基于三代全长转录组测序的极边扁咽齿鱼SSR分子标记开发

SSR molecular maker development based on three-generation full-length transcriptome sequencing of Platypharodon extremus

    摘要
  • 摘要: 【目的】基于三代全长转录组测序数据,开发极边扁咽齿鱼SSR分子标记,积累和丰富该物种遗传信息,为后期指导和优化人工繁殖技术提供理论参考。【方法】基于极边扁咽齿鱼三代全长转录组测序结果(PRJNA792686),分析SSR分布情况,筛选出具有多态性的SSR引物,并对2个流域37尾边扁咽齿鱼的遗传多样性进行分析。【结果】极边扁咽齿鱼单核苷酸重复SSR个数最多,占37.87%,二核苷酸~六核苷酸重复序列依次占31.77%、10.32%、1.29%、0.25%和0.10%;二核苷酸、三核苷酸、四核苷酸和六核苷酸重复序列均以重复次数5~8次居多,分别占各自SSR总数的55.26%、94.90%、70.92%和89.52%;之后随重复次数增加而逐渐减少,当重复次数为21~24次时最低,分别占各自SSR总数的2.63%、0.04%、2.68%和0。单核苷酸重复序列以重复次数9~12次居多,占其SSR总数的71.43%;五核苷酸重复序列的重复次数主要集中在5~8次,占其SSR总数的83.33%;三核苷酸、四核苷酸和五核苷酸重复序列中,排名前5的序列分别是TA(17.07%)、AT(16.95%)、TG(15.29%)、AC(12.51%)和CA(9.71%);TTA(7.53%)、GAT(6.07%)、TAT(5.93%)、CAG(5.50%)和GAG(5.50%);TATC(4.83%)、TTTA(4.32%)、TTCT(3.81%)、TATT(3.56%)和TGTT(3.18%)。共设计38对SSR引物,最终筛选得到9对SSR多态性引物。2个流域的极边扁咽齿鱼等位基因数(Na)为3~29,其中,A流域极边扁咽齿鱼的期望杂合度(He)为0.484~0.931,香农指数(H′)为0.677~2.793;B流域极边扁咽齿鱼的He为0.315~0.948,H′为0.578~3.075。【结论】2个流域的极边扁咽齿鱼具有较高的遗传多样性。开发的9个多态性SSR分子标记可用于极边扁咽齿鱼育种和种质保护。

     

    Abstract: 【Objective】The purpose of the study was to develop SSR molecular markers for Platypharodon extremus based on three-generation full-length transcriptome sequencing data, in order to accumulate and enrich the genetic information of this species,and to provide theoretical reference for guiding and optimizing artificial breeding techniques in the later stage. 【Method】Based on the three-generation full-length transcriptome sequencing results of P. extremus(PRJNA 792686), the distribution of SSRs was analyzed, and polymorphic SSR primers were selected. The genetic diversity of 37 P. extremus individuals in two watersheds was analyzed. 【Result】The results showed that the number of mononucleotide repeat SSRs was the largest in P. extremus, accounting for 37.87%. The dinucleotide-hexanucleotide repeat sequences accounted for 31.77%, 10.32%, 1.29%, 0.25% and 0.10% in order. The dinucleotide, trinucleotide, tetranucleotide and hexanucleotide repeat sequences were predominant at 5-8 repeats, accounting for 55.26%, 94.90%, 70.92% and 89.52%of the total number of their respective SSRs, respectively. After that, they gradually decreased with the increase of repeat number, and they were the lowest when the number of repeats was 21-24, accounting for 2.63%, 0.04%, 2.68% and 0 of the total number of their respective SSRs, respectively. The mononucleotide repeat sequences were predominantly repeated 9-12 times, accounting for 71.43% of their total SSRs.The pentanucleotide repeats were mainly concentrated in 5-8times, accounting for 83.33% of their total SSRs. The top 5 sequences in trinucleotide, tetranucleotide and pentanucleotide repeats were TA(17.07%),AT(16.95%), TG(15.29%), AC(12.51%)and CA(9.71%); TTA(7.53%),GAT(6.07%), TAT(5.93%), CAG(5.50%)and GAG(5.50%); TATC(4.83%), TTTA(4.32%), TTCT(3.81%), TATT(3.56%) and TGTT(3.18%), respectively. A total of 38 pairs of SSR primers were designed, and 9 pairs of SSR polymorphic primers were ultimately screened. The number of alleles(Na) in the two watersheds was 3-29, in which the expected heterozygosity(He) of P. extremus in watershed A was 0.484-0.931 and the Shannon index(H') was 0.677 to 2.793. The He of the watershed B was 0.315-0.948 and H' was 0.578-3.075. 【Conclusion】P. extremus in the two watersheds have high genetic diversities. The nine polymorphic SSR molecular markers developed can be used for breeding and germplasm protection of P. extremus.

     

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