基因芯片在畜禽遺傳育種中的應(yīng)用及展望

汪佳豪，趙卿堯，周月玲，史良玉，王楚端，俞英

綜 述

基因芯片在畜禽遺傳育種中的應(yīng)用及展望

汪佳豪1，趙卿堯1，周月玲1，史良玉2，王楚端1，俞英1

1. 中國(guó)農(nóng)業(yè)大學(xué)動(dòng)物科學(xué)技術(shù)學(xué)院，北京 100193 2. 武漢輕工大學(xué)動(dòng)物科學(xué)與營(yíng)養(yǎng)工程學(xué)院，武漢 430023

基因芯片是一種通過(guò)DNA雙鏈或DNA-RNA互補(bǔ)雜交檢測(cè)特定DNA序列的高通量技術(shù)，其中SNP基因分型芯片已經(jīng)廣泛用于畜禽的遺傳育種工作，在牛()、豬()、羊()、雞()等畜禽中取得了重大成就。但是在實(shí)際生產(chǎn)中使用的基因組選擇僅利用了基因組信息，無(wú)法完全解釋復(fù)雜性狀的分子遺傳基礎(chǔ)，限制了基因組選擇的準(zhǔn)確性。隨著表觀遺傳學(xué)研究的不斷深入、商用甲基化芯片的推出、表觀基因組關(guān)聯(lián)分析(epigenome-wide association study，EWAS)的提出，DNA甲基化已被廣泛用于解釋遺傳與表型的因果關(guān)系。未來(lái)，有望開(kāi)發(fā)專門(mén)針對(duì)畜禽的甲基化芯片，通過(guò)EWAS探索與畜禽經(jīng)濟(jì)性狀顯著相關(guān)的甲基化位點(diǎn)，深化對(duì)復(fù)雜性狀因果變異的理解。結(jié)合甲基化芯片與SNP芯片捕獲畜禽表觀基因組和基因組信息，更準(zhǔn)確地解讀遺傳變異，提高基因組選擇的準(zhǔn)確性，推動(dòng)畜禽分子遺傳育種工作的精細(xì)化發(fā)展。本文綜述了SNP芯片在畜禽上的應(yīng)用，并對(duì)甲基化芯片在畜禽上的應(yīng)用進(jìn)行了展望，以期為基因芯片在動(dòng)物育種中的進(jìn)一步應(yīng)用提供借鑒和參考。

基因芯片；SNPs；DNA甲基化；畜禽遺傳育種

基因芯片技術(shù)是20世紀(jì)90年代發(fā)展起來(lái)的分子生物學(xué)高新技術(shù)，經(jīng)過(guò)30多年的快速發(fā)展，現(xiàn)已在功能上大致分為3個(gè)主要類別：基因表達(dá)譜芯片、SNP基因分型芯片和甲基化芯片。隨著基因組選擇的提出，SNP基因分型芯片被廣泛應(yīng)用于畜禽遺傳育種工作，大大提高了畜禽的遺傳進(jìn)展[1]。目前，基因組選擇模型主要使用基因組信息[2]，不能完全解釋復(fù)雜性狀的分子遺傳基礎(chǔ)。畜禽的育種值定義為親代可以穩(wěn)定遺傳給子代的部分[3～5]。隨著遺傳學(xué)的不斷發(fā)展，科研人員發(fā)現(xiàn)不僅DNA序列的信息可以穩(wěn)定遺傳給后代，DNA序列上的表觀遺傳修飾如DNA甲基化也能遺傳給子代并影響子代的表型[6～11]。此外，表觀遺傳修飾也可以作為環(huán)境與基因之間的橋梁，調(diào)控基因在特定環(huán)境下的表達(dá)[12]。未來(lái)，有望結(jié)合基因組和表觀基因組信息，更深入地解釋畜禽的遺傳變異及環(huán)境與基因之間的相互作用，為畜禽遺傳育種提供更全面的視角。

本文綜述了基因芯片的發(fā)展，探討了其在畜禽遺傳育種領(lǐng)域的關(guān)鍵應(yīng)用。此外，本文對(duì)未來(lái)如何開(kāi)發(fā)專門(mén)針對(duì)畜禽的甲基化芯片，以及如何將DNA甲基化信息與基因組信息相結(jié)合，從而提高基因組選擇的準(zhǔn)確性實(shí)現(xiàn)更為精細(xì)的分子遺傳育種進(jìn)行了深入的探討。

1 基因芯片

20世紀(jì)90年代初，F(xiàn)odor等[13]組合固相化學(xué)、光不穩(wěn)定保護(hù)基團(tuán)、光刻等技術(shù)研發(fā)出第一款基因芯片。1995年，Schena等[14]合成了第一款基因表達(dá)譜芯片，基因芯片開(kāi)始進(jìn)入發(fā)展的黃金時(shí)期。此后，熒光微球技術(shù)[15]、光纖微球技術(shù)[16]、靶向捕獲測(cè)序技術(shù)[17]等相繼提出并得到實(shí)際應(yīng)用。目前，基因芯片技術(shù)主要聚焦于SNP基因分型和DNA甲基化檢測(cè)。

1.1 SNP芯片

1994年，單核苷酸多態(tài)性(single nucleotide polymorphisms，SNPs)的概念首次被提出[18]。1996年，Lander[19]正式提出并認(rèn)定其為繼限制性片段長(zhǎng)度多態(tài)性(restriction fragment length polymorphisms，RFLP)和簡(jiǎn)單重復(fù)序列(simple sequence repeat，SSR)之后的第三代分子遺傳標(biāo)記。SNPs作為新一代分子遺傳標(biāo)記，不僅遺傳穩(wěn)定性高、在基因組內(nèi)廣泛分布，并且絕大部分是二等位基因(單個(gè)位點(diǎn)有兩種堿基)，便于實(shí)現(xiàn)自動(dòng)化的基因分型[20,21]。Delahunty等[22]于1996年結(jié)合PCR與寡核苷酸連接檢測(cè)(oligonucleotide ligation assay，OLA)技術(shù)實(shí)現(xiàn)了半自動(dòng)化的SNPs基因分型。2000年，Affymetrix公司通過(guò)單堿基延伸(single base extension，SBE)原理設(shè)計(jì)出對(duì)SNPs進(jìn)行自動(dòng)化、高通量基因分型的寡核苷酸陣列[23,24]。2002年，Illumina公司基于微球矩陣技術(shù)也實(shí)現(xiàn)準(zhǔn)確、高性價(jià)比的高通量SNPs基因分型[25,26]。此后，隨著下一代測(cè)序技術(shù)的發(fā)展，測(cè)序速度得到了顯著提高。但許多研究和疾病診斷只需測(cè)序基因組中的特殊序列就能實(shí)現(xiàn)，基于該需求，靶向捕獲測(cè)序技術(shù)被提出并獲得了廣泛的應(yīng)用[17]。與具有極高工藝門(mén)檻的固相基因芯片不同，基于靶向捕獲測(cè)序技術(shù)的液相芯片只需設(shè)計(jì)相應(yīng)的探針即可，使得該技術(shù)得到很多公司的青睞。如博瑞迪公司靶向測(cè)序基因型檢測(cè)(genotyping by target sequencing，GBTS)技術(shù)[27]、華智公司基于目標(biāo)區(qū)域基因組序列液相捕獲的精準(zhǔn)定位測(cè)序分型技術(shù)(genotyping by pinpoint sequencing of captured target，cGPS)(https:// www.higentec.com/)等均是基于靶向捕獲測(cè)序技術(shù)進(jìn)一步發(fā)展而來(lái)，此類技術(shù)的不斷創(chuàng)新和完善使得SNP芯片的種類日益豐富，目前已逐漸形成固相芯片和液相芯片兩大類。

1.2 甲基化芯片

DNA甲基化是表觀遺傳修飾中研究最深入的一種修飾，一般發(fā)生在胞嘧啶-磷酸-鳥(niǎo)嘌呤(CpG)中胞嘧啶的第五位碳原子上[28]。DNA甲基化不僅能夠調(diào)控基因表達(dá)，影響細(xì)胞分化[29]，而且與各種疾病、生理狀態(tài)有關(guān)，被廣泛作為生物標(biāo)志物[30,31]。鑒于其在遺傳和分子生物學(xué)中的重要性，DNA甲基化也被人們稱為“第五堿基”。1992年，F(xiàn)rommer等[32]對(duì)DNA進(jìn)行亞硫酸氫鹽處理，將非甲基化的胞嘧啶(C)轉(zhuǎn)化為尿嘧啶(U)，PCR擴(kuò)增中轉(zhuǎn)化為胸腺嘧啶(T)，可以實(shí)現(xiàn)對(duì)DNA甲基化的精確檢測(cè)。此后，隨著對(duì)該方法的不斷完善，已成為DNA甲基化檢測(cè)的金標(biāo)準(zhǔn)[33]。DNA甲基化的檢測(cè)與SNP相似：檢測(cè)亞硫酸氫鹽轉(zhuǎn)化后的CpG中C的位置為C還是T即可。2006年，Illumina在原有SNP基因分型技術(shù)上開(kāi)發(fā)出高通量的甲基化檢測(cè)芯片[34]。此后，在靶向捕獲測(cè)序技術(shù)基礎(chǔ)上也開(kāi)發(fā)出靶向亞硫酸氫鹽測(cè)序(targeted bisulfite sequencing，TBS)技術(shù)，用于檢測(cè)DNA甲基化。其主要分為兩種策略：(1)亞硫酸氫鹽處理后再對(duì)目標(biāo)序列捕獲測(cè)序[35]；(2)對(duì)目標(biāo)序列進(jìn)行捕獲、亞硫酸氫鹽轉(zhuǎn)化、上機(jī)測(cè)序[36]。經(jīng)亞硫酸氫鹽處理及PCR擴(kuò)增后使序列的復(fù)雜度大大降低，難以在高密度的CpGs區(qū)域設(shè)計(jì)最佳的捕獲探針。因此，現(xiàn)行的技術(shù)流程主要采用策略(2)。甲基化芯片不僅能對(duì)特定區(qū)域的甲基化水平進(jìn)行檢測(cè)，而且顯著降低甲基化檢測(cè)成本，已成為甲基化檢測(cè)的主要方法之一[37]。

2 SNP芯片在畜禽上的應(yīng)用

隨著新一代分子遺傳標(biāo)記SNPs的發(fā)現(xiàn)及應(yīng)用、基因芯片的發(fā)展，使應(yīng)用于動(dòng)物上的商業(yè)化SNP芯片成為可能(圖1)。Meuwissen等[38]于2001年提出基因組選擇：假設(shè)覆蓋全基因組的高密度標(biāo)記中有些標(biāo)記與影響該目標(biāo)性狀的數(shù)量性狀基因座(quantitative trait locus，QTL)的位置非常接近，處于連鎖不平衡，這樣使得每個(gè)QTL的效應(yīng)都可以通過(guò)標(biāo)記得到反映，即全基因組范圍內(nèi)的標(biāo)記輔助選擇?；蚪M選擇相比于傳統(tǒng)的育種方法具有更高的估計(jì)準(zhǔn)確性，能大大縮短世代間隔，對(duì)傳統(tǒng)方法難以實(shí)施選擇的性狀也能有較好選擇[39,40]。但是，由于缺乏對(duì)動(dòng)植物進(jìn)行基因分型的工具而無(wú)法有效實(shí)施。

2007年，Illumina發(fā)布第一款畜禽的商業(yè)化SNP基因分型芯片——BovineSNP50 Genotyping BeadChip[41]，基因組選擇率先應(yīng)用于奶牛的遺傳育種工作，拉開(kāi)了畜禽遺傳育種的新篇章。VanRaden等[42]于2008年使用BovineSNP50 Genotyping BeadChip對(duì)5335頭公牛進(jìn)行基因分型，驗(yàn)證基因組選擇的可行性。相比于傳統(tǒng)的預(yù)測(cè)方法，基因組預(yù)測(cè)的可靠性更高。2009年，美國(guó)首次公開(kāi)發(fā)布奶?；蚪M選擇的結(jié)果，并完全接納基因組信息作為官方種公牛遺傳評(píng)定發(fā)布的信息來(lái)源，綜合傳統(tǒng)評(píng)定結(jié)果與基因組育種值對(duì)公牛進(jìn)行排名[43]。自此，基因組選擇被廣泛應(yīng)用于奶牛的遺傳育種工作。2014年，Hutchison等[44]對(duì)美國(guó)荷斯坦牛、娟姍牛的數(shù)據(jù)研究發(fā)現(xiàn)：年輕公牛用于育種的數(shù)量越來(lái)越多，顯著縮短了奶牛的世代間隔。

基因組選擇也廣泛應(yīng)用于其他畜禽動(dòng)物[45]。2009年，首款豬的SNP基因分型芯片Illumina Porcine60 Genotyping BeadChip問(wèn)世[46]，豬的基因組選擇育種工作在全球范圍內(nèi)也逐漸開(kāi)展起來(lái)。溫氏集團(tuán)作為我國(guó)最大的豬育種與肉豬生產(chǎn)公司，于2011年開(kāi)始進(jìn)行基因組選擇育種[39]，2013年成功選育出一頭杜洛克特級(jí)種公豬[47]。Genus公司通過(guò)基因組選擇顯著提高了豬的出生仔豬總數(shù)、日增重、出生到斷奶階段的死亡率、采食量、眼肌高度等5個(gè)性狀的估計(jì)育種值準(zhǔn)確性[48]。2009年，綿羊()SNP基因分型芯片OvineSNP50 BeadChip問(wèn)世，基因組技術(shù)也逐漸應(yīng)用在綿羊的遺傳育種工作[49]。

隨著我國(guó)對(duì)種業(yè)工作的不斷重視，適用于我國(guó)本地品種的SNP芯片陸續(xù)被開(kāi)發(fā)出來(lái)。自主研發(fā)的廣泛適用于我國(guó)地方雞種的基因組育種芯片“京芯一號(hào)”，不僅擺脫了對(duì)國(guó)外基因芯片的依賴，而且能更好的適用于本地雞品種的遺傳育種工作[50,51]。此后，在豬[52,53]、牛[54～56]、羊[57,58]上自主研發(fā)的SNP芯片的相繼問(wèn)世大大推進(jìn)了我國(guó)畜禽的遺傳育種工作(表1)。

3 表觀遺傳學(xué)在畜禽遺傳育種上的應(yīng)用

表觀遺傳學(xué)是指DNA序列不變的情況下，基因的表達(dá)卻發(fā)生了可遺傳的變化。造成這些變化的表觀遺傳修飾包括DNA/RNA甲基化、組蛋白修飾(甲基化、乙?；?、磷酸化等)和非編碼RNA (miRNAs、lncRNAs、circRNAs等)。目前，關(guān)于表觀遺傳的研究在DNA甲基化方面最為成熟[69]。

表1 畜禽上常用的SNP芯片

研究表明，DNA甲基化對(duì)畜禽繁殖、骨骼肌發(fā)育、免疫等性狀均有顯著影響。例如：差異甲基化基因使同卵雙生的兩頭公牛的表型、繁殖性狀的估計(jì)育種值均存在差異[70]；位于啟動(dòng)子的DNA甲基化能夠抑制該基因表達(dá)，促進(jìn)肌細(xì)胞增殖、抑制其分化，以調(diào)控骨骼肌的生長(zhǎng)發(fā)育[71]；DNA甲基化對(duì)T細(xì)胞亞群的分化調(diào)控等方面具有重要作用，特別是基因的DNA甲基化顯著影響CD4+T細(xì)胞數(shù)量和功能，能夠在一定程度上改變家畜的免疫應(yīng)答能力或抗病性能，以對(duì)抗或適應(yīng)外界環(huán)境改變[72]。

目前被廣泛應(yīng)用的GBLUP (genomic best linear unbiased prediction)、ssGBLUP (single-step genomic best linear unbiased prediction)模型僅使用了系譜和DNA序列的信息。雖然將SNPs的功能作為先驗(yàn)信息可以提高模型估計(jì)育種值的準(zhǔn)確性[2]，但是忽略了可以穩(wěn)定遺傳的表觀遺傳變異對(duì)性狀的影響。所估計(jì)的育種值只捕捉到真實(shí)育種值的部分信息，大大限制了基因組選擇準(zhǔn)確性的提升。

隨著表觀遺傳學(xué)研究的深入，數(shù)量表觀遺傳學(xué)[12,73]、群體表觀遺傳學(xué)[74,75]不斷發(fā)展并完善。數(shù)量表觀遺傳學(xué)研究表明：DNA甲基化等表觀遺傳修飾通過(guò)影響基因的表達(dá)，進(jìn)而影響等位基因之間的顯性效應(yīng)、非等位基因之間的上位效應(yīng)，導(dǎo)致遺傳方差組分發(fā)生變化[12]。群體表觀遺傳學(xué)對(duì)經(jīng)典群體遺傳學(xué)進(jìn)行拓展，引入單甲基化多態(tài)性(single methylation polymorphism，SMP)的概念，即胞嘧啶位點(diǎn)是否發(fā)生甲基化修飾[75]。Hu等[76]根據(jù)位點(diǎn)甲基化水平對(duì)甲基化信息進(jìn)行轉(zhuǎn)化，構(gòu)建與GBLUP中G矩陣類似的epi-G矩陣，結(jié)果顯示表觀遺傳變異能夠解釋65%的表型變異。陳思倩[77]根據(jù)SNPs是否位于表觀功能基因組區(qū)域進(jìn)行分類，將表觀基因組信息引入GFBLUP (genomic feature best linear unbiased prediction)模型，其預(yù)測(cè)準(zhǔn)確性相比于傳統(tǒng)GBLUP有所提高。這些都表明將表觀基因組信息納入基因組選擇模型中對(duì)提高復(fù)雜性狀預(yù)測(cè)準(zhǔn)確性的重要性。

4 甲基化芯片的應(yīng)用及展望

畜禽遺傳育種工作的核心是選擇優(yōu)秀的個(gè)體，而選擇的關(guān)鍵是提高選種的準(zhǔn)確性。基因組選擇已極大的縮短了畜禽的世代間隔，未來(lái)提高遺傳進(jìn)展的關(guān)鍵在于提高基因組選擇育種值估計(jì)的準(zhǔn)確性。畜禽的表型并非完全由基因決定，DNA甲基化對(duì)畜禽的各種性狀都能產(chǎn)生影響。未來(lái)通過(guò)整合表觀遺傳學(xué)的信息，有望提高基因組選擇的準(zhǔn)確性，實(shí)現(xiàn)更精準(zhǔn)的分子遺傳育種工作[78]。

4.1 全基因組甲基化芯片

DNA甲基化在遺傳調(diào)控方面的重要性引起了人們的廣泛關(guān)注。但是常規(guī)的全基因組甲基化測(cè)序(whole genome bisulfite sequencing，WGBS)成本極高，不易實(shí)現(xiàn)大規(guī)模的甲基化分析；簡(jiǎn)化代表性亞硫酸氫鹽測(cè)序(reduced representation bisulfite sequ-en--cing，RRBS)只能檢測(cè)CpGs富集的區(qū)域[37]。甲基化芯片則可以自主設(shè)計(jì)檢測(cè)特定區(qū)域甲基化的探針，實(shí)現(xiàn)對(duì)更多具有生物學(xué)意義的區(qū)域進(jìn)行高通量低成本的檢測(cè)，進(jìn)行大規(guī)模的甲基化研究[79]。

最早的甲基化芯片可以追溯到21世紀(jì)初，其主要用于檢測(cè)癌癥[34]。2009年，Illumina公司開(kāi)發(fā)了商業(yè)化的人甲基化芯片HumanMethylation27K BeadChip (HM27)，該芯片能夠檢測(cè)超過(guò)27K個(gè)CpG位點(diǎn)的甲基化水平，被廣泛用于甲基化研究[80～82]。隨著表觀基因組關(guān)聯(lián)分析(epigenome-wide associa-tion study，EWAS)被人們提出并廣泛用于檢測(cè)與人類疾病相關(guān)的CpG位點(diǎn)，解釋GWAS中無(wú)法解釋的因果關(guān)系[83]，甲基化芯片逐漸成為EWAS的重要工具。此后進(jìn)一步開(kāi)發(fā)出更高密度的甲基化芯片HumanMethylation450 BeadChip[84]、MethylationEPIC (EPIC) BeadChip[81]，并開(kāi)發(fā)出專門(mén)進(jìn)行EWAS的軟件[85]和數(shù)據(jù)平臺(tái)[86]。在人類研究中，利用甲基化芯片進(jìn)行EWAS在肥胖[87]、初生重[88]等性狀鑒定到顯著相關(guān)的CpGs位點(diǎn)，進(jìn)一步解釋了遺傳與性狀的因果關(guān)系。目前，EWAS也逐漸應(yīng)用于動(dòng)植物上[89,90]。已有研究利用甲基化芯片鑒定到與畜禽重要性狀相關(guān)的甲基化位點(diǎn)，并進(jìn)一步開(kāi)發(fā)出生物標(biāo)志物[90,91]。但針對(duì)畜禽的甲基化芯片種類較少，在一定程度上限制了畜禽的甲基化研究。未來(lái)，通過(guò)開(kāi)發(fā)畜禽的全基因組甲基化芯片，將大大降低甲基化檢測(cè)的費(fèi)用，實(shí)現(xiàn)更大規(guī)模的甲基化分析，進(jìn)一步解釋遺傳與表型之間的分子機(jī)制，為功能性甲基化芯片的開(kāi)發(fā)提供基礎(chǔ)(圖2)。

4.2 精子甲基化芯片

在現(xiàn)代畜牧業(yè)中，家畜的交配大部分由人工授精完成[92]。一頭種公畜每年可以產(chǎn)生成百上千的后代，對(duì)群體的貢獻(xiàn)遠(yuǎn)大于母畜[93,94]。因此，種公畜性能的好壞以及精子質(zhì)量的優(yōu)劣在很大程度上決定了畜禽遺傳改良的進(jìn)展。而精子中含有豐富的表觀基因組信息，更好的利用這些信息有望選擇更優(yōu)秀的種公畜[95]?；蚪M印記是表觀遺傳的重要現(xiàn)象之一：基因組上部分基因的表達(dá)具有親本選擇特性，即只有一個(gè)親本的等位基因表達(dá)，另一個(gè)親本的等位基因不表達(dá)或很少表達(dá)[96]。畜禽精子中含有大量可以穩(wěn)定遺傳給后代并影響子代生長(zhǎng)發(fā)育的印記基因[97,98]。這些印記基因通常在染色體上成簇存在，并且大部分由不同親本來(lái)源的等位基因調(diào)控區(qū)的差異甲基化引起[97～100]。例如最典型的印記基因，印記通過(guò)的差異甲基化區(qū)域(differentially methylated region，DMR)進(jìn)行維持，該區(qū)域低甲基化將導(dǎo)致的雙等位基因表達(dá)，引發(fā)胎兒過(guò)度生長(zhǎng)[8]。Nishio等[101]將父母雙方的等位基因根據(jù)印記效應(yīng)重新賦予加性效應(yīng)并創(chuàng)建GBLUP-I模型，相比傳統(tǒng)的GBLUP模型，GBLUP-I模型估計(jì)遺傳方差的準(zhǔn)確性更高。

圖2 甲基化芯片原理及在畜禽遺傳育種上的應(yīng)用

精子DNA甲基化信息不僅包含印記基因修飾，也包含可以指示精子生育能力以及影響后代表型的甲基化修飾。Costes等[102]對(duì)100頭生育能力有差異的牛的精子進(jìn)行甲基化分析，共鑒定到490個(gè)與生育力相關(guān)的差異甲基化胞嘧啶位點(diǎn)(differentially methylated cytosines，DMCs)，使用這些甲基化位點(diǎn)構(gòu)建模型能夠很好的區(qū)分低生育力和正常生育力的公牛。Takeda等[91]通過(guò)甲基化分析鑒定到多個(gè)與公牛繁殖力相關(guān)的DMCs和DMRs，通過(guò)這些DMRs構(gòu)建模型能夠很好的預(yù)測(cè)公牛的生育力。Liu等[70]和Shojaei Saadi等[103]均發(fā)現(xiàn)：盡管同卵雙胞胎牛具有相同的基因組信息，但不同的環(huán)境可以導(dǎo)致其DNA甲基化模式的差異，從而影響其精子的表型和公牛的繁殖能力，甚至進(jìn)一步影響其后代的表型。

隨著不同密度SNP芯片的發(fā)布，根據(jù)目標(biāo)和需求可以結(jié)合不同SNP芯片對(duì)畜禽實(shí)施更有效的選擇策略[104]。未來(lái)，開(kāi)發(fā)精子甲基化芯片具有巨大的應(yīng)用潛力，例如：畜禽出生后首周利用低密度SNP芯片進(jìn)行初篩，在后備種公畜生產(chǎn)性能測(cè)定期，結(jié)合中/高密度SNP芯片和精子甲基化芯片數(shù)據(jù)進(jìn)行第二次篩選(圖3)；構(gòu)建種公畜繁殖力、后代表型與精子DNA甲基化的函數(shù)，結(jié)合基因組選擇指數(shù)進(jìn)一步構(gòu)建新的綜合選擇指數(shù)以更準(zhǔn)確的估計(jì)種公畜的繁殖力和育種值，以期選出性能更優(yōu)異的種公畜。

4.3 游離DNA甲基化芯片

盡管精子甲基化芯片能夠檢測(cè)印記基因、影響公畜繁殖力以及后代表型的甲基化位點(diǎn)，但是精子經(jīng)歷重編程，使得部分在原始生殖細(xì)胞階段被擦除的甲基化信息不能被檢測(cè)出來(lái)[11]。此外，不同細(xì)胞、組織均有其特異的啟動(dòng)子甲基化區(qū)域集，以決定其功能并維持機(jī)體的生理狀態(tài)，這無(wú)疑增加了對(duì)整體甲基化信息檢測(cè)的難度[105]。

圖3 種公畜選擇新策略

近年來(lái)，游離DNA(cell-free DNA，cfDNA)成為研究熱點(diǎn)，使檢測(cè)畜禽整體的甲基化成為可能。cfDNA是由細(xì)胞主動(dòng)或被動(dòng)死亡時(shí)將胞內(nèi)的DNA釋放到體液中產(chǎn)生的，攜帶有其起源細(xì)胞、組織的遺傳、表觀遺傳信息，可以反映細(xì)胞、組織的情況[106～108]。通過(guò)反卷積算法能夠確定cfDNA的來(lái)源、對(duì)cfDNA進(jìn)行甲基化分析可以檢測(cè)特定組織的甲基化水平是否正常[109]。Lehmann-Werman等[110]基于cfDNA中組織特異性甲基化模式開(kāi)發(fā)出在血液中檢測(cè)特定組織細(xì)胞死亡情況的方法，可通過(guò)特定細(xì)胞死亡的情況檢測(cè)疾病。Loyfer等[111]對(duì)不同細(xì)胞類型的甲基化進(jìn)行分析，找到了各種細(xì)胞內(nèi)特異的甲基化標(biāo)記，并基于這些甲基化標(biāo)記設(shè)計(jì)的反卷積算法能夠在不同來(lái)源的cfDNA中很好的檢測(cè)出其細(xì)胞來(lái)源。目前，cfDNA的提取、檢測(cè)流程較為成熟[112]，其甲基化模式的研究主要集中在對(duì)人類癌癥進(jìn)行無(wú)創(chuàng)檢測(cè)[105,113]。隨著對(duì)cfDNA甲基化模式研究的不斷深入，預(yù)期未來(lái)可能開(kāi)發(fā)畜禽的cfDNA甲基化芯片，以檢測(cè)畜禽整體的甲基化狀態(tài)，進(jìn)行健康預(yù)測(cè)。

5 結(jié)語(yǔ)與展望

在畜禽分子遺傳育種研究中，表觀遺傳學(xué)的重要性日益凸顯。開(kāi)發(fā)專門(mén)針對(duì)畜禽的甲基化基因芯片，將為畜禽甲基化研究提供有力工具，更好的解釋復(fù)雜性狀的分子調(diào)控機(jī)制。此外，應(yīng)用精子甲基化芯片可以剖析環(huán)境對(duì)種公畜繁殖性狀以及后代表型的影響，進(jìn)一步提高種公畜的選擇準(zhǔn)確性；應(yīng)用cfDNA甲基化芯片精確探究影響畜禽性狀的各個(gè)組織甚至各個(gè)細(xì)胞類型的甲基化模式，揭示復(fù)雜性狀的深層次分子機(jī)制，更全面地了解畜禽的生理生化及健康狀態(tài)。

因此，全面整合DNA甲基化信息、基因組信息、系譜信息將為畜禽分子遺傳育種提供新的視角，實(shí)現(xiàn)更精準(zhǔn)的分子遺傳育種，提高育種選擇工作的準(zhǔn)確性，選擇性能更優(yōu)良的種畜，推動(dòng)畜禽遺傳育種技術(shù)的發(fā)展。

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Application and prospect of gene chip in genetic breeding of livestock and poultry

Jiahao Wang1, Qingyao Zhao1, Yueling Zhou1, Liangyu Shi2, Chuduan Wang1, Ying Yu1

Gene chip is a high-throughput technique for detecting specific DNA sequences by DNA or DNA-RNA complementary hybridization, among which SNP genotyping chips have been widely employed in the animal genetics and breeding, and have made great achievements in cattle, pigs, sheep, chickensand other livestock.However, genomic selection applied in production merely uses genomic information and cannot fully explain the molecular mechanism of complex traits genetics, which limits the accuracy of genomic selection.With the continuous progresses in epigenetic research, the development of commercial methylation chips and the application of the epigenome-wide association study (EWAS), DNA methylation has been extensively used to draw the causal connections between genetics and phenotypes.In the future, it is hopefully expected to develop methylation chips customized for livestock and poultry and explore methylation sites significantly related to economic traits of livestock and poultry through EWAS thereby extending the understanding of causal variation of complex traits.Combining methylation chips and SNP chips, we can capture the epigenomic and genomic information of livestock and poultry, interpret genetic variation more precisely, improve the accuracy of genome selection, and promote the fine evolution of molecular genetic breeding of livestock and poultry.In this review, we summarize the application of SNP chips and depict the prospects of the application of methylation chips in livestock and poultry. This review will provide valuable insights for further application of gene chips in farm animal breeding.

gene chip; SNPs; DNA methylation; genetic breeding

2023-09-06;

2023-10-05;

2023-11-08

國(guó)家重點(diǎn)研發(fā)計(jì)劃項(xiàng)目(編號(hào)：2021YFD1200903，2021YFD1200900)，國(guó)家奶牛產(chǎn)業(yè)技術(shù)體系項(xiàng)目(編號(hào)：CARS-36)和國(guó)家自然科學(xué)基金國(guó)際(地區(qū))合作項(xiàng)目(編號(hào)：31961143009)資助[Supported by the National Key R&D Program of China (Nos.2021YFD1200903, 2021YFD1200900), the National Dairy Industry Technology System Project (No.CARS-36) and the National Natural Science Foundation of China-Pakistan Science Foundation Join Project (No.31961143009)]

汪佳豪，碩士研究生，專業(yè)方向：動(dòng)物遺傳育種與繁殖。E-mail: 2423280404@qq.com

俞英，教授，博士生導(dǎo)師，研究方向：表觀遺傳抗病育種。E-mail: yuying@cau.edu.cn

王楚端，教授，博士生導(dǎo)師，研究方向：豬遺傳育種與規(guī)?；a(chǎn)。E-mail: cdwang@cau.edu.cn

10.16288/j.yczz.23-233

(責(zé)任編委: 李明洲)