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3種白鯽雜交子代的轉(zhuǎn)錄組學(xué)分析

2019-09-10 07:22周大顏張志新黃彩林招志杰莫飛龍
關(guān)鍵詞:轉(zhuǎn)錄組信號(hào)通路

周大顏 張志新 黃彩林 招志杰 莫飛龍

摘要:【目的】研究3種白鯽雜交子代轉(zhuǎn)錄組學(xué)特征,為揭示鯽鯉雜交優(yōu)勢(shì)的分子機(jī)理提供理論依據(jù),同時(shí)為在生產(chǎn)上培育出生長(zhǎng)速度快、肉質(zhì)好、適應(yīng)能力強(qiáng)的雜交品種提供技術(shù)參考?!痉椒ā恳园做a(♀)×黑龍江野鯉(♂)雜交子代(簡(jiǎn)稱HB)、白鯽(♀)×散鱗鏡鯉(♂)雜交子代(簡(jiǎn)稱SB)和白鯽(♀)×興國(guó)紅鯉(♂)雜交子代(簡(jiǎn)稱XB)為研究對(duì)象,利用RNA-seq高通量測(cè)序技術(shù)構(gòu)建3種白鯽雜交子代轉(zhuǎn)錄組文庫(kù),以HiSeq PE150進(jìn)行測(cè)序分析,原始序列經(jīng)Trinity組裝后進(jìn)行功能注釋(E-value<1e-5);以DESeq2 R鑒定差異表達(dá)基因,利用GOseq R和KOBAS分別對(duì)差異表達(dá)的基因進(jìn)行GO和KEGG富集分析;并采用MicroSAtellite對(duì)轉(zhuǎn)錄本中的SSR位點(diǎn)進(jìn)行挖掘?!窘Y(jié)果】共組裝得225858條unigenes,平均長(zhǎng)度為668 bp,N50為938 bp,有171461條unigenes可注釋到蛋白質(zhì)數(shù)據(jù)庫(kù)(Nr)、非冗余核苷酸數(shù)據(jù)庫(kù)(Nt)、蛋白質(zhì)序列數(shù)據(jù)庫(kù)(SwissPort)、基因本體論(GO)、直系同源基因簇(COG/KOG)和京都基因與基因組百科全書(KEGG)數(shù)據(jù)庫(kù)中,注釋比例為75.92%。其中,52630條unigenes注釋到NR數(shù)據(jù)庫(kù),43659條unigenes注釋到SwissPort數(shù)據(jù)庫(kù),35756條unigenes注釋到COG/KOG數(shù)據(jù)庫(kù),包括生化代謝、信號(hào)轉(zhuǎn)導(dǎo)機(jī)制、防御系統(tǒng)和細(xì)胞結(jié)構(gòu)等。差異表達(dá)基因KEGG分析結(jié)果顯示,較多的差異表達(dá)基因注釋到內(nèi)吞作用、Jak-STAT信號(hào)通路、溶酶體、吞噬體和Wnt信號(hào)通路等免疫相關(guān)及與生長(zhǎng)發(fā)育相關(guān)的MAPK信號(hào)通路、Hippo信號(hào)通路和背腹軸形成等通路中。此外,從獲得的轉(zhuǎn)錄組序列中共鑒定出20272個(gè)SSR位點(diǎn),大多數(shù)為二核苷酸重復(fù)基元(占62.15%)?!窘Y(jié)論】不同白鯽雜交子代間存在較多的差異表達(dá)基因,從中獲得參與抗氧化、免疫和生長(zhǎng)發(fā)育相關(guān)的通路和基因序列,且挖掘出20272個(gè)SSR位點(diǎn),有助于選擇性育種、分子標(biāo)記開(kāi)發(fā)及開(kāi)展遺傳多樣性、遺傳圖譜構(gòu)建和QTL定位等研究。

關(guān)鍵詞: 白鯽;雜交子代;轉(zhuǎn)錄組;信號(hào)通路;SSR位點(diǎn)

中圖分類號(hào):? S965.117? ? ? ? ? ? ? ? ? ? ? ? ? ? 文獻(xiàn)標(biāo)志碼: A 文章編號(hào):2095-1191(2019)06-1328-11

Abstract:【Objective】Transcriptomics characteristics of three Carassius auratus cuvieri hybrids were studied to provide reference for molecular mechanism of hybrid heterosis of the hybrids, and also to offer technical support for breeding hybrids with rapid growth, quality meat and strong adaptability. 【Method】Three white crucian carp hybrids i.e. C. auratus cuvieri(♀)×Cyprinus carpio haermatopterus(♂)(abbreviated as HB), C. auratus cuvieri(♀)×C. carpio L.(♂)(abbreviated as SB) and C. auratus cuvieri(♀)×C. carpio var. singuonensis(♂) (abbreviated as XB) were used as research objectives. RNA-seq high-throughput sequencing was used to construct C. auratus cuvieri hybrids transcriptome library.? HiSeq PE150 was used to conduct sequencing analysis, and the function annotation on original sequence after Trinity assembling was carried out(E-value<1e-5). Differentially expressed genes was identified by DESeq2 R, then GO and KEGG enrichment analysis of differentially expressed genes were conducted by GOseq R and KOBAS respectively. MicroSAtellite was used to dig SSR loci in transcript. 【Result】A total of 225858 unigenes, with average length of 668 bp and N50 of 938 bp were generated. Of these, a total of 171461 unigenes could be annotated in protein sequence database(Nr), non-redundant nucleotide database(Nt), protein sequence database(SwissProt), Gene Ontology database(GO), Clusters of Ortho-logous Groups(COG/KOG) and Kyoto Encyclopedia of Genes and Genomes(KEGG) database. The annotation ratio was 75.92%. There were 52630 unigenes annotated in NR database, 43659 unigenes annotated in SwissPort database, 35756 unigenes annotated in COG/KOG database, including biochemical metabolism, signal transduction mechanism, defensive system and cell structure. KEGG analysis of differentially expressed genes showed that more differentially expressed genes were involved in immune-related pathways such as endocytosis, Jak-STAT signaling pathway, lysosome, phagosome and Wnt signaling pathways, and growth and development related pathways such as MAPK signaling pathway, Hippo signaling pathway and dorso-ventral axis formation. In addition, 20272 SSR loci were identified from transcriptome sequence and majority of which were dinucleotide repeats units(accounted for 62.15%). 【Conclusion】There are many differentially expressed genes in different hybrids. Some pathways and gene sequences involved in antioxidation, immunity, growth and development are preliminarily obtained. A total of 20272 SSR loci are dug. This result is helpful for selective breeding, molecular markers development, researches in genetic diversity, genetic map construction and QTL mapping.

Key words: Carassius auratus cuvieri; hybrid; transcriptome; signal pathway; SSR locus

收稿日期:2018-10-13

基金項(xiàng)目:農(nóng)業(yè)部物種品種資源(漁業(yè))保護(hù)費(fèi)項(xiàng)目(171721301354052099);廣西海洋和漁業(yè)廳預(yù)算項(xiàng)目(桂海漁財(cái)〔2018〕97號(hào))

作者簡(jiǎn)介:周大顏(1981-),主要從事水產(chǎn)動(dòng)物遺傳育種與養(yǎng)殖推廣研究工作,E-mail:4948738@qq.com

0 引言

【研究意義】我國(guó)水生生物多樣性豐富,許多種類經(jīng)人工馴化及繁育后已發(fā)展成為重要的經(jīng)濟(jì)水產(chǎn)品種。其中,鯉科(Cyprinidae)的多樣性尤為突出,在現(xiàn)存的800余種淡水魚中,鯉科魚類約占50%。鯉魚養(yǎng)殖迄今已有2400余年歷史,是池塘、稻田和網(wǎng)箱等養(yǎng)殖的主要對(duì)象,在天然水域產(chǎn)量中也占有很高比例(朱健等,2000)。遺傳組成不同的個(gè)體或群體結(jié)合后,其遺傳物質(zhì)重新組合而形成新品種的現(xiàn)象稱為雜交(Zhang et al.,2014),在自然界中普遍存在。雜交育種作為一種常規(guī)的育種手段,在水生生物品種改良和生產(chǎn)中發(fā)揮重要作用,是獲得新品種的重要途徑之一(Arcella et al.,2014),且雜交獲得的F1代通常在生產(chǎn)、生活、繁殖和適應(yīng)性等方面具有優(yōu)于雙親均值或超過(guò)親本的雜交優(yōu)勢(shì)(Jin et al.,2017)。因此,基于轉(zhuǎn)錄組學(xué)分析技術(shù)開(kāi)展雜交優(yōu)勢(shì)分子機(jī)理研究,對(duì)加速水產(chǎn)動(dòng)物選擇性育種、分子標(biāo)記開(kāi)發(fā)及遺傳多樣性分析等具有重要意義?!厩叭搜芯窟M(jìn)展】目前,國(guó)內(nèi)外關(guān)于水產(chǎn)動(dòng)物雜交的研究已有大量報(bào)道,如梭子蟹(Portunus trituberculatus)(Gao et al.,2014)、牙鲆(Paralichthys olivaceus)(Liu et al.,2014)、鯰魚雜交種(Pseudoplatystoma sp.)(Sinhorin et al.,2014)、石斑魚(Epinephelus fuscoguttatus ♀×E. lanceolatus ♂)(Firdaus et al.,2016)、鮑魚(Haliotis discus hannai)(Li et al.,2017)、牡蠣(Crass-ostrea sikamea×C. angulata)(Yan et al.,2017)和中華鱉(Pelodiscus sinensis)(Zhang et al.,2017b)等,其中又以不同鯉魚品種間雜交的效果最好(樓允東,1999)。我國(guó)已培育出具有顯著雜種優(yōu)勢(shì)的豐鯉(Cyprinus carpio var. singuonensis ♀×C. carpio L.♂)、荷元鯉(C. carpio var. wuyuanensis ♀×C. carpio var. yuankiang ♂)、荷花鯉(C. carpio L. ♀×C. carpio var. singuonensis ♂)和三雜交鯉(Heyuan carp ♀×C. carpio L. ♂)等雜交種(Liu et al.,2017),且均已得到推廣養(yǎng)殖,但目前針對(duì)雜交鯉的研究仍停留在形態(tài)學(xué)特征、主要經(jīng)濟(jì)性狀、遺傳變異及親子鑒定等方面(劉義新等,2007;武耀等,2012;Liu et al.,2018)。隨著基因組測(cè)序技術(shù)的快速發(fā)展,轉(zhuǎn)錄組測(cè)序技術(shù)已成為挖掘缺乏基因組信息物種功能基因的重要手段(唐玉娟等,2018),在經(jīng)濟(jì)魚類雜交后代的雜種優(yōu)勢(shì)研究中得到廣泛應(yīng)用。Bougas等(2010)對(duì)3個(gè)美洲紅點(diǎn)鮭(Salvelinus fontinalis Mitchill)群體及其雜交后代進(jìn)行轉(zhuǎn)錄組學(xué)分析,結(jié)果顯示不同群體雜交后代表現(xiàn)出不同的基因表達(dá)模式和生長(zhǎng)優(yōu)勢(shì)。Gao等(2013)對(duì)雜交河豚(Jiyan-1 Puffer)及其親本(Takifugu rubripes ♀×T. flavidus ♂)進(jìn)行轉(zhuǎn)錄組測(cè)序分析,結(jié)果發(fā)現(xiàn)雜種優(yōu)勢(shì)的形成可能與能量代謝、離子結(jié)合和激酶激活等過(guò)程有關(guān)。Liu等(2018)對(duì)白鯽(Carassius auratus ♀)和紅鯽(C. auratus red var. ♂)及其雜交F1代的轉(zhuǎn)錄組進(jìn)行測(cè)序,GO分析結(jié)果顯示,F(xiàn)1代的雜合基因與代謝過(guò)程、免疫系統(tǒng)和生長(zhǎng)發(fā)育有關(guān)。【本研究切入點(diǎn)】白鯽(C. auratus cuvieri)又稱日本白鯽,隸屬于鯉形目(Cypriniformes)鯉科(Cyprinidae)鯉亞科(Cyprinae)鯽屬(Carassius),原產(chǎn)于日本琵琶湖,于1976年引進(jìn)我國(guó),體色為白色,具有繁殖力強(qiáng)、食性廣、生長(zhǎng)速度快、適應(yīng)性強(qiáng)等優(yōu)點(diǎn),但其肉質(zhì)欠佳(王靜等,2015)。黑龍江野鯉(C. carpio haermatopterus)與黃河、長(zhǎng)江、遼河等野鯉同屬一個(gè)亞種,起源于歐洲野鯉,在長(zhǎng)期的進(jìn)化過(guò)程中黑龍江野鯉對(duì)于黑龍江流域多變的水溫環(huán)境已完全適應(yīng),具有極強(qiáng)的抗寒能力和抗病能力(朱健等,2014)。散鱗鏡鯉(C. carpio L.)原產(chǎn)于前蘇聯(lián),于1959年引入我國(guó),抗逆性強(qiáng)、生長(zhǎng)速度快,是雜交選育的重要親本來(lái)源(李盛文等,2014)。興國(guó)紅鯉(C. carpio var. singuonensis)主要分布在江西興國(guó)縣,已有超過(guò)1300年的養(yǎng)殖歷史,其溫度適應(yīng)范圍廣、易于飼養(yǎng)、產(chǎn)卵量大,是重要的雜交親本,在我國(guó)魚類雜交育種中占據(jù)重要地位(岳華梅等,2016)。以白鯽為母本,分別與黑龍江野鯉、散鱗鏡鯉和興國(guó)紅鯉雜交,均可獲得具有明顯雜交優(yōu)勢(shì)的雜交F1代,但其雜交優(yōu)勢(shì)的分子機(jī)理尚未明確,因此有必要利用轉(zhuǎn)錄組測(cè)序技術(shù)從基因?qū)用娼沂觉庺~雜交優(yōu)勢(shì)的分子機(jī)理。【擬解決的關(guān)鍵問(wèn)題】通過(guò)RNA-seq高通量轉(zhuǎn)錄組測(cè)序、組裝和分析,對(duì)3種白鯽雜交子代進(jìn)行轉(zhuǎn)錄組學(xué)特征研究,以期為揭示鯽鯉雜交優(yōu)勢(shì)分子機(jī)理提供理論依據(jù),同時(shí)為在生產(chǎn)上培育出生長(zhǎng)速度快、肉質(zhì)好、適應(yīng)能力強(qiáng)的雜交品種提供技術(shù)參考。

1 材料與方法

1. 1 試驗(yàn)材料

供試魚為白鯽(♀)×黑龍江野鯉(♂)雜交子代(簡(jiǎn)稱HB)、白鯽(♀)×散鱗鏡鯉(♂)雜交子代(簡(jiǎn)稱SB)和白鯽(♀)×興國(guó)紅鯉(♂)雜交子代(簡(jiǎn)稱XB),由廣西水產(chǎn)引育種中心武鳴基地提供。試驗(yàn)前,先在室內(nèi)養(yǎng)殖池中隔離暫養(yǎng)10 d,饑餓48 h后從不同雜交子代中分別挑選200尾體質(zhì)健康、規(guī)格一致的供試魚。經(jīng)測(cè)量發(fā)現(xiàn),HB、SB和XB供試魚的平均體重分別為31.9、32.0和32.3 g/尾,并分別在腹腔注射PIT電子標(biāo)記,記錄標(biāo)記號(hào)后放入池塘殖。

1. 2 養(yǎng)殖管理

每天上、下午各投喂1次;每晚開(kāi)增氧機(jī)增氧;每隔25 d潑灑聚維酮碘(水產(chǎn)用)1次,進(jìn)行水體消毒和疾病預(yù)防。養(yǎng)殖周期共90 d(2017年7月25日—2017年10月24日)。

1. 3 樣品采集

養(yǎng)殖結(jié)束后,從HB、SB和XB供試魚中隨機(jī)抽取樣品魚各30尾,用無(wú)菌手術(shù)刀剪取肌肉、魚肝和魚皮,分別裝入標(biāo)識(shí)好的無(wú)RNA酶試管中,經(jīng)液氮速凍后置于-80 ℃冰箱中保存?zhèn)溆谩?/p>

1. 4 RNA提取

取樣品魚的肌肉、肝臟和魚皮,置于經(jīng)高壓滅菌的研缽中加液氮研磨成粉狀,然后參照Invitrogen公司的TRIzol Reagent說(shuō)明提取組織總RNA。以TURBO DNA-FreeTM Kit(Ambion,Thermo Fisher Scientific,USA)消除總RNA中的DNA后,分別用1%非變性瓊脂糖凝膠電泳(120 V,10 min)檢測(cè)RNA的完整性、NanoDrop 2000超微量分光光度計(jì)(Thermo Fisher Scientific,USA)檢測(cè)RNA純度和濃度。

1. 5 轉(zhuǎn)錄組文庫(kù)構(gòu)建及測(cè)序

取檢測(cè)合格的各組織RNA等量混合(總量1 μg)后進(jìn)行文庫(kù)構(gòu)建和測(cè)序。用攜帶Oligo(dT)的磁珠富集含poly(A)尾巴的mRNA,隨后加入Fragmentation Buffer打斷mRNA,以片段化的mRNA為模板,采用六堿基隨機(jī)引物合成單鏈cDNA;再加入緩沖液、dNTPs、RNase H和DNA聚合酶I合成cDNA第二鏈,經(jīng)QiaQuick PCR試劑盒純化并加入EB緩沖液洗脫后進(jìn)行末端修復(fù)及加poly(A)尾巴,并連接測(cè)序接頭;以瓊脂糖凝膠電泳進(jìn)行片段大小篩選,最后進(jìn)行PCR擴(kuò)增,構(gòu)建好的文庫(kù)采用HiSeq PE150進(jìn)行測(cè)序分析。

1. 6 序列組裝及功能注釋

由測(cè)序獲得的數(shù)據(jù)稱為raw reads,先對(duì)raw reads進(jìn)行質(zhì)控(QC),去除測(cè)序過(guò)程中低質(zhì)量序列及不確定序列;采用轉(zhuǎn)錄組組裝軟件Trinity對(duì)過(guò)濾后的clean reads進(jìn)行組裝,選取每個(gè)transcript cluster中最長(zhǎng)的轉(zhuǎn)錄本作為unigene。以NCBI蛋白質(zhì)數(shù)據(jù)庫(kù)(Nr)、非冗余核苷酸數(shù)據(jù)庫(kù)(Nt)、蛋白質(zhì)序列數(shù)據(jù)庫(kù)(SwissPort)、基因本體論(GO)、直系同源基因簇(COG/KOG)和京都基因與基因組百科全書(KEGG)數(shù)據(jù)庫(kù)作為參考,對(duì)所獲得的unigene進(jìn)行功能注釋(E-value<1e-5)(Zhang et al.,2017a)。

1. 7 差異表達(dá)基因分析

采用RPKM(Reads per kilo bases per million reads)衡量基因表達(dá)量(Li and Dewey,2011),并以DESeq2 R進(jìn)行差異表達(dá)基因篩選,篩選標(biāo)準(zhǔn)為錯(cuò)誤發(fā)現(xiàn)率P≤0.05,|log2ratio|≥1(Yan et al.,2017)。利用GOseq R(Young et al.,2010)和KOBAS(Mao et al.,2005)分別對(duì)差異表達(dá)的基因進(jìn)行GO和KEGG富集分析,其中,GO富集分析以corrected-pvalue≤0.05為閾值,滿足此條件即定義為在差異表達(dá)基因中顯著富集的GO term;Pathway顯著性富集分析以KEGG Pathway為單位,應(yīng)用超幾何檢驗(yàn),當(dāng)Q value≤0.05即定義為在差異表達(dá)基因中顯著富集的Pathway。

1. 8 SSR分子標(biāo)記鑒定

利用MicroSAtellite(MISA,http://pgrc.ipk-gater-sleben.de/misa/)對(duì)轉(zhuǎn)錄本中的SSR位點(diǎn)進(jìn)行挖掘,為鑒定二、三、四、五和六核苷酸重復(fù)基元,對(duì)應(yīng)的閾值分別被設(shè)為 6、5、4、4和4(Zhou et al.,2013)。根據(jù)堿基互補(bǔ)配對(duì)原則,將所有互補(bǔ)的簡(jiǎn)單重復(fù)序列視為一類,其中,二核苷酸重復(fù)基元4種(AT、AG、AC和CG),三核苷酸重復(fù)基元10種,四核苷酸重復(fù)基元33種,五核苷酸重復(fù)基元102種,六核苷酸重復(fù)基元350種。

2 結(jié)果與分析

2. 1 測(cè)序數(shù)據(jù)的產(chǎn)出和組裝結(jié)果

為保證數(shù)據(jù)質(zhì)量,對(duì)各測(cè)序樣品的原始數(shù)據(jù)進(jìn)行質(zhì)控和過(guò)濾。去除低質(zhì)量、含接頭序列的reads后,得到的clean reads如表1所示。各樣品測(cè)序質(zhì)量不低于20(Q20)的堿基占總堿基比例均在96.00%以上,質(zhì)量不低于30(Q30)的堿基占比在90.00%以上,GC含量則低于50.00%。采用Trinity對(duì)所有clean reads進(jìn)行從頭組裝后,共得到225858條unigenes,總長(zhǎng)150956990 bp,平均長(zhǎng)度668 bp,N50為938 bp。其中,有102591條(45.42%)的unigenes長(zhǎng)度在200~400 bp,42555條(18.84%)的unigenes長(zhǎng)度在400~600 bp,長(zhǎng)度在1000 bp以上的unigenes有42723條(18.91%)(圖1)。

2. 2 功能注釋及分類結(jié)果

將所有unigenes分別與NR、SwissProt、KEGG和COG/KOG(E-value<1e-5)等數(shù)據(jù)庫(kù)中的相關(guān)序列進(jìn)行BLASTx比對(duì)分析,找出與指定unigene具有最高序列相似性的蛋白,從而獲知unigene的蛋白功能注釋信息。結(jié)果顯示,在225858條unigenes中有171461條獲得蛋白功能注釋信息,注釋比例為75.92%。其中,52630條unigenes注釋到NR數(shù)據(jù)庫(kù),43659條unigenes注釋到SwissPort數(shù)據(jù)庫(kù),35756條unigenes注釋到COG/KOG數(shù)據(jù)庫(kù),包括生化代謝、信號(hào)轉(zhuǎn)導(dǎo)機(jī)制、防御系統(tǒng)和細(xì)胞結(jié)構(gòu)等(圖2)。此外,有28164條unigenes注釋到不同的KEGG通路中(圖3),最有代表性的是信號(hào)轉(zhuǎn)導(dǎo)通路(4215條)、細(xì)胞通訊(2253條)、內(nèi)分泌系統(tǒng)(2059條)及轉(zhuǎn)運(yùn)和分解代謝(2058條)(表2)。

2. 3 差異表達(dá)基因的篩選及其功能注釋

HB、SB和XB 3種雜交子代兩兩進(jìn)行差異表達(dá)基因篩選分析,結(jié)果顯示,HB和XB間(HB-XB)的差異表達(dá)基因有6448個(gè),其中上調(diào)基因2064個(gè)、下調(diào)基因4384個(gè);SB和HB間(SB-HB)的差異表達(dá)基因有1615個(gè),其中上調(diào)基因1053個(gè)、下調(diào)基因562個(gè);SB和XB間(SB-XB)的差異表達(dá)基因有4249個(gè),其中上調(diào)基因2290個(gè)、下調(diào)基因1959個(gè)。對(duì)這些差異表達(dá)基因進(jìn)行KEGG通路分析,結(jié)果發(fā)現(xiàn),在HB-XB的差異表達(dá)基因中,有較多基因注釋到內(nèi)吞作用、Jak-STAT信號(hào)通路、溶酶體和吞噬體(圖4);在SB-HB的差異表達(dá)基因中,以注釋到內(nèi)吞作用、mRNA監(jiān)測(cè)通路、RNA轉(zhuǎn)運(yùn)和粘著連接中注釋的差異表達(dá)基因較多(圖5);在SB-XB的差異表達(dá)基因中,較多基因注釋到內(nèi)吞作用、吞噬作用、Wnt信號(hào)通路和RNA轉(zhuǎn)運(yùn)通路中(圖6)。

2. 4 SSR位點(diǎn)鑒定結(jié)果

將拼接所得序列提交至MISA進(jìn)行分析,共得到20272個(gè)SSR位點(diǎn),其中有2359條序列包含1個(gè)以上的SSR位點(diǎn)。根據(jù)重復(fù)單元的類型和重復(fù)次數(shù)對(duì)SSR位點(diǎn)進(jìn)行分類,結(jié)果發(fā)現(xiàn)二核苷酸重復(fù)基元的SSR位點(diǎn)數(shù)最多,為12594個(gè),占全部重復(fù)序列的62.15%;其次是三核苷酸重復(fù)基元的SSR位點(diǎn)(5318個(gè)),占26.25%;四、五和六核苷酸重復(fù)基元的SSR位點(diǎn)較少(1826、371和163個(gè)),占比分別為9.00%、1.80%和0.80%。

2. 5 重復(fù)性檢驗(yàn)結(jié)果

在轉(zhuǎn)錄組學(xué)研究中通常以相關(guān)性熱圖展示樣品間的關(guān)系,而樣品間基因表達(dá)水平相關(guān)性是檢驗(yàn)試驗(yàn)可靠性和樣品選擇是否合理的重要指標(biāo)。根據(jù)RPKM定量結(jié)果,計(jì)算出所有樣品(每個(gè)樣品3個(gè)重復(fù))兩兩間的相關(guān)系數(shù)(圖7),發(fā)現(xiàn)相關(guān)系數(shù)均在0.5900以上,說(shuō)明樣品檢測(cè)的重復(fù)性較好,結(jié)果可信度較高。

3 討論

近年來(lái),高通量測(cè)序技術(shù)已廣泛應(yīng)用于模式和非模式生物的相關(guān)研究領(lǐng)域,如在興國(guó)紅鯉、白鯽、紅鯽及紅白鯽雜交子代中均有利用高通量測(cè)序技術(shù)進(jìn)行轉(zhuǎn)錄組學(xué)分析的研究報(bào)道(岳華梅等,2016;Zhang et al.,2017c;Liu et al.,2018)。在轉(zhuǎn)錄組學(xué)研究中,為獲取較多轉(zhuǎn)錄本以避免個(gè)體變異而產(chǎn)生的負(fù)面影響,通常從不同組織或個(gè)體中提取RNA,等體積混合后進(jìn)行轉(zhuǎn)錄組測(cè)序分析(Huang et al.,2012;Li et al.,2012;Liao et al.,2013;李冰冰等,2017)。本研究利用RNA-seq高通量測(cè)序技術(shù)對(duì)3種白鯽雜交子代進(jìn)行轉(zhuǎn)錄組學(xué)測(cè)序分析,共得到225858條unigenes,平均長(zhǎng)度為668 bp,N50為938 bp。與紅白鯽雜交子代轉(zhuǎn)錄組學(xué)分析結(jié)果(Liu et al.,2018)相比,本研究中的unigene數(shù)目較多,且質(zhì)量較好,可能與本研究利用多個(gè)樣本多個(gè)組織的總RNA進(jìn)行測(cè)序有關(guān),所獲得的轉(zhuǎn)錄組數(shù)據(jù)相比單個(gè)組織或單個(gè)樣品測(cè)序更全面。

目前,有關(guān)水產(chǎn)動(dòng)物雜交分子機(jī)理的研究主要集中在差異表達(dá)基因與雜種優(yōu)勢(shì)的相關(guān)性方面(Zhai et al.,2013;Zhang et al.,2017a)。孫穎(2016)通過(guò)對(duì)棕點(diǎn)石斑魚(E. fuscogutatus ♀)×鞍帶石斑魚(E. lanceolatus ♂)雜交F1代(虎龍斑)生長(zhǎng)優(yōu)勢(shì)的轉(zhuǎn)錄組學(xué)進(jìn)行研究,結(jié)果在虎龍斑中找到大量的差異表達(dá)基因,且這些差異表達(dá)基因主要分布在GH/IGF軸及其下游涉及蛋白與糖原合成的信號(hào)通路上,對(duì)虎龍斑生長(zhǎng)優(yōu)勢(shì)有重要貢獻(xiàn)。本研究結(jié)果表明,在白鯽(♀)×黑龍江野鯉(♂)與白鯽(♀)×興國(guó)紅鯉(♂)的雜交子代中差異表達(dá)基因最多,為6448個(gè);其次是白鯽(♀)×散鱗鏡鯉(♂)與白鯽(♀)×興國(guó)紅鯉(♂)的雜交子代,為4249個(gè);差異表達(dá)基因最少的是白鯽(♀)×散鱗鏡鯉(♂)與白鯽(♀)×黑龍江野鯉(♂)的雜交子代,僅有1615個(gè);3個(gè)轉(zhuǎn)錄組的共有基因?yàn)?3個(gè)。差異表達(dá)基因的KEGG分析結(jié)果顯示,大多數(shù)差異表達(dá)基因注釋到免疫相關(guān)通路中,包括內(nèi)吞作用、Jak-STAT信號(hào)通路、溶酶體、吞噬體和Wnt信號(hào)通路等;也有差異表達(dá)基因注釋到Toll樣受體信號(hào)通路和NOD樣受體信號(hào)通路中。此外,從差異表達(dá)基因中鑒定出過(guò)氧化氫酶(unigene071729)、鐵蛋白(unigene045233)、peroxiredoxin-6(unigene058224)及α-2M(unigene079502)等抗氧化和免疫相關(guān)的基因,在3個(gè)白鯽雜交子代中這些基因的表達(dá)量均呈不同程度上調(diào)趨勢(shì)。免疫系統(tǒng)可保護(hù)生物體免受自然環(huán)境中寄生蟲或致病菌的感染,是機(jī)體產(chǎn)生抗病性的生理基礎(chǔ),也是育種過(guò)程中進(jìn)行人工選擇的重要指標(biāo)之一(Rauw,2012)。免疫相關(guān)基因或通路的鑒定,既可為鯽鯉雜交的人工選育提供依據(jù),又能佐證這些基因或通路對(duì)3個(gè)白鯽雜交子代的生長(zhǎng)優(yōu)勢(shì)具有重要貢獻(xiàn)。

生長(zhǎng)速度和體重一直是選育過(guò)程中的關(guān)鍵指標(biāo)。本課題組的前期研究結(jié)果表明,3種白鯽雜交子代間的體重存在顯著差異。本研究也鑒定出與生長(zhǎng)和發(fā)育相關(guān)的差異表達(dá)基因通路,如MAPK信號(hào)通路(ko04010)、Hippo信號(hào)通路(ko04390)和背腹軸形成(ko04320)等。Zhang等(2017b)對(duì)中華鱉(Pelodiscus sinensis)的雜交子代進(jìn)行轉(zhuǎn)錄組學(xué)分析,也曾鑒定出這3條通路,說(shuō)明這3條通路在促進(jìn)雜交子代生長(zhǎng)和發(fā)育的過(guò)程中發(fā)揮重要作用。其中,MAPK信號(hào)通路(ko04010)和Hippo信號(hào)通路(ko04390)在調(diào)節(jié)器官發(fā)育及器官發(fā)育過(guò)程中發(fā)揮重要作用(Schaeffer and Weber,1999;Yu et al.,2012)。此外,與生長(zhǎng)相關(guān)的表皮生長(zhǎng)因子受體(unigene078218)、微管相關(guān)蛋白(unigene024234)、血小板源生長(zhǎng)因子受體(unigene038197和unigene068685)等基因均明顯上調(diào),而生長(zhǎng)抑制蛋白(unigene067656)明顯下調(diào)。這些基因的差異表達(dá)有助于開(kāi)展雜交子代的生長(zhǎng)性狀比較分析。

分子標(biāo)記是進(jìn)行種群遺傳學(xué)、生物地理學(xué)及進(jìn)化關(guān)系研究的關(guān)鍵(Li and Gui,2007;Jia et al.,2008;Wang et al.,2011;陳春林等,2018)。通過(guò)高通量測(cè)序技術(shù)獲得的轉(zhuǎn)錄組序列,可為分子標(biāo)記的挖掘和開(kāi)發(fā)提供一種更直觀、高效的方法(Davey et al.,2011;Fu and He,2012;Ji et al.,2012)。至今,利用轉(zhuǎn)錄組測(cè)序技術(shù)進(jìn)行SSR分子標(biāo)記挖掘在國(guó)際上已有很多成功的報(bào)道(Tian et al.,2014;Chen et al.,2015;Xiao et al.,2015)。Liao等(2013)從鯽魚的轉(zhuǎn)錄組數(shù)據(jù)庫(kù)中挖掘到11295個(gè)SSR位點(diǎn),且大多數(shù)SSR位點(diǎn)為二核苷酸重復(fù)基元。岳華梅等(2016)從興國(guó)紅鯉的轉(zhuǎn)錄組中鑒定出13652個(gè)SSR位點(diǎn),并從中隨機(jī)篩選出30個(gè)進(jìn)行PCR驗(yàn)證,發(fā)現(xiàn)有20對(duì)SSR分子標(biāo)記可擴(kuò)增出清晰穩(wěn)定的條帶。本研究從3個(gè)白鯽雜交子代中鑒定出20272個(gè)SSR位點(diǎn),其中有62.15%的SSR位點(diǎn)為二核苷酸重復(fù)基元,與Liao等(2013)的研究結(jié)果相似。這些挖掘獲得的SSR位點(diǎn)可為后續(xù)的遺傳多樣性、遺傳圖譜構(gòu)建及QTL定位等研究打下基礎(chǔ)。

4 結(jié)論

不同白鯽雜交子代間存在較多的差異表達(dá)基因,從中獲得參與抗氧化、免疫和生長(zhǎng)發(fā)育相關(guān)的通路和基因序列,且挖掘出20272個(gè)SSR位點(diǎn),有助于選擇性育種、分子標(biāo)記開(kāi)發(fā)及開(kāi)展遺傳多樣性、遺傳圖譜構(gòu)建和QTL定位等研究。

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