高廣亮，張克山，趙獻(xiàn)芝，許國洋，謝友慧，周莉，張昌蓮，王啟貴

全基因組關(guān)聯(lián)分析篩選鵝蛋品質(zhì)相關(guān)分子標(biāo)記

高廣亮1, 2，張克山1, 2，趙獻(xiàn)芝1, 2，許國洋1，謝友慧1, 2，周莉3，張昌蓮1, 2，王啟貴

1重慶市畜牧科學(xué)院，重慶 402460；2重慶市肉鵝遺傳改良工程技術(shù)研究中心，重慶 402460；3重慶綦江區(qū)動物疫病預(yù)防控制中心，重慶 401420

【目的】通過篩選與鵝蛋品質(zhì)性狀相關(guān)的分子標(biāo)記和候選基因，為解析蛋品質(zhì)性狀的遺傳機制及分子標(biāo)記輔助選擇提供理論支撐?！痉椒ā坎捎猛谓】邓拇ò座Z群體（209只）作為研究對象。收集了每只鵝在產(chǎn)蛋高峰期連續(xù)生產(chǎn)的5枚蛋，并測定了蛋重、蛋形指數(shù)、蛋殼強度、蛋殼厚度、蛋殼重和蛋黃重量等6個蛋品質(zhì)性狀。基于前期209只四川白鵝（母鵝）2.896 Tb全基因組重測序數(shù)據(jù)（12.44×/個體），采用全基因組關(guān)聯(lián)分析的方法，篩選與蛋品質(zhì)性狀相關(guān)的SNP位點和重要候選基因，并通過核酸飛行時間質(zhì)譜方法檢測了這些SNP位點的基因型頻率。 【結(jié)果】經(jīng)過篩選過濾，共有9 279 339個SNPs和209個個體用于后續(xù)研究。GWAS研究發(fā)現(xiàn)，48個SNP位點與6個鵝蛋品質(zhì)性狀顯著或建議性顯著相關(guān)（閾值分別為5.43×10-9和1.09×10-7），并注釋出27個蛋品質(zhì)性狀相關(guān)的候選基因，包括妊娠相關(guān)血漿蛋白A（pappalysin1, PAPPA）、蛋白絲氨酸/蘇氨酸磷酸酶調(diào)節(jié)亞基2基因（serine/threonine-protein phosphatase 4 regulatory subunit 2，PP4R2）、乙醇胺磷酸轉(zhuǎn)移酶1（ethanolamine phosphotransferase 1, EPT1）和離子型谷氨酸受體K2（glutamate receptor ionotropic, kainate 2，GRIK2）等，其中候選基因參與蛋白質(zhì)代謝，促進(jìn)生長因子IGF生成，在的11bp范圍內(nèi)存在5個SNPs與蛋殼厚度顯著相關(guān)，另外在上有6個SNPs與蛋黃重量顯著相關(guān)，和分別與機體血鈣維持功能以及膽固醇代謝相關(guān)。功能富集研究發(fā)現(xiàn)，候選基因主要參與了response to growth factor（GO:0070848）、intracellular chemical homeostasis（GO:0055082）、response to hormone（GO:0009725）和regulation of the monoatomic ion transport（GO:43269）等代謝通路。【結(jié)論】經(jīng)GWAS方法篩選出和分別作為蛋重、蛋黃重、蛋殼強度等蛋品質(zhì)性狀潛在功能基因，為鵝蛋品質(zhì)性狀的改良提供了分子遺傳標(biāo)記的理論參考。

全基因組關(guān)聯(lián)分析；分子標(biāo)記輔助選擇；鵝；蛋品質(zhì)性狀

0 引言

【研究意義】我國是世界上養(yǎng)鵝量最大的國家，國家水禽產(chǎn)業(yè)體系2022年報告顯示我國鵝年出欄量已達(dá)4.68億只，產(chǎn)值526.73億元，因此針對直接影響鵝蛋經(jīng)濟效益的蛋品質(zhì)性狀研究具有重要的現(xiàn)實意義[1]?！厩叭搜芯窟M(jìn)展】目前研究表明：品種、年齡、營養(yǎng)、環(huán)境、疾病等都可影響蛋品質(zhì)性狀[2-7]。全基因組關(guān)聯(lián)分析（genome-wide association study, GWAS）方法是在全基因組水平上關(guān)聯(lián)定位分析重要經(jīng)濟性狀的分子遺傳標(biāo)記，篩選有生物學(xué)意義的候選基因。目前GWAS已廣泛運用到家禽的分子遺傳標(biāo)記選擇中，如：ZHU等[8-9]對北京鴨體尺和屠體性狀等進(jìn)行GWAS分析，發(fā)現(xiàn)高皮脂北京鴨抗脂肪生成基因存在特定的突變位點，并發(fā)現(xiàn)屠體重、全凈膛重等5個體組成性狀都有1個相同的全基因組顯著位點，候選基因為；Zhang等[10]在兩個高低腹脂專門化肉雞品系中用60K SNP芯片作單倍型GWAS研究，定位了7個可能控制腹脂含量的候選基因，包括、1、和等；LIU等采用600 K高密度SNP陣列，研究了產(chǎn)蛋后期72和80周齡的母雞蛋品質(zhì)的全基因關(guān)聯(lián)分析，結(jié)果發(fā)現(xiàn)上的8.95—9.31 Mb（約0.36 Mb）的基因組區(qū)域與白蛋白高度和白蛋白單位密切相關(guān)，兩個最重要的SNPs占了3.12%—5.75%的表型變異，并篩選了3個與蛋殼顏色的相關(guān)的候選基因、和[11]；前期本課題組通過GWAS分析篩選了與48周齡鵝蛋數(shù)、60周齡鵝蛋數(shù)候選基因以及與鵝蛋黃顏色顯著相關(guān)的、等4個基因[12]?！颈狙芯壳腥朦c】由于鵝蛋品質(zhì)性狀是一個復(fù)雜的多因素組成數(shù)量性狀，其蛋重、蛋形指數(shù)、蛋比重、蛋殼質(zhì)量等性狀的遺傳力都較高，可通過遺傳分析來定位主效基因區(qū)間?！緮M解決的關(guān)鍵問題】在本研究筆者通過GWAS挖掘鵝蛋品質(zhì)性狀的相關(guān)分子標(biāo)記，定位功能基因區(qū)間，為解析蛋品質(zhì)性狀的遺傳機制及分子標(biāo)記輔助選擇提供理論支撐。

1 材料與方法

1.1 實驗動物和表型測定

以重慶市家禽科研基地同批次健康四川白鵝（母鵝）群體（209只）為實驗動物。為準(zhǔn)確記錄鵝蛋品質(zhì)性狀，每只鵝從出雛后進(jìn)行系統(tǒng)的腳號和翅號標(biāo)記，鵝群28周齡時轉(zhuǎn)移到個體籠（600 mm×800 mm×900 mm）中飼養(yǎng)至65周（休產(chǎn)期），飼養(yǎng)過程自由飲水和采食全價飼料，收集產(chǎn)蛋高峰期每只鵝連續(xù)生產(chǎn)的5個鵝蛋，測定其蛋品質(zhì)性狀（蛋重、蛋形指數(shù)、相對密度、蛋殼強度、蛋殼重、蛋殼厚度和蛋黃重）并統(tǒng)計其平均值作為后續(xù)GWAS研究的表型性狀。利用電子天平稱量蛋殼重、蛋黃重；利用游標(biāo)卡尺測量蛋殼的鈍部、尖部和中間部位，并取上述三者的平均值計算蛋殼厚度；利用游標(biāo)卡尺檢測蛋長直徑和短徑，計算蛋形指數(shù)；利用羅氏比色卡進(jìn)行蛋黃顏色的測定；蛋殼強度利用蛋殼測力計（EFG-0502，Robotmation 公司）進(jìn)行檢測。

1.2 DNA抽提及GWAS測序

抗凝真空管采集上述209只鵝翅靜脈血液2 mL，利用血液基因組提取試劑盒（北京天根公司，DP332）提取血液基因組DNA，NanoDrop 2000分光光度計質(zhì)檢通過后，委托天津諾禾生物有限公司利用Illumina HiSeq X Ten平臺進(jìn)行全基因重測序。數(shù)據(jù)過濾后，BWA軟件比對鵝基因組（ASM1303099v1）[13]，GATK軟件進(jìn)行SNP 基因型數(shù)據(jù)檢出[14]；Plink軟件對獲得的SNP數(shù)據(jù)進(jìn)行質(zhì)量控制（參數(shù)設(shè)置：geno 0.1 mind 0.1 MAF 0.05 hwe 0.0000001）和主成分分析方法檢測群體結(jié)構(gòu)分層情況[15]。

1.3 表型數(shù)據(jù)關(guān)聯(lián)與單倍型分析

表1 驗證篩選的SNPs用引物序列表

1.4 SNPs注釋

BEDTools軟件[18]在鵝染色體基因組上提取鑒定的顯著SNPs位點上下游500 kb區(qū)域序列，Annovar軟件注釋SNP所在基因及鄰近基因[19]，Metascape（http://metascape.org/）在線對候選基因進(jìn)行功能分析[20]。

2 結(jié)果

2.1 蛋品質(zhì)表型數(shù)據(jù)統(tǒng)計分析

209只四川白鵝個體高峰期平均蛋重為132.20 g；蛋形指數(shù)在1.24—1.60之間，平均蛋形指數(shù)為1.45；蛋殼強度平均值為67.20 kg·cm?2；蛋殼重平均為22.87 g；蛋殼厚度平均在0.42 cm；蛋黃重量平均值為41.20 g（表2）。

表2 統(tǒng)計分析鵝蛋品質(zhì)性狀

2.2 測序數(shù)據(jù)分析

209只四川白鵝母鵝全基因組重測序共獲得2.896 Tb數(shù)據(jù)，測序平均覆蓋深度為12.44×。經(jīng)數(shù)據(jù)過濾，2.891 Tb高質(zhì)量測序數(shù)據(jù)比對到鵝參考基因組序列，比對率為96.58%—98.38%，共檢出了16 687 310個初始SNP位點；經(jīng)過質(zhì)量控制，最終檢出9 279 339個SNPs[12]。GWAS結(jié)果顯示，48個SNPs與上述6個蛋品質(zhì)性狀顯著或建議性顯著關(guān)聯(lián)，并定位到27個基因（表3，圖1）。

以各性狀顯著關(guān)聯(lián)SNPs上下游500 k范圍內(nèi)注釋基因，功能富集分析發(fā)現(xiàn)：候選基因主要聚類到response to growth factor（GO:0070848, Log(P)=-8.49）、intracellular chemical homeostasis（GO:0055082, Log(P)= -7.77）、response to hormone（GO:0009725, Log(P)=-7.44）和regulation of the monoatomic ion transport（GO: 43269, Log(P)= -6.08）。富集差異基因P值前20的GO分類條目如圖2所示。

表3 篩選與蛋品質(zhì)性狀顯著相關(guān)的SNPs表

續(xù)表3 Continued table 3

: Pregnancy-associated plasma protein A;: Alpha-2C adrenergic receptor;: Epsilon-sarcoglycan;: Thioredoxin-like protein 1;: BCL-6 corepressor;: Mid1-interacting protein 1;: Glutaredoxin-3;: Pre-mRNA-splicing factor ATP-dependent RNA helicase DHX15;: TBC1 domain family member 14;: Dystrobrevin beta;: Ethanolamine phosphotransferase 1;: Nuclear factor of activated T-cells, cytoplasmic 1;: Cytoplasmic dynein 1 intermediate chain 1;: Protein bassoon;: NADPH oxidase 5;: KAT8 regulatory NSL complex subunit 1;: Uncharacterized protein KIAA1257;: Cytochrome P450 4V2;: Kinesin-like protein KIF16B;: Tribbles homolog 2;: Gamma-aminobutyric acid receptor subunit beta-3;: Uncharacterized protein KIAA1522;: Serine/threonine-protein phosphatase 4 regulatory subunit 2;: Activating signal cointegrator 1 complex subunit 3;: Glutamate receptor ionotropic, kainate 2;: Transcription elongation factor B polypeptide 1;:Transcription factor SOX-13

顯著關(guān)聯(lián)（閾值5.43×10-9），*潛在顯著關(guān)聯(lián)（閾值1.09×10-7）

Significant correlation (p-value 5.43×10-9), *potential significant correlation (p-value 1.09×10-7)

圖1 鵝蛋品質(zhì)性狀全基因組關(guān)聯(lián)分析的曼哈頓圖和Q-Q plot圖

圖2 差異顯著SNPs上下游500k注釋基因聚類分析

2.3 顯著相關(guān)SNPs的MALDI-TOF MS驗證結(jié)果

針對重要候選SNPs合成合適的引物作MALDI- TOF MS驗證，結(jié)果如表4所示：對于蛋重性狀，chr29: 3653854 AA基因型個體極顯著（＜0.01）高于AG和GG基因型個體；對于蛋形指數(shù)性狀，chr15: 20262147 GG基因型極顯著（＜0.01）高于GT和TT基因型個體；對于蛋殼厚度性狀，chr18:18353514 CC基因型個體極顯著（＜0.01）高于其他基因型個體；對于蛋黃重量性狀，chr34:3206857 GG基因型個體極顯著（＜0.01）低于其他基因型個體；chr1:36829464 AA基因型個體極顯著（＜0.01）高于其他基因型個體。

3 討論

3.1 鵝蛋品質(zhì)性狀表型與其他家禽蛋品質(zhì)的比較分析

蛋品質(zhì)性狀是鵝蛋經(jīng)濟效益的直接影響因素，蛋重決定了產(chǎn)蛋總重的大小，蛋形指數(shù)、蛋黃比例影響消費者對商品蛋的選擇，蛋重、蛋形指數(shù)、蛋殼質(zhì)量等均會影響種蛋的合格率及孵化率[21-23]。本研究測定了四川白鵝產(chǎn)蛋高峰期蛋品質(zhì)性狀，發(fā)現(xiàn)檢測群體的平均蛋重（132.20±18.90）g，顯著高于雞蛋和鴨蛋，但蛋形指數(shù)平均值為1.45，與雞、鴨蛋相當(dāng)，正常雞蛋的蛋形指數(shù)一般為1.32—1.39，鴨蛋為1.20—1.58[24]。鵝蛋的平均蛋殼強度為（67.20±10.19）kg·cm?2，平均蛋殼厚度為（0.42±0.06）cm，明顯高于雞蛋，這可能解釋了生產(chǎn)中相同數(shù)量受精蛋雞蛋的孵化率可達(dá)90%以上，而鵝蛋孵化率一般在80%左右，更厚和強度更高的蛋殼增加了新生雛鵝的破殼難度而導(dǎo)致破殼失敗，降低了孵化率。鵝蛋的蛋黃平均重為（41.20±6.73）g，蛋黃比例達(dá)到30%以上，最大的蛋黃重量達(dá)到62.75 g。早成鳥的蛋黃比例一般比鴿子、企鵝等后代需要照顧的晚成鳥蛋黃比例高，鵝是早成鳥的一種，鵝蛋的高蛋黃比例可為幼雛供應(yīng)更多的營養(yǎng)物質(zhì)，使其出殼后能夠獨立生存[25-26]。

3.2 影響鵝蛋品質(zhì)性狀表型的GWAS分析

蛋品質(zhì)性狀是一個復(fù)雜的多因素控制數(shù)量性狀，遺傳因素是主要因素之一，已有的研究顯示：雞蛋的蛋重遺傳力約為0.5，殼顏色遺傳力為 0.45—0.75，蛋殼強度遺傳力約為0.3[21]。全基因組關(guān)聯(lián)技術(shù)能對數(shù)量性狀遺傳控制區(qū)間作有效定位。本研究通過GWAS在全基因組水平分析了鵝蛋品質(zhì)遺傳控制因素，筆者對209只四川白鵝血樣DNA進(jìn)行全基因組重測序，基于前期本課題組組裝了一個染色體水平的鵝基因組[24]，有利于將高質(zhì)量的全基因組重測序數(shù)據(jù)比對到鵝基因組序列并進(jìn)行了GWAS分析。最終篩選并使用MALDI-TOF MS驗證了48個與蛋重、蛋殼厚度、蛋殼強度等蛋品質(zhì)性狀顯著相關(guān)的SNPs。

表4 部分SNPs的基因型對鵝6個蛋品質(zhì)性狀的影響

不同字母代表差異顯著(＜0.05)；相同字母代表差異不顯著(＞0.05)

Different letters in the same row indicate a significant difference between the genotypes (＜0.05); the same letter indicates no significant difference between genotypes (＞0.05)

通過GWAS分析在12、15以及29號染色體上共鑒定4個SNPs與蛋重顯著相關(guān)，分別位于、、和。妊娠相關(guān)血漿蛋白A（pregnancy-associated plasma protein A,）基因編碼一種分泌型金屬蛋白酶，可裂解胰島素樣生長因子結(jié)合蛋白（），而激活I(lǐng)GF信號通路，參與蛋白質(zhì)代謝以及對胰島素樣生長因子（IGF）運輸和攝取的調(diào)節(jié)過程[27]。在卵泡發(fā)育過程中卵丘顆粒細(xì)胞中mRNA表達(dá)水平上調(diào)是卵母細(xì)胞成熟的標(biāo)志之一[28]。MAROULI等[29]通過全基因組關(guān)聯(lián)研究發(fā)現(xiàn)了83個與人身高相關(guān)的編碼變體，其中增高等位基因促進(jìn)了的蛋白酶活性，增加了對的裂解，從而導(dǎo)致的生物利用度提高。在家禽中，體型和蛋重一般有著正向相關(guān)的關(guān)系，能夠促進(jìn)身高，這提示可能作為蛋重正向選擇的候選基因。值得注意的是，在篩選的48個候選SNPs中有5個與蛋殼厚度相關(guān)的SNPs都定位在chr18的蛋白絲氨酸/蘇氨酸磷酸酶調(diào)節(jié)亞基2基因（serine/ threonine-protein phosphatase 4 regulatory subunit 2，），是PP4復(fù)合體5個調(diào)節(jié)亞基中的一個，參與許多關(guān)鍵的細(xì)胞途徑，包括DNA損傷反應(yīng)（DNA修復(fù)、細(xì)胞周期調(diào)節(jié)和細(xì)胞凋亡）、葡萄糖代謝、細(xì)胞遷移、腫瘤發(fā)生和免疫反應(yīng)等[30-31]。家禽無論在排卵還是在蛋殼形成過程中都存在著明確的時序性，這種時序性受周期調(diào)節(jié)基因的控制，而調(diào)控核心節(jié)律基因/活性[32]，另外，高濃度的葡萄糖影響Ca2+吸收[33]，而參與葡萄糖代謝，由此推測可能參與了蛋殼形成。通過GWAS分析發(fā)現(xiàn)chr1:1466542和chr1: 1467878與蛋殼強度顯著相關(guān)的SNPs都定位乙醇胺磷酸轉(zhuǎn)移酶1（ethanolamine phosphotransferase 1, EPT1）基因上，該基因編碼蛋白將CDP-乙醇胺催化形成磷脂酰乙醇胺，是磷脂代謝的重要組成部分[34]。如表3所示，6個SNPs定位在chr1上的（glutamate receptor ionotropic, kainate 2）與蛋黃重量顯著相關(guān)（P=3.46*10-8—2.87* 10-9），ZEMUNIK等[35]通過全基因組關(guān)聯(lián)分析方法在一群孤立的人群中鑒別了與生化性狀（總膽固醇、低密度脂蛋白膽固醇、高密度脂蛋白膽固醇和甘油三酯等）相關(guān)的遺傳變異基礎(chǔ)，發(fā)現(xiàn)、和等是重要的候選基因。另外，兩個定位到（activating signal cointegrator 1 complex subunit 3, ASSC3）上的SNPs chr1 rs36803702C/T和rs36829464 A/G 同樣與蛋黃重量顯著相關(guān)，前期研究發(fā)現(xiàn)蛋白酶前體轉(zhuǎn)化酶亞硫酸酯酶/切割酶9型（Proprotein convertase subtilisin/kexin type 9，PCSK9）是最主要的低密度脂蛋白膽固醇調(diào)節(jié)子，可減少低密度脂蛋白膽固醇的含量，而和能作用核糖體，保護(hù)細(xì)胞免受因PCSK9抑制劑（PF8503）引起的細(xì)胞毒性[36]。蛋黃主要由膽固醇和磷脂組成，本研究發(fā)現(xiàn)的候選基因、等均參與膽固醇代謝，由此推測、可作為蛋黃重量相關(guān)的候選基因。

3.3 候選基因的功能聚類分析

家禽的產(chǎn)蛋行為受激素調(diào)控，F(xiàn)SH促進(jìn)卵泡生長成熟，LH促進(jìn)排卵， PRL與抱窩性相關(guān)，性激素維持鵝的生殖特征，甲狀旁腺激素和降鈣素調(diào)控蛋殼中鈣的沉積[37-39]。在本研究中筆者通過對注釋的功能基因進(jìn)行聚類分析也發(fā)現(xiàn)response to hormone（GO: 0009725）是P值最高的GO分類條目之一。每個雞蛋中的鈣量通常占雞體內(nèi)鈣儲存總量的10%左右，身體中絕大多數(shù)鈣存在于細(xì)胞內(nèi)，僅有不足0.1%位于細(xì)胞外，這部分包括電離鈣、與蛋白質(zhì)結(jié)合的鈣以及與陰離子結(jié)合的鈣三種形式，其中電離鈣是生理活性形式，家禽能在24 h內(nèi)將身體10%的鈣形成蛋殼[40- 41]，而不引起機體出現(xiàn)低血鈣癥，這與家禽獨特的血清鈣平衡維持密切相關(guān)，在進(jìn)行GWAS分析的時候筆者也發(fā)現(xiàn)與蛋品質(zhì)顯著相關(guān)的候選基因明顯聚到intracellular chemical homeostasis （GO:0055082）、regulation of the monoatomic ion transport（GO:0043269）等通路。

4 結(jié)論

本研究通過GWAS方法在全基因組水平上篩選與蛋品質(zhì)性狀顯著相關(guān)的分子遺傳標(biāo)記，注釋篩選了重要候選基因、、和分別作為蛋重、蛋黃重、蛋殼強度等蛋品質(zhì)性狀潛在功能基因。研究結(jié)果促進(jìn)了鵝蛋品質(zhì)性狀分子標(biāo)記選擇研究與運用，也將為四川白鵝蛋品質(zhì)性能的選育提供理論支撐。

[1] 侯水生, 劉靈芝. 2022年水禽產(chǎn)業(yè)現(xiàn)狀、未來發(fā)展趨勢與建議. 中國畜牧雜志, 2023, 59(3): 274-280.

HOU S S, LIU L Z. Present situation, future development trend and suggestions of waterfowl industry in 2022. Chinese Journal of Animal Science, 2023, 59(3): 274-280. (in Chinese)

[2] 王一冰, 陳芳, 茍鐘勇, 李龍, 林廈菁, 張盛, 蔣守群. 快大型黃羽肉種雞VD3需要量研究. 中國農(nóng)業(yè)科學(xué), 2021, 54(16): 3549-3560.

WANG Y B, CHEN F, GOU Z Y, LI L, LIN X J, ZHANG S, JIANG S Q. Requirement of vitamin D3on fast-growing yellow-feathered breeder hens. Scientia Agricultura Sinica, 2021, 54(16): 3549-3560. (in Chinese)

[3] KOWALSKA E, KUCHARSKA-GACA J, KU?NIACKA J, LEWKO L, GORNOWICZ E, BIESEK J, ADAMSKI M. Egg quality depending on the diet with different sources of protein and age of the hens. Scientific Reports, 2021, 11: 2638.

[4] NASRI H, VAN DEN BRAND H, NAJJAR T, BOUZOUAIA M. Egg storage and breeder age impact on egg quality and embryo development. Journal of Animal Physiology and Animal Nutrition, 2020, 104(1): 257-268.

[5] SUN C J, LIU J N, YANG N, XU G Y. Egg quality and egg albumen property of domestic chicken, duck, goose, Turkey, quail, and pigeon. Poultry Science, 2019, 98(10): 4516-4521.

[6] DA SILVA TEIXEIRA M, TRIGINELLI M V, DE ATAíDE COSTA T, LARA L J C, SOTO-BLANCO B. Effects of caffeine on egg quality and performance of laying hens. Frontiers in Veterinary Science, 2020, 7: 545359.

[7] WANG J, YUE H Y, WU S G, ZHANG H J, QI G H. Nutritional modulation of health, egg quality and environmental pollution of the layers. Animal Nutrition, 2017, 3(2): 91-96.

[8] ZHU F, CUI Q Q, HOU Z C. SNP discovery and genotyping using Genotyping-by-Sequencing in Pekin ducks. Scientific Reports, 2016, 6: 36223.

[9] ZHU F, YIN Z T, WANG Z, SMITH J, ZHANG F, MARTIN F, OGEH D, HINCKE M, LIN F B, BURT D W, ZHOU Z K, HOU S S, ZHAO Q S, LI X Q, DING S R, LI G S, YANG F X, HAO J P, ZHANG Z D, LU L Z, YANG N, HOU Z C. Three chromosome-level duck genome assemblies provide insights into genomic variation during domestication. Nature Communications, 2021, 12: 5932.

[10] ZHANG H, SHEN L Y, XU Z C, KRAMER L M, YU J Q, ZHANG X Y, NA W, YANG L L, CAO Z P, LUAN P, REECY J M, LI H. Haplotype-based genome-wide association studies for carcass and growth traits in chicken. Poultry Science, 2020, 99(5): 2349-2361.

[11] LIU Z, SUN C J, YAN Y Y, LI G Q, SHI F Y, WU G Q, LIU A Q, YANG N. Genetic variations for egg quality of chickens at late laying period revealed by genome-wide association study. Scientific Reports, 2018, 8: 10832.

[12] GAO G L, GAO D F, ZHAO X Z, XU S S, ZHANG K S, WU R, YIN C H, LI J, XIE Y H, HU S L, WANG Q G. Genome-wide association study-based identification of SNPs and haplotypes associated with goose reproductive performance and egg quality. Frontiers in Genetics, 2021, 12: 602583.

[13] LI H, DURBIN R. Fast and accurate long-read alignment with Burrows-Wheeler transform. Bioinformatics, 2010, 26(5): 589-595.

[14] MCKENNA A, HANNA M, BANKS E, SIVACHENKO A, CIBULSKIS K, KERNYTSKY A, GARIMELLA K, ALTSHULER D, GABRIEL S, DALY M, DEPRISTO M A. The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Research, 2010, 20(9): 1297-1303.

[15] PURCELL S, NEALE B, TODD-BROWN K, THOMAS L, FERREIRA M A R, BENDER D, MALLER J, SKLAR P, DE BAKKER P I W, DALY M J, SHAM P C. PLINK: a tool set for whole-genome association and population-based linkage analyses. The American Journal of Human Genetics, 2007, 81(3): 559-575.

[16] ZHOU X, STEPHENS M. Genome-wide efficient mixed-model analysis for association studies. Nature Genetics, 2012, 44(7): 821-824.

[17] KARSSEN L C, VAN DUIJN C M, AULCHENKO Y S. The GenABEL project for statistical genomics. F1000Research, 2016, 5: 914.

[18] QUINLAN A R. BEDTools: the swiss-army tool for genome feature analysis. Current Protocols in Bioinformatics, 2014, 47: 11.12.1- 11.1234.

[19] WANG K, LI M Y, HAKONARSON H. ANNOVAR: functional annotation of genetic variants from high-throughput sequencing data. Nucleic Acids Research, 2010, 38(16): e164.

[20] ZHOU Y Y, ZHOU B, PACHE L, CHANG M, KHODABAKHSHI A H, TANASEICHUK O, BENNER C, CHANDA S K. Metascape provides a biologist-oriented resource for the analysis of systems-level datasets. Nature Communications, 2019, 10: 1523.

[21] KETTA M, T?MOVá E. Eggshell structure, measurements, and quality-affecting factors in laying hens: a review. Czech Journal of Animal Science, 2016, 61(7): 299-309.

[22] HISASAGA C, GRIFFIN S E, TARRANT K J. Survey of egg quality in commercially available table eggs. Poultry Science, 2020, 99(12): 7202-7206.

[23] PICARD DRUET D, VARENNE A, HERRY F, HéRAULT F, ALLAIS S, BURLOT T, LE ROY P. Reliability of genomic evaluation for egg quality traits in layers. BMC Genetics, 2020, 21(1): 17.

[24] LI Y, GAO G L, LIN Y, HU S L, LUO Y, WANG G S, JIN L, WANG Q G, WANG J W, TANG Q Z, LI M Z. Pacific Biosciences assembly with Hi-C mapping generates an improved, chromosome- level goose genome. GigaScience, 2020, 9(10): 114.

[25] LIKER A, SZéKELY T. Mortality costs of sexual selection and parental care in natural populations of birds. International Journal of Organic Evolution, 2005, 59(4): 890-897.

[26] WILLIAMS T D. Physiology, activity and costs of parental care in birds. The Journal of Experimental Biology, 2018, 221(Pt 17): jeb169433.

[27] GYRUP C, OXVIG C. Quantitative analysis of insulin-like growth factor-modulated proteolysis of insulin-like growth factor binding protein-4 and -5 by pregnancy-associated plasma protein-A. Biochemistry, 2007, 46(7): 1972-1980.

[28] KORDUS R J, HOSSAIN A, CORSO M C, CHAKRABORTY H, WHITMAN-ELIA G F, LAVOIE H A. Cumulus cell pappalysin-1, luteinizing hormone/choriogonadotropin receptor, amphiregulin and hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta- isomerase 1 mRNA levels associate with oocyte developmental competence and embryo outcomes. Journal of Assisted Reproduction and Genetics, 2019, 36(7): 1457-1469.

[29] MAROULI E, GRAFF M, MEDINA-GOMEZ C, LO K S, WOOD A R, KJAER T R, FINE R S, LU Y C, SCHURMANN C, HIGHLAND H M, RüEGER S, et al. Rare and low-frequency coding variants alter human adult height. Nature, 2017, 542(7640): 186-190.

[30] LEE J, ADELMANT G, MARTO J A, LEE D H. Dephosphorylation of DBC1 by protein phosphatase 4 is important for p53-mediated cellular functions. Molecules and Cells, 2015, 38(8): 697-704.

[31] PARK J, LEE D H. Functional roles of protein phosphatase 4 in multiple aspects of cellular physiology: a friend and a foe. BMB Reports, 2020, 53(4): 181-190.

[32] KLEMZ S, WALLACH T, KORGE S, ROSING M, KLEMZ R, MAIER B, FIORENZA N C, KAYMAK I, FRITZSCHE A K, HERZOG E D, STANEWSKY R, KRAMER A. Protein phosphatase 4 controls circadian clock dynamics by modulating CLOCK/BMAL1 activity. Genes & Development, 2021, 35(15/16): 1161-1174.

[33] POHOREC V, KRI?AN?I? BOMBEK L, SKELIN KLEMEN M, DOLEN?EK J, STO?ER A. Glucose-stimulated calcium dynamics in beta cells from male C57BL/6J, C57BL/6N, and NMRI mice: a comparison of activation, activity, and deactivation properties in tissue slices. Frontiers in Endocrinology, 2022, 13: 867663.

[34] AHMED M Y, AL-KHAYAT A, AL-MURSHEDI F, AL-FUTAISI A, CHIOZA B A, PEDRO FERNANDEZ-MURRAY J, SELF J E, SALTER C G, HARLALKA G V, RAWLINS L E, et al. A mutation ofunderlies a new disorder of Kennedy pathway phospholipid biosynthesis. Brain, 2017: aww318.

[35] ZEMUNIK T, BOBAN M, LAUC G, JANKOVI? S, ROTIM K, VATAVUK Z, BENCI? G, DOGAS Z, BORASKA V, TORLAK V, SUSAC J, ZOBI? I, RUDAN D A, PULANI? D, MODUN D, MUDNI? I, GUNJACA G, BUDIMIR D, HAYWARD C, VITART V, WRIGHT A F, CAMPBELL H, RUDAN I. Genome-wide association study of biochemical traits in Korcula Island, Croatia. Croatian Medical Journal, 2009, 50(1): 23-33.

[36] LIAUD N, HORLBECK M A, GILBERT L A, GJONI K, WEISSMAN J S, CATE J H D. Cellular response to small molecules that selectively stall protein synthesis by the ribosome. PLoS Genetics, 2019, 15(3): e1008057.

[37] CHEN Q Y, DUAN J D, WU H Z, LI J B, JIANG Y L, TANG H, LI X Y, KANG L. Expression dynamics of gonadotropin-releasing hormone-I and its mutual regulation with luteinizing hormone in chicken ovary and follicles. General and Comparative Endocrinology, 2019, 270: 96-102.

[38] GHANEM K, JOHNSON A L. Follicle dynamics and granulosa cell differentiation in the Turkey hen ovary. Poultry Science, 2018, 97(10): 3755-3761.

[39] TANIMOTO R, SEKII K, MOROHAKU K, LI J Z, PéPIN D, OBATA Y. Blocking estrogen-induced AMH expression is crucial for normal follicle formation. Development, 2021, 148(6): dev197459.

[40] DE MATOS R. Calcium metabolism in birds. Veterinary Clinics of North America: Exotic Animal Practice, 2008, 11(1): 59-82.

[41] DESCALZO E, CAMARERO P R, SáNCHEZ-BARBUDO I S, MARTINEZ-HARO M, ORTIZ-SANTALIESTRA M E, MORENO- OPO R, MATEO R. Integrating active and passive monitoring to assess sublethal effects and mortality from lead poisoning in birds of prey. The Science of the Total Environment, 2021, 750: 142260.

Identification of Molecular Markers Associated with Goose Egg Quality Through Genome-Wide Association Analysis

1Chongqing Academy of Animal Science, Chongqing 402460;2Chongqing Engineering Research Center of Goose Genetic Improvement, Chongqing 402460;3Chongqing Qijiang Animal Disease Control Center, Chongqing 401420

【Objective】 The objective of this study was to screen molecular markers and candidate genes related to goose egg quality traits, to provide a theoretical support for the analysis of the genetic mechanism of egg quality traits and marker-assisted selection. 【Method】 In this study, a batch of healthy Sichuan White Geese (209 individuals) was selected as the research subjects. Five eggs from each goose during the peak egg production period were collected, and then six egg quality traits were measured, including egg weight, egg shape index, eggshell strength, eggshell thickness, eggshell weight, and egg yolk weight. Based on the whole-genome resequencing data (2.896 Tb, 12.44×/individual) of 209 Sichuan White Geese (female geese), a genome-wide association analysis was conducted to identify SNP loci and important candidate genes associated with egg quality traits. The genotype frequencies of the SNP loci were determined using the nucleic acid flight time mass spectrometry method. 【Result】After filtering, a total of 9 279 339 SNPs and 209 individuals were included for further analysis. The GWAS analysis identified 48 SNP loci significantly or suggestively associated with six egg quality traits (thresholds: 5.43×10-9and 1.09×10-7). These loci were annotated to 27 candidate genes related to egg quality traits, including Pregnancy-associated plasma protein A (), Serine/threonine-protein phosphatase 4 regulatory subunit 2 (), Ethanolamine phosphotransferase 1 (), and Glutamate receptor ionotropic, kainate 2 (). Among them, the candidate genewas involved in protein metabolism and promotes the generation of insulin-like growth factor. Five SNPs within the 11 bp range ofwere significantly associated with eggshell thickness. Additionally, six SNPs on thegene were significantly associated with yolk weight.andwere respectively associated with blood calcium homeostasis and cholesterol metabolism in organisms. Functional enrichment analysis revealed that the candidate genes were mainly annotated to “response to growth factor” (GO:0070848), “intracellular chemical homeostasis” (GO:0055082), “response to hormone” (GO:0009725), and “regulation of monoatomic ion transport” (GO:43269). 【Conclusion】 The GWAS analysis showed that theandare potential functional genes associated with various egg quality traits, such as egg weight, egg yolk weight, and shell strength, providing theoretical references for molecular genetic marker-assisted selection of goose egg quality traits.

genome-wide association analysis; molecular marker-assisted selection; goose; egg quality traits

10.3864/j.issn.0578-1752.2023.19.015

2022-05-10；

2023-08-08

重慶市科研機構(gòu)績效激勵引導(dǎo)專項項目（cstc2022jxjl80007）、重慶市科研院所績效激勵引導(dǎo)專項項目（22527J）、財政部和農(nóng)業(yè)部：國家現(xiàn)代農(nóng)業(yè)產(chǎn)業(yè)技術(shù)體系（CARS-42-51）、重慶市技術(shù)創(chuàng)新與應(yīng)用發(fā)展專項重點項目（cstc2019jscx-gksbX0097）

高廣亮，E-mail：guanglianggaocq@hotmail.com。張克山，E-mail：zhangkshLK1988@163.com。高廣亮和張克山為同等貢獻(xiàn)作者。通信00作者王啟貴，E-mail：wangqigui@hotmail.com

（責(zé)任編輯 林鑒非）