2023, 38(2): 196-205. doi: 10.16535/j.cnki.dlhyxb.2022-205
Keywords： Coilia nasus , transcriptome , microsatellite , molecular marker
为加速长江刀鲚(Coilia nasus)分子标记辅助育种的研究进程,对养殖长江刀鲚雌、雄个体分别进行转录组测序,利用MISA软件挖掘长江刀鲚脑转录组Unigenes中的EST-SSRs,采用SSRMMD软件规模化开发多态性EST-SSRs,并对3个长江刀鲚养殖群体开展遗传多样性研究。结果表明:从雌性长江刀鲚脑转录组143 374条Unigenes中获得了 95 905个EST-SSRs,从雄性脑转录组135 215条Unigenes中获得了88 774个EST-SSRs;雌、雄转录组EST-SSRs均以二核苷酸重复基序数量最多,其他核苷酸重复基序的数量均呈逐级减少的趋势;单核苷酸到三核苷酸重复基序的优势重复基序分别为A/T、AC/GT和AGG/CCT;从1 082个多态性EST-SSRs中随机选取20个位点设计基因特异性PCR扩增引物,并进行有效性和多态性验证,获得了18对多态性较好的EST-SSRs(多态率约为90%),用其评估长江刀鲚不同年份(2018、2019和2020年)养殖群体的遗传多样性,结果显示,2018、2019、2020养殖群体的平均观测杂合度(Ho)、平均期望杂合度(He)和多态信息含量(PIC)分别为0.445 2、0.485 4和0.420 9,表明长江刀鲚养殖群体的遗传变异较为丰富。研究表明,基于长江刀鲚脑转录组数据所开发的多态性EST-SSRs能够应用于不同年份养殖群体的种质资源评估和遗传育种研究。
In order to accelerate the research process of marker-assisted selection (MAS) in breeding of tapertail anchovy (Coilia nasus), the brain transcriptome sequencing was carried out in 12 individuals of 2 years old including 6 females and 6 males of cultured tapertail anchovy, respectively. The MISA software was used to mining expressed sequence tag-simple sequence repeats (EST-SSRs) from unigenes in the brain transcriptome of tapertail anchovy, and large-scale development of polymorphic EST-SSRs was carried out by SSRMMD tool. The genetic diversity of three cultured populations of tapertail anchovy was studied based on the selected polymorphic EST-SSR markers. Results showed that 95 905 EST-SSRs were identified from 143 374 unigenes in the brain transcriptome of female tapertail anchovy, and 88 774 EST-SSRs were identified from 135 215 unigenes in the brain transcriptome of male tapertail anchovy, with the maximal number of di-nucleotide EST-SSRs, and decreasing trend of the number of the other nucleotide EST-SSRs, accompanied by an increase in the number of repeat base. The dominant repeating units were found to be A/T in mononucleotide, dinucleotide and trinucleotide EST-SSRs, AC/GT in dinucleotide and AGG/CCT in trinucleotide EST-SSRs. Twenty EST-SSRs sequences from 1 082 polymorphic EST-SSRs were randomly selected to design specific PCR amplification primers, in order to evaluate their validity and polymorphism. Eighteen pairs of primers were proved to have better polymorphism and were used to evaluate the genetic diversity of three cultured tapertail anchovy populations in different years (2018, 2019 and 2020). The tapertail anchovy had average values of 0.445 2 in observed heterozygosity (Ho), 0.485 4 in expected heterozygosity (He) and 0.485 4 in polymorphic information content (PIC) in the three cultured populations, which proved that the genetic variation of cultured tapertail anchovy was still abundant. The finding indicated that these polymorphic EST-SRRs developed from the brain transcriptome data were applied to germplasm resource evaluation of the cultured population in different years and further genetic breeding research of tapertail anchovy.
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