劉玉萍 王棋 黃新芯 徐開達 王忠明 高天翔 楊天燕
摘要:【目的】通過高通量測序平臺對帶魚肌肉組織進行轉(zhuǎn)錄組測序,從海量數(shù)據(jù)中查找微衛(wèi)星(SSR)位點并進行生物信息學分析,為科學制定帶魚種質(zhì)資源保護對策和管理措施提供參考依據(jù)?!痉椒ā炕贗llumina HiSeqTM 2500高通量測序平臺對采自浙江舟山近海的帶魚肌肉組織進行轉(zhuǎn)錄組測序,經(jīng)FastQC和Trimmomatic進行數(shù)據(jù)質(zhì)量評估和過濾后,使用Trinity組裝、去冗余得到Unigenes,再運用Micro-Satellite(MISA)挖掘SSR信息,并以Excel 2010計算SSR的數(shù)目、發(fā)生頻率、出現(xiàn)頻率、分布距離與密度、重復基元類別及重復區(qū)段長度等信息。【結(jié)果】帶魚肌肉組織轉(zhuǎn)錄組測序共獲得40424018條Raw reads,經(jīng)Trinity從頭組裝獲得70113條Transcripts,去冗余后得到50482條Unigenes,其總長度為33886190 bp,平均長度為671.25 bp。使用MISA進行篩選,結(jié)果共發(fā)現(xiàn)18873個SSR位點,且這些SSR位點僅分布在其中的13082條Unigenes上,發(fā)生頻率為25.91%,出現(xiàn)頻率為37.39%。SSR按核苷酸重復類型進行分類,可分為單核苷酸重復、二核苷酸重復、三核苷酸重復、四核苷酸重復、五核苷酸重復和六核苷酸重復6種類型,以單核苷酸重復SSR數(shù)最多(10763個),出現(xiàn)頻率高達21.32%;帶魚肌肉組織轉(zhuǎn)錄組SSR中共檢測出173種重復基元,以四核苷酸重復基元種類最多(66種)、單核苷酸重復基元種類最少(4種)。單核苷酸重復中以A堿基的數(shù)量最多,有5167個(占48.09%);二核苷酸重復基元以TG為主,有692個(占19.55%);三核苷酸重復基元中以GAG為主,有206個(占10.08%);四核苷酸重復基元出現(xiàn)頻率較高的有AAAC、ATGG、ATGT、CTGT、CTTT和TCCA,出現(xiàn)頻率均為3.64%;五核苷酸重復和六核苷酸重復的基元類型數(shù)量分布較均勻,無明顯優(yōu)勢重復基元。SSR基元重復次數(shù)主要分布在5~6次和10~12次,共9864個,占SSR總數(shù)的52.27%?!窘Y(jié)論】經(jīng)高通量測序獲得的帶魚肌肉轉(zhuǎn)錄組SSR可用性高且具有較高的多態(tài)性潛能,在此基礎(chǔ)上可有針對性進行引物設(shè)計,為帶魚遺傳多樣性評價、遺傳結(jié)構(gòu)分析及功能基因克隆等研究提供有效的分子標記,進而為其種質(zhì)資源的保護與利用提供遺傳學數(shù)據(jù)資料。
關(guān)鍵詞: 帶魚;肌肉組織;微衛(wèi)星(SSR);轉(zhuǎn)錄組;高通量測序
中圖分類號: S917.4;S965.326? ? ? ? ? ? ? ? ? ? ? ? ? ?文獻標志碼: A 文章編號:2095-1191(2022)03-0725-10
Bioinformatic analysis of microsatellite loci in the muscle transcriptome of Trichiurus lepturus based on
high-throughput sequencing
LIU Yu-ping WANG Qi HUANG Xin-xin XU Kai-da WANG Zhong-ming GAO Tian-xiang YANG Tian-yan
(1 Fisheries College, Zhejiang Ocean University, Zhoushan, Zhejiang? 316022, China; 2 Marine Fishery Research
Institute of Zhejiang Province, Zhoushan, Zhejiang? 316021, China)
Abstract:【Objective】Transcriptome sequencing of Trichiurus lepturus muscle tissue was carried out based on high-throughput sequencing platform. Microsatellite (SSR) loci were searched from massive data and bioinformatics analysis was conducted to provide references for germplasm resources protection and management of T. lepturus. 【Method】The Illumina HiSeqTM 2500 high-throughput sequencing platform was used to sequence the muscle transcriptome of T. lepturus collected from the coastal waters of Zhoushan, Zhejiang Province. The quality of high throughput sequencing data was evaluated and filtered by software FastQC and Trimmomatic. Unigenes were obtained through assembly and redundancy removal using software Trinity. Micro-Satellite (MISA) tool was performed to explore the SSR information. Excel 2010 was used to calculate the number, occurring frequency, appearing frequency, distribution distance and density, repeat motif and repeat length of SSR. 【Result】A total of 40424018 raw reads were generated from muscle transcriptome sequencing. About 70113 transcripts were obtained by de novo assembly using Trinity, and 50482 unigenes were retained after deduplication, with a total length of 33886190 bp and an average length of 671.25 bp. A total of 18873 SSR loci distributing among 13082 unigenes were screened by MISA, with the occurrence frequency of 25.91% and the occurrence frequency of 37.39%, respectively. SSR was classified according to the types of nucleotide repeats, which could be divided into six types:Mononucleotide repeat, dinucleotide repeat, trinucleotide repeat, tetranucleotide repeat, pentanucleotide repeat and hexanucleotide repeat. The number of mononucleotide repeat was the largest (10763), with the occurrence frequency up to 21.32%. A total of 173 repeat motifs were detected, in which the number of tetranucleotide repeat motifs (66) was the largest and the number of mononucleotide repeat motifs (4) was the smallest. Among the mononucleotide repeats, the number of A was the largest (5167, 48.09%); TG was the main dinucleotide repeat (692, 19.55%); GAG was the main trinucleotide repeat (206, 10.08%); AAAC, ATGG, ATGT, CTGT, CTTT and TCCA were the most frequent repeats with the same frequency of 3.64%. Pentanucleotide repeats and hexanucleotide repeats were evenly distributed, and no obvious dominant repeat motifs were observed. The number of repeat motifs (9864) was mainly distributed in 5-6 and 10-12 times, accounting for 52.27% of the total number of SSR. 【Conclusion】The SSRs obtained by high-throughput sequencing have higher availability and polymorphism potential. On this basis, researchers can targetedly design primers and provide effective molecular markers for the studies of genetic diversity evaluation, genetic structure analysis and functional genes cloning of T. lepturus, and then provide genetic data for further protection and utilization of its germplasm resources.FF6A4387-7446-4F61-9047-3F7726652A6C
Key words: Trichiurus lepturus; muscle tissue; microsatellite (SSR); transcriptome; high-throughput sequencing
Foundation items: Zhejiang Key Research and Development Project(2019C02056); Zhoushan Science and Technology Project (2022C41022); Open Foundation for Marine Sciences of Zhejiang Ocean University in the First-Class Subjects of Zhejiang (20180017)
0 引言
【研究意義】帶魚(Trichiurus lepturus)又稱刀魚或肥帶等,隸屬于鱸形目(Perciformes)帶魚科(Tri-chiuridae)帶魚屬(Trichiurus),生活在溫暖水域,我國的渤海、黃海、東海及南海海區(qū)均有分布,曾與曼氏無針烏賊(Sepiella maindroni)、大黃魚(Larimichthys crocea)和小黃魚(L. polyactis)并稱為四大海產(chǎn),是我國單魚種產(chǎn)量最高的經(jīng)濟魚類(嚴利平等,2005;林龍山等,2006;李發(fā)凱等,2016)。但自20世紀80年代以來,由于過度捕撈和產(chǎn)卵場環(huán)境遭到破壞,東海帶魚種群一度出現(xiàn)小型化、低齡化和低質(zhì)化現(xiàn)象(徐漢祥等,1997;周永東等,2002)。近年來,隨著東海產(chǎn)卵帶魚保護區(qū)、東海帶魚國家級水產(chǎn)種質(zhì)資源保護區(qū)和伏期休漁制度的建立,其資源衰退趨勢有所減緩,但整體形勢仍不容樂觀(杜萍等,2020)。因此,開展帶魚種質(zhì)資源現(xiàn)狀及遺傳背景的調(diào)查研究,對于帶魚種群資源的恢復和開發(fā)管理具有重要意義。【前人研究進展】至今,國內(nèi)外有關(guān)帶魚的研究主要集中在資源變動、漁業(yè)生物學及種群生態(tài)學等方面(徐漢祥等,2003;凌建忠等,2004;林龍山等,2006;陳云龍等,2013;金鑫等,2014)。關(guān)于帶魚遺傳多樣性和種群鑒別的常見技術(shù)手段有同工酶(王可玲等,1994;楊天燕和高天翔,2007)、隨機擴增DNA多態(tài)性(蒙子寧等,2003)、線粒體DNA(張繼民等,2009;鄭文娟等,2015;卞光明等,2017;吳仁協(xié)等,2019)及微衛(wèi)星(Yang et al.,2007;An et al.,2010;畢金貞,2010)等。其中,微衛(wèi)星DNA(Microsatellites DNA)又稱為簡單重復序列(Simple sequence repeat,SSR),是由1~6個核苷酸組成的串聯(lián)重復片段所構(gòu)成,每個單元長度在1~10 bp,而單個微衛(wèi)星DNA的總長度多在200 bp內(nèi)(趙彥花等,2019;周康奇等,2020)。SSR分子標記廣泛分布于真核生物和少許原核生物基因組中,具有多態(tài)性豐富、穩(wěn)定性良好、PCR擴增重復性高、雜合率高且遵循孟德爾遺傳定律呈共顯性遺傳等優(yōu)點,已成為魚類種群遺傳多樣性、遺傳連鎖圖譜構(gòu)建、增殖放流效果評估等研究領(lǐng)域的重要分子標記之一(何平,1998;朱濱和常劍波,1999;郭寶英等,2007)。近年來,隨著高通量測序技術(shù)的發(fā)展,基于轉(zhuǎn)錄組(RNA-Seq)數(shù)據(jù)大規(guī)模開發(fā)SSR分子標記的研究已十分成熟和便捷(肖韻錚等,2020;楊利艷等,2020)。Mikheyev等(2010)從斑蝶(Euphydryas editha)轉(zhuǎn)錄組中快速挖掘出大量SSR位點,有效解決了鱗翅目(Lepidoptera)昆蟲從基因組中開發(fā)微衛(wèi)星困難的問題。Yuan等(2014)對列入世界自然保護聯(lián)盟(IUCN)名錄中的瀕危物種棘腹蛙(Quasipaa boulengeri)進行轉(zhuǎn)錄組測序,成功分離獲得32個SSR位點,與采用傳統(tǒng)的富集文庫法開發(fā)SSR分子標記相比更便利。Hu等(2015)利用高通量測序技術(shù)從中國特有植物麻核桃(Juglans hopeiensis)的葉片、花蕾和果實組織轉(zhuǎn)錄組中開發(fā)出25對多態(tài)性SSR引物,并對2個地理種群的植株進行遺傳差異分析。Zhao等(2019)從我國北方常見淡水小型蝦類中華小長臂蝦(Palaemonetes sinensis)轉(zhuǎn)錄組中檢測出16對多態(tài)性SSR引物,進而對7個地理種群的319尾個體進行遺傳多樣性和遺傳結(jié)構(gòu)研究。此外,章霞等(2019)對日本帶魚(T. japanicus)肝臟轉(zhuǎn)錄組SSR特征進行統(tǒng)計,并開發(fā)出10對可于今后多態(tài)性分析和驗證的SSR引物?!颈狙芯壳腥朦c】傳統(tǒng)的SSR分子標記開發(fā)多采用生物信息法、鏈霉親和素磁珠富集法、硝酸纖維素膜雜交法等方式,其開發(fā)過程復雜、費時費力,且多態(tài)位點數(shù)量不多,能揭示的遺傳信息十分有限,難以滿足精準的種群遺傳結(jié)構(gòu)分析和種質(zhì)資源評估(賈小平等,2009)。因此,亟待通過高通量測序技術(shù)大量開發(fā)帶魚SSR分子標記,為其資源保護與利用提供遺傳學數(shù)據(jù)資料?!緮M解決的關(guān)鍵問題】基于Illumina HiSeqTM 2500高通量測序平臺對采自浙江舟山近海的帶魚樣本肌肉組織進行轉(zhuǎn)錄組測序,從海量數(shù)據(jù)中查找SSR位點并進行生物信息學分析,以期為科學制定帶魚種質(zhì)資源保護對策和管理措施提供參考依據(jù)。
1 材料與方法
1. 1 試驗材料
用于轉(zhuǎn)錄組測序的10尾帶魚樣品于2020年3月采自浙江近海的東海帶魚國家級水產(chǎn)種質(zhì)資源保護區(qū)(東經(jīng)123°15′,北緯30°10′)。剪取適量背部肌肉裝入含RNAhold保存液(北京全式金生物技術(shù)股份有限公司)的2 mL凍存管(美國Corning公司)中,置于-80 ℃超低溫冰箱中冷凍保存。
1. 2 RNA提取及轉(zhuǎn)錄組測序
采用常規(guī)TRIzol法提取總RNA(美國Invitrogen公司),使用Agilent 2100(美國Agilent公司)和NanoDrop 2000(美國Thermo Fisher公司)檢測RNA濃度和純度。合格的RNA樣品送至生工生物工程(上海)股份有限公司構(gòu)建cDNA混合文庫,然后基于Illumina HiSeqTM 2500平臺進行轉(zhuǎn)錄組測序。測序獲得的帶魚肌肉組織轉(zhuǎn)錄組Raw reads經(jīng)FastQC和Trimmomatic進行質(zhì)量評估和剪切,去除接頭、樣品標識序列、低質(zhì)量的Reads及帶N堿基較多的Reads,以獲取高質(zhì)量的Clean reads(Bolger et al.,2014)。FF6A4387-7446-4F61-9047-3F7726652A6C
1. 3 數(shù)據(jù)拼接組裝及SSR位點篩選和統(tǒng)計
使用Trinity對Clean reads進行De nove組裝以獲得轉(zhuǎn)錄本(Transcript),參數(shù)設(shè)為“min_kmer_cov 2”,其余默認(Haas et al.,2013),去冗余后取每個轉(zhuǎn)錄本聚類中最長的轉(zhuǎn)錄本作為Unigenes用于后續(xù)分析。以Micro-Satellite(MISA)對帶魚Unigenes中潛在的SSR進行搜索(Beier et al.,2017),篩選條件參數(shù)設(shè)為:基元長度1~6 bp,單核苷酸重復次數(shù)不小于10次,二核苷酸重復次數(shù)大于6次,三核苷酸、四核苷酸、五核苷酸、六核苷酸重復次數(shù)至少5次。若2個SSR間的距離小于100 bp,則記為1個復合微衛(wèi)星。使用Excel 2010統(tǒng)計并計算SSR的數(shù)目、發(fā)生頻率、出現(xiàn)頻率、分布距離與密度、重復基元類別及重復區(qū)段長度等信息。具體統(tǒng)計公式如下:
SSR發(fā)生頻率(%)=含SSR的Unigenes總數(shù)/Unigenes總數(shù)×100
SSR相對豐度(個/Mb)=篩選獲得的SSR總數(shù)/Unigenes總長度
SSR平均距離(bp)=Unigenes總長度/篩選獲得的SSR總數(shù)
SSR出現(xiàn)頻率(%)=檢測所得SSR總數(shù)/Unigenes總數(shù)×100
2 結(jié)果與分析
2. 1 轉(zhuǎn)錄組測序、拼接及組裝結(jié)果
基于Illumina HiSeqTM 2500平臺測序共獲得41886302條Raw reads,經(jīng)質(zhì)控后得到40424018條Raw reads,共計5643766129個核苷酸,平均為139.61 bp。GC含量為50.62%,N堿基數(shù)量為888823個,所占比例僅為0.02%。Q10、Q20和Q30分別為99.60%、98.15%和92.34%(表1),以上數(shù)據(jù)表明獲得的測序結(jié)果較好。
經(jīng)Trinity從頭組裝獲得70113條Transcripts,去冗余后得到50482條Unigenes,總長度為33886190 bp,平均長度為671.25 bp。N50和N90(累加轉(zhuǎn)錄本長度為不小于總長50%或90%的拼接轉(zhuǎn)錄本長度)分別為1078和268 bp,其中最大長度為29197 bp、最小長度為201 bp。長度大于1000 bp的Unigens有8868條,占總Unigenes的17.57%;長度在500 bp以上的有18216條,占比36.08%(表2)。
2. 2 轉(zhuǎn)錄組SSR重復類型及其分布情況
使用MISA對總長33886190 bp的50482條Unigenes進行篩選,結(jié)果共發(fā)現(xiàn)18873個SSR位點,且這些SSR位點僅分布在其中的13082條Unigenes上,發(fā)生頻率為25.91%,出現(xiàn)頻率為37.39%。將這些SSR位點按核苷酸重復類型進行分類,可分為單核苷酸重復、二核苷酸重復、三核苷酸重復、四核苷酸重復、五核苷酸重復和六核苷酸重復6種類型。其中,以單核苷酸重復SSR數(shù)最多,有10763個,存在于8252條Unigenes上,出現(xiàn)頻率最高(21.32%);然后依次是二核苷酸重復、三核苷酸重復、四核苷酸重復、六核苷酸重復和五核苷酸重復SSR,出現(xiàn)頻率分別為7.01%、4.05%、0.22%、0.04%和0.03%。SSR位點相對豐度排序為單核苷酸重復(317.62個/Mb)>二核苷酸重復(104.44個/Mb)>三核苷酸重復(60.32個/Mb)>四堿基重復(3.25個/Mb)>六核苷酸重復(0.59個/Mb)>五核苷酸重復(0.38個/Mb);SSR平均長度以六核苷酸重復最長,為37.50 bp,然后依次為五核苷酸重復(34.60 bp)、四核苷酸重復(24.73 bp)、二核苷酸重復(20.55 bp)、三核苷酸重復(18.41 bp)和單核苷酸重復(13.14 bp)(表3)。此外,以復合形式存在的SSR位點有2384個,出現(xiàn)頻率為4.72%。
2. 3 轉(zhuǎn)錄組SSR重復基元類型
帶魚肌肉組織轉(zhuǎn)錄組SSR中共檢測出173種重復基元,其中以四核苷酸重復基元種類最多,有66種,然后依次為三核苷酸重復(58種)、六核苷酸重復(20種)、五核苷酸重復(13種)和二核苷酸重復(12種),單核苷酸重復因為受堿基數(shù)量的限制,重復基元種類最少(僅4種)。在66種四核苷酸重復基元類型中,數(shù)量和分布特征變化較小,SSR的優(yōu)勢重復基元為AAAC、ATGG、ATGT、CTGT、CTTT和 TCCA等6種。在重復基元中,單核苷酸重復依舊在數(shù)量上占據(jù)絕對優(yōu)勢,其中A、T堿基重復基元占單核苷酸重復SSR位點數(shù)量的94.50%;相對而言,五核苷酸重復和六核苷酸重復的基元類型與數(shù)量分布均較少。
從不同重復基元類型的含量(表4)來看,單核苷酸重復中以A堿基數(shù)量最多,有5167個(占48.09%),而C堿基數(shù)量最少,為251個(僅占2.33%);二核苷酸重復基元以TG為主,有692個(占19.55%),其次是GT(19.02%)、AC(17.77%)和CA(15.91%)等3種重復基元,而CG和GC的數(shù)量分別只有4和5個;在三核苷酸重復基元中,所占比例最高的是GAG,有206個(占10.08%),其次是TCC(5.19%)、CAG(4.55%)、CCT(4.40%)和GGA(4.31%),而CGA的數(shù)量僅有2個(占0.10%);四核苷酸重復基元出現(xiàn)頻率較高的有AAAC(3.63%)、ATGG(3.63%)、ATGT(3.63%)、CTGT(3.63%)和TCCA(3.64%);五核苷酸重復和六核苷酸重復的基元類型數(shù)量分布較均勻,無明顯的優(yōu)勢重復基元(圖1)。
在帶魚肉組織轉(zhuǎn)錄組SSR中,以10次重復的SSR數(shù)量最多,達3659個,占SSR總數(shù)的19.39%;其次是11、12、6和5次重復,SSR數(shù)量在1000~2300個,約占總SSR數(shù)目的52.27%。其中,單核苷酸重復次數(shù)分布在10~50次,主要集中在10~25次,占單核苷酸重復總數(shù)的97.71%;二核苷酸重復次數(shù)分布在6~42次,且主要集中在6~12次,共2887個,占該類型核苷酸重復總數(shù)的81.58%;三核苷酸重復次數(shù)分布在5~22次,其中2~8次重復占三核苷酸重復總數(shù)的93.74%;四核苷酸重復次數(shù)分布在5~24次,其中5~6次重復占四核苷酸重復總數(shù)的85.45%;五核苷酸重復、六核苷酸重復次數(shù)均在15次以內(nèi),以5~10次居多(表5)。FF6A4387-7446-4F61-9047-3F7726652A6C
綜上所述,SSR基元重復次數(shù)主要分布在5~6次和10~12次,共9864個,占SSR總數(shù)的52.27%;其次是7~9次和13~20次,共5772個,占SSR總數(shù)30.58%;重復次數(shù)大于25次的SSR相對較少,主要由單核苷酸和二核苷酸重復基元組成,共342個,占SSR總數(shù)的1.81%,且單核苷酸和二核苷酸基元無重復5次的相關(guān)數(shù)據(jù)。此外,SSR基元重復次數(shù)增至10次后,隨著重復次數(shù)的增加SSR數(shù)量依次遞減,而單核苷酸重復所占的比例逐漸增加。
2. 4 轉(zhuǎn)錄組SSR長度分布及多態(tài)性評價結(jié)果
帶魚肌肉組織SSR序列長度區(qū)間跨度較大,范圍在10~96 bp。其中,六核苷酸重復SSR長度變化最小,在30~60 bp;二核苷酸重復SSR長度變化范圍最大,為12~84 bp。單核苷酸重復、三核苷酸重復、四核苷酸重復、五核苷酸重復的SSR長度范圍分別為10~51 bp、15~66 bp、20~96 bp和25~65 bp。根據(jù)SSR長度可為兩大類:第一類是重復序列長度在20 bp及其以上的高度多態(tài)性I型,第二類是重復序列長度在12~20 bp的中度多態(tài)性II型(Wang et al.,2010)。本研究中,I型SSR僅2558條,約占SSR總數(shù)的18%;II型SSR共8663條,約占SSR總數(shù)的50%(圖2)。
3 討論
充分了解和掌握漁業(yè)對象的生物學特征和遺傳學背景,是開展?jié)O業(yè)資源科學管理及可持續(xù)開發(fā)利用的前提(Ward,2020)。帶魚作為我國重要的海洋經(jīng)濟魚類,線粒體和SSR分子標記仍是其種群遺傳學研究的主要手段。已報道的帶魚SSR分子標記篩選方法主要有鏈霉素磁珠捕獲法和富集文庫法,但這2種方法不僅耗時長、花費高,開發(fā)的SSR分子標記數(shù)量還十分有限。隨著高通量測序技術(shù)的發(fā)展和測序成本的降低,基于轉(zhuǎn)錄組學數(shù)據(jù)大量開發(fā)具有高多態(tài)性的帶魚SSR位點變得更加高效、便捷。本研究對采自浙江舟山近海的帶魚肌肉組織進行轉(zhuǎn)錄組高通量測序,使用SSR識別工具MISA在50482條Unigenes中搜索出18873個SSR位點,出現(xiàn)頻率為37.39%,與其他海洋魚類轉(zhuǎn)錄組中SSR的出現(xiàn)頻率相比,高于牙鲆(Paralichthys olivaceus,27.12%)(李超等,2015)、刀鱭(Coilia nasus,8.76%)(Fang et al.,2015)、銀鯧(Pampus argenteus,2.62%)(劉磊等,2016)和龍頭魚(Harpadon nehereus,18.99%)(黃新芯等,2021),但略低于黃姑魚(Nibea albiflora,39.30%)(龔詩琦等,2016),說明帶魚屬于轉(zhuǎn)錄組中SSR數(shù)量較豐富的魚類,帶魚轉(zhuǎn)錄組測序質(zhì)量和SSR含量均處于較高水平,能為后續(xù)群體遺傳學研究提供充足的序列資源。相對于帶魚肝臟轉(zhuǎn)錄組測序數(shù)據(jù)中的發(fā)生頻率(40.95%)(章霞等,2019),本研究所得的數(shù)據(jù)略低,且與肝臟中單核苷酸重復~六核苷酸重復SSR的出現(xiàn)頻率(53.79%、30.63%、14.34%、1.12%、0.08%和0.03%)相比也偏低。此外,帶魚肝臟轉(zhuǎn)錄組SSR中單核苷酸和二核苷酸在重復次數(shù)分別達23和11次時突然增加,而在肌肉組織中單核苷酸重復~六核苷酸重復的出現(xiàn)次數(shù)隨著重復次數(shù)的增加依次遞減,并無突增現(xiàn)象,究其原因可能與選擇的組織不同有一定關(guān)系,但不排除測序平臺及檢索參數(shù)設(shè)置不同帶來的影響。
相對于序列長度長且重復性低的串聯(lián)重復單元,序列長度短且重復性高的串聯(lián)重復單元則表現(xiàn)出更高的突變率,其多態(tài)性比例也較高(Kelkar et al.,2008),但重復序列長度小于12 bp時SSR多態(tài)性反而降低,因此,長度在20 bp及其以上的I型SSR和長度在12~20 bp的II型SSR是可用性較高的潛在分子標記(Temnykh et al.,2001)。從核苷酸重復次數(shù)來看,本研究中帶魚肌肉組織SSR核苷酸重復次數(shù)在5~50次,剔除易發(fā)生錯配的單核苷酸重復類型,重復數(shù)最高可達50次。從SSR長度來看,重復長度在12~20 bp的SSR為2558個,重復長度大于20 bp的SSR有8663個,即超過75%的SSR具有中度及以上水平的多樣性??梢?,帶魚肌肉轉(zhuǎn)錄組SSR可用性高且具有較高的多態(tài)性潛能,在此基礎(chǔ)上可有針對性進行引物設(shè)計,為帶魚的種質(zhì)鑒定、遺傳差異檢測及功能基因定位等研究提供理論依據(jù)和實踐基礎(chǔ)。
二核苷酸重復是帶魚基因組SSR的主要類型(張浩冉等,2019),而本研究發(fā)現(xiàn)帶魚肌肉組織轉(zhuǎn)錄組中的SSR重復類型以單核苷酸重復為主。已有研究表明,二核苷酸重復類型的SSR主要位于非編碼區(qū)(虞杭等,2018)。由于簡化基因組測序是采取對基因組特定區(qū)域進行測序以反映物種全基因組信息的策略,能檢測到廣泛分布于編碼區(qū)、非編碼區(qū)和調(diào)控區(qū)的SSR位點信息,而真核生物在轉(zhuǎn)錄本RNA加工過程中會對內(nèi)含子進行剪切,丟失部分非編碼區(qū)序列信息。因此,在依托轉(zhuǎn)錄組和基因組數(shù)據(jù)開發(fā)的SSR中其主導重復類型和重復數(shù)量存在一定差異。考慮到基因組信息有助于轉(zhuǎn)錄組注釋,而轉(zhuǎn)錄組數(shù)據(jù)也可對基因組序列進行補充和校正,在今后開展帶魚遺傳多樣性分析時可將這2種技術(shù)手段結(jié)合起來,為分子標記的挖掘和應(yīng)用提供更全面的參考信息。Harr和Schl?tterer(2000)對黑腹果蠅(Drosophila melanogaster)基因組SSR結(jié)構(gòu)和組成的研究發(fā)現(xiàn),復雜的多基元重復SSR出現(xiàn)頻率較高,通常暗示著物種進化水平較低或具有較低的變異頻率。在本研究中,帶魚肌肉組織轉(zhuǎn)錄組中低級重復基元(單核苷酸重復~三核苷酸重復)約占SSR總數(shù)的86.61%,出現(xiàn)頻率也較高(32.38%),而高級重復基元SSR的多態(tài)性相對較低,表明帶魚可能具有較長的進化歷史,且積累了較多的遺傳變異。
4 結(jié)論
經(jīng)高通量測序獲得的帶魚肌肉轉(zhuǎn)錄組SSR可用性高且具有較高的多態(tài)性潛能,在此基礎(chǔ)上可有針對性進行引物設(shè)計,為帶魚遺傳多樣性評價、遺傳結(jié)構(gòu)分析及功能基因克隆等研究提供有效的分子標記,進而為其種質(zhì)資源的保護與利用提供遺傳學數(shù)據(jù)資料。FF6A4387-7446-4F61-9047-3F7726652A6C
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