for more than 20 years has seriously degraded the germplasm.It is fundamental to maintain the sustainable development of its aquaculture industry are introduced the new genetic resources and achieved the breakthrough in the research on the genetic improvement of it.Argopecten ‘Bohaihong' was produced by genetically purifying from hybrid generation of Argopecten irradias and Argopecten purpuratus.It is a new species that has been popularized and cultivated in the sea areas of northern China in recent years.In September 2015,A.'Bohaihong' was introduced from Qingdao,Shandong,and Qinhuangdao,Hebei by our research team.Our group has hybridized it with the selected A.irradians concentricus and succeeded in it.The feasibility of hybridization has been confirmed.However,A.irradians concentricus and t A.'Bohaihong' belong to the hermaphrodite shellfish,homozygous fertilization is likely to cause genetic decline.In order to ensure the effect of cross breeding,it is necessary to analyze the molecular markers of genetic differences between the two scallops.With the maturation and popularity of next-generation high-throughput sequencing technology,transcriptomics has emerged as an important source of molecular marker development and functional gene mining.In view of this,we have started related research.The main results are as follows:1.Transcriptome Illumina sequencing and bioinformatics analysis of Argopecten irradians concentricusThis study was based on the Illumina Hi Seq ^TM 2000 platform to perform transcriptome sequencing for rapid and slow growth group of A.i.concentricus.By de nove assembly,120 563 and 118 794 high-quality unigenes sequences were obtained in the fast-growing and slow-growing groups,respectively,and the average length of this unigene was 944 bp and 969 bp.According to the principle of FDR?0.001 and |log2Ratio|?1,a total of 9 901 differentially expressed genes?DEGs?were screened.Among them,the fast-growing group compared to the slow-growing group,4976?50.26%?were up-regulated genes and 4925?49.74%?were down-regulated genes.A total of 47 growth-related genes were screened from them,involving 17 types about muscle growth proteins,digestive enzymes,growth factors and their receptors.KEGG metabolic pathway analysis has revealed several MAPK signal transduction,TGF-? and other growth-related pathways involved in cell proliferation and differentiation.Nine randomly selected growth-related genes were used for q RT-PCR validation.The results showed that the overall trend of q PCR expression profiles was consistent with RNA-Seq sequencing results,indicating that RNA-Seq accurately quantified gene expression.In the GO functional classification,most of the differentially expressed genes enriched by biological processes?53%?involved 26 subclasses,suggesting that the growth of A.irradians concentricus is a omnidirectional response to biological processes.In the rapid growth group,a total of 10 870 SSRs and 242 551 SNPs were identified,and 10 857 SSRs and 228 121 SNPs were identified in the slow growth group.2.Screening of growth-related genes and SNPs of Argopecten irradians concentricus based on transcriptome sequencingUnigene23250 and CL4903.Contig2 are two growth-related sequences screened from transcriptome of A.i.concentricus and annotated as alpha-amylase gene and multiple epidermal growth factor domain 6.The potential SNPs predicted from the two sequences were verified by PCR amplification and direct sequencing.Five SNPs were detected,including four transitions and one transversion.The three full linkage sites on the CL4903.Contig2 sequence?A-6167-T,G-6178-A,G-6185-A?and the G-1142-A site on the Unigene 23250 sequence are non-synonymous mutation SNPs point.One-way analysis of variance?ANOVA?was performed to determine the associations between the identified SNP markers and body weight,shell weight,shell length,shell width,and shell height.The results showed that the weight,muscle weight and shell length,shell width and shell height were significantly higher with genotype of AA on G-1142-A locus than the genotype of AG and GG?P<0.05?.The body weight and average weight of adductor muscle were significantly higher with AA genotype than AG and GG genotypes of CL4903.Contig2 G-6085-A site?P<0.05?.The genotype AA is presumed to be the dominant genotype of these sites.The results of the study indicate that these SNP loci screened from the transcriptome of A.i.concentricus have the potential for molecular assisted breeding on growth traits.3.SSR development and hybrid F1 identification of Argopecten irradians concentricus and Argopecten ‘Bohaihong'To identify hybrid offspring of A.irradians concentricus and A.'Bohaihong'and assess their heterosis,SSR markers developed based on the transcriptome of A.irradians concentricus.A total of 26 polymorphic loci were screened out from 117 loci,and the polymorphic rate was 22.2%.The common rate for the scallops A.'Bohaihong' was 80.77%,for Argopecten irradias was 92.31%.These polymorphic sites are dominated by two-base 6 repeats,and the type of primitive with the highest amplification efficiency was AT.Eight pairs of polymorphic universal primers were randomly selected to analyze the genetic diversity of four populations in Qingdao,Qinhuangdao scallop A.'Bohaihong',A.irradians concentricus,and A.irradias.The results showed that the average allele number of the four populations was 5.250 to 6.375.The average number of effective alleles was 2.205-3.126,the average heterozygosity was 0.388-0.641,and the average expected heterozygosity was 0.494-0.643.The scallop A.'Bohaihong' had the highest polymorphism information content,and both geographic groups were at higher levels?P>0.50?.While the scallops A.irradians concentricus have the lowest genetic diversity and the polymorphic information content was at a moderate level?0.25<P<0.50?.The genetic distance cluster analysis indicating that there are certain genetic differences between the two scallops.If these two scallops are crossed,a greater heterosis will be expected.This study also developed three interspecific SSR markers for A.irradians concentricus and A.'Bohaihong',These three pairs of specific markers were applied to the hybridization progeny for identification.It was found that the 30 offspring tested contained both the corresponding specific bands of both parents,which proved that they were all progeny of interspecific hybrids.