基于全基因组重测序的山羊产羔数性状关键调控基因的筛选

doi:10.3864/j.issn.0578-1752.2022.23.015

摘要/Abstract

摘要： 目的对产羔数不同的山羊进行全基因组重测序分析，挖掘参与调控川中黑山羊产羔数性状关键调控基因，为解析山羊产羔数性状遗传机制及分子遗传改良提供理论依据。方法选择6只产4—6羔的川中黑山羊为高繁组（high fecundity, HF）和6只产单羔的川中黑山羊为低繁组（low fecundity, LF），采集颈静脉血液样本提取基因组DNA，构建350 bp双末端测序文库，利用Illumina HiSeq PE150平台对12个文库进行全基因组重测序。测序产出的净数据经BWA软件比对至山羊参考基因组ARS1，所获得的高质量SNPs通过两种全基因组扫描分析方法（Fst、Hp）的综合分析确定候选区域，候选区域的注释基因分别利用g:Profiler和KOBAS在线数据库进行GO分析与KEGG通路分析，以筛选调节川中黑山羊产羔数性状候选基因。为了进一步鉴定调节山羊产羔数目的关键遗传标记，根据基因组重测序变异分析，对繁殖候选基因的同义与非同义SNPs进行定位筛选，后续将12个山羊样本的扩增产物进行Sanger测序以验证重测序结果。结果 12只山羊共获得431.50 Gb 净数据，经变异检测与注释发现，HF组山羊共发现7 771 417个单核苷酸多态性(single nucleotide polymorphism, SNPs)，LF组山羊检测到8 935 907个SNPs，且LF组各类SNPs 均多于HF组。设置同时达到top 5%最大ZFst值和top 5%最小ZHp值的窗口为候选区域，在低杂合性、高遗传分化的区域共注释130个强选择信号，其中HF组、LF组以及共享窗口的注释基因分别为84、59和13个，经GO富集与KEGG通路分析发现，19个候选基因参与川中黑山羊的繁殖、繁殖过程和胚胎发育等调控，包括11个HF组特异性候选基因（ADCY10、DRD1、HS6ST1、IGFBP7、MSX2、NOG、NPHP4、PAPPA、PRLHR、TDRP和XYLT1），5个LF组特异性候选基因（ANXA5、IGF1、EDNRA、FANCL 和TAC1）和3个HF组与LF组共享窗口基因（AKR1B3、HDAC4和OPRM1）。同时，大多数GO分析，如G蛋白偶联受体活性、激素反应和神经肽信号通路等，都包含这19个候选基因。此外，14个HF候选基因有9个显著富集在代谢途径、神经活性配体-受体相互作用、糖胺聚糖-硫酸乙酰肝素/肝素的生物合成、钙离子信号通路、cAMP信号通路和叶酸生物合成等KEGG通路中（P<0.05）。19个繁殖候选基因中共有2个同义突变（MSX2 G771T，ADCY10 A4662G）与2个非同义突变（PRLHR G529A，DRD1 A281T），且仅定位于HF候选基因中。Sanger测序发现，MSX2、PRLHR和DRD1突变位点均可检测到多态性，与基因组重测序结果一致，其中PRLHR G529A多态性导致第177位丙氨酸突变为苏氨酸，DRD1 A281T多态性导致翻译提前终止。结论本研究共发现11个HF组特异性候选基因，推测是川中黑山羊多羔性状的关键调控基因，PRLHR外显子G529A与DRD1外显子A281T突变可能是调控山羊多羔性状的关键遗传标记，在改良山羊繁殖性能方面具有较大的应用价值。

关键词: 川中黑山羊, 基因组重测序, 多羔性状, 候选基因

Abstract:

【Objective】 The purpose of this study was to analyze the genome of different fecundity populations of goats (Capra hircus) and to explore the key regulatory genes involved in the regulation of litter size traits of Chuanzhong black goats (CBGs), and to provide the theoretical reference for analyzing the genetic mechanism of litter size traits and molecular genetic improvement of fecundity in goats. 【Method】 The high fecundity (HF) CBG does (n = 6) that produced 4-6 kids per doe kidding and low fecundity (LF) does (n = 6) that produced only one kid per doe kidding were chosen in this study. The jugular blood samples were collected to extract genomic DNA. The 350 bp double-terminal sequencing library was constructed, and then 12 whole genome libraries were resequenced by IlluminaHiSeqPE150 platform. The clean data from sequencing were mapped to goat reference genome ARS1 by using BWA software, and two whole-genome scanning analysis methods (Fst and Hp) were used to comprehensively analyze the high-quality SNPs obtained to identify candidate regions. GO analysis and KEGG pathway analysis were performed on the G:Profiler and KOBAS online databases, respectively, to screen candidate genes for regulating the number of kids in CBGs. To further identify the key genetic markers that regulate the number of kids, the synonymous and non-synonymous single nucleotide polymorphisms (SNPs) of reproductive candidate genes were mapped and screened according to the variation analysis report of genome resequencing. The amplified products of 12 goat samples were sequenced by Sanger sequencing to verify the resequencing results.【Result】 A total of 431.50 Gb clean data were obtained from the genome resequencing study of 12 CBGs. Through mutation detection and annotation, 7 771 417 SNPs were detected in HF group and 8 935 907 SNPs were detected in LF group, and all types of the LF group SNPs were more than those in HF group. The windows that reach the maximum ZFst value of top 5% and the minimum ZHp value of top 5% were set as candidate regions. A total of 130 strong selection signals were annotated in the regions with low heterozygosity and high genetic differentiation, of which 84, 59 and 13 genes were annotated in HF group, LF group and shared window, respectively. GO enrichment analysis and KEGG pathway showed that 19 candidate genes were involved in the regulation of reproduction, reproduction and embryonic development of CBG, including 11 HF group-specific candidate genes (ADCY10, DRD1, HS6ST1, IGFBP7, MSX2, NOG, NPHP4, PAPPA, PRLHR, TDRP, and XYLT1), and five strong selection signal genes (ANXA5, IGF1, EDNRA, FANCL, and TAC1) in LF group, and three window genes (AKR1B3, HDAC4 and OPRM1) in HF group shared with LF group. The most GO terms, such as G-protein-coupled receptor activity, hormone response and neuropeptide signal pathway, contained these 19 candidate genes. In addition, nine of the 14 HF candidate genes were significantly enriched in metabolic pathway, neuroactive ligand-receptor interaction, glycosaminoglycan-heparan sulfate/heparin biosynthesis, calcium signal pathway, cAMP signal pathway and folate biosynthesis KEGG pathways (P<0.05). Among the 19 reproductive candidate genes, there were two synonymous mutations (MSX2 G771T, ADCY10 A4662G) and two non-synonymous mutations (PRLHR G529, DRD1 A281T), which were only located in the HF candidate genes. The Sanger sequencing showed that polymorphisms of MSX2, PRLHR and DRD1 gene mutations could be detected, and this result was consistent with the results of genome resequencing, in which PRLHR G529A polymorphism led to alanine mutation to threonine, and DRD1 A281T polymorphism led to early termination of translation.【Conclusion】 A total of 11 HF group-specific candidate genes were found in this study, which were speculated to be the key regulatory genes for fecundity trait. The mutations of PRLHR gene exon G529A and DRD1 exon A281T might be the key genetic markers for regulating prolificacy traits in goats, which had great application value in improving reproductive performance of goats.

Key words: Chuanzhong black goat, genome resequencing, fecundity, candidate genes

李恒,字向东,王会,熊燕,吕明杰,刘宇,蒋旭东. 基于全基因组重测序的山羊产羔数性状关键调控基因的筛选[J]. 中国农业科学, 2022, 55(23): 4753-4768.

LI Heng,ZI XiangDong,WANG Hui,XIONG Yan,LÜ MingJie,LIU Yu,JIANG XuDong. Screening of Key Regulatory Genes for Litter Size Trait Based on Whole Genome Re-Sequencing in Goats (Capra hircus)[J]. Scientia Agricultura Sinica, 2022, 55(23): 4753-4768.

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参考文献 83

[1]	DE LIMA L G, DE SOUZA N O B, RIOS R R, DE MELO B A, DOS SANTOS L T A, DE MORAES SILVA K, MURPHY T W, FRAGA A B. Advances in molecular genetic techniques applied to selection for litter size in goats (Capra hircus): A review. Journal of Applied Animal Research, 2020, 48(1): 38-44. doi:10.1080/09712119.2020.1717497. doi: 10.1080/09712119.2020.1717497
[2]	MULSANT P, LECERF F, FABRE S, SCHIBLER L, MONGET P, LANNELUC I, PISSELET C, RIQUET J, MONNIAUX D, CALLEBAUT I, CRIBIU E, THIMONIER J, TEYSSIER J, BODIN L, COGNIÉ Y, CHITOUR N, ELSEN J M. Mutation in bone morphogenetic protein receptor-IB is associated with increased ovulation rate in Booroola Mérino ewes. Proceedings of the National Academy of Sciences of the United States of America, 2001, 98(9): 5104-5109. doi:10.1073/pnas.091577598. doi: 10.1073/pnas.091577598 pmid: 11320249
[3]	GALLOWAY S M, MCNATTY K P, CAMBRIDGE L M, LAITINEN M P E, JUENGEL J L, JOKIRANTA T S, MCLAREN R J, LUIRO K, DODDS K G, MONTGOMERY G W, BEATTIE A E, DAVIS G H, RITVOS O. Mutations in an oocyte-derived growth factor gene (BMP15) cause increased ovulation rate and infertility in a dosage- sensitive manner. Nature Genetics, 2000, 25(3): 279-283. doi:10.1038/77033. doi: 10.1038/77033
[4]	MARTINEZ-ROYO A, JURADO J J, SMULDERS J P, MARTÍ J I, ALABART J L, ROCHE A, FANTOVA E, BODIN L, MULSANT P, SERRANO M, FOLCH J, CALVO J H. A deletion in the bone morphogenetic protein 15 gene causes sterility and increased prolificacy in Rasa Aragonesa sheep. Animal Genetics, 2008, 39(3): 294-297. doi:10.1111/j.1365-2052.2008.01707.x. doi: 10.1111/j.1365-2052.2008.01707.x
[5]	HANRAHAN J P, GREGAN S M, MULSANT P, MULLEN M, DAVIS G H, POWELL R, GALLOWAY S M. Mutations in the genes for oocyte-derived growth factors GDF9 and BMP15 are associated with both increased ovulation rate and sterility in Cambridge and belclare sheep (Ovis aries). Biology of Reproduction, 2004, 70(4): 900-909. doi:10.1095/biolreprod.103.023093. doi: 10.1095/biolreprod.103.023093
[6]	NICOL L, BISHOP S C, PONG-WONG R, BENDIXEN C, HOLM L E, RHIND S M, MCNEILLY A S. Homozygosity for a single base-pair mutation in the oocyte-specific GDF9 gene results in sterility in Thoka sheep. Reproduction (Cambridge, England), 2009, 138(6): 921-933. doi:10.1530/rep-09-0193. doi: 10.1530/rep-09-0193
[7]	AHLAWAT S, SHARMA R, MAITRA A. Screening of indigenous goats for prolificacy associated DNA markers of sheep. Gene, 2013, 517(1): 128-131. doi:10.1016/j.gene.2012.12.015. doi: 10.1016/j.gene.2012.12.015 pmid: 23299026
[8]	TEJANGOOKEH H M, SHAHNEH A Z, ZAMIRI M J, DALIRI M, KOHRAM H, JAVAREMI A N. Study of BMP 15 gene polymorphism in Iranian goats. African Journal of Biotechnology, 2009, 8(13), 2929-2932.
[9]	SUPAKORN C, PRALOMKARN W.Sheep FecB gene polymorphism role in Thai meat goat proliferation rate//Proceedings of 9th World Congress Genetics Applied to Livestock Production, Leipizig Germany, 2010.
[10]	HUA G H, CHEN S L, AI J T, YANG L G. None of polymorphism of ovine fecundity major genes FecB and FecX was tested in goat. Animal Reproduction Science, 2008, 108(3/4): 279-286. doi:10.1016/j.anireprosci.2007.08.013. doi: 10.1016/j.anireprosci.2007.08.013
[11]	HE Y Q, MA X K, LIU X Y, ZHANG C X, LI J. Candidate genes polymorphism and its association to prolificacy in Chinese goats. Journal of Agricultural Science, 2010, 2(1): 88-92. doi:10.5539/jas.v2n1p88. doi: 10.5539/jas.v2n1p88
[12]	李恒, 字向东. 全基因组测序在山羊上的研究进展. 中国畜牧杂志, 2021, 57(10): 29-34. doi:10.19556/j.0258-7033.20200910-01. doi: 10.19556/j.0258-7033.20200910-01
	LI H, ZI X D. Research progress on whole-genome sequencing on goat. Chinese Journal of Animal Science, 2021, 57(10): 29-34. doi:10.19556/j.0258-7033.20200910-01. (in Chinese) doi: 10.19556/j.0258-7033.20200910-01
[13]	LAI F N, ZHAI H L, CHENG M, MA J Y, CHENG S F, GE W, ZHANG G L, WANG J J, ZHANG R Q, WANG X, MIN L J, SONG J Z, SHEN W. Whole-genome scanning for the litter size trait associated genes and SNPs under selection in dairy goat (Capra hircus). Scientific Reports, 2016, 6: 38096. doi:10.1038/srep38096. doi: 10.1038/srep38096
[14]	GUANG-XIN E, ZHAO Y J, HUANG Y F. Selection signatures of litter size in Dazu black goats based on a whole genome sequencing mixed pools strategy. Molecular Biology Reports, 2019, 46(5): 5517-5523. doi:10.1007/s11033-019-04904-6. doi: 10.1007/s11033-019-04904-6
[15]	ISLAM R, LIU X X, GEBRESELASSIE G, ABIED A, MA Q, MA Y H. Genome-wide association analysis reveals the genetic locus for high reproduction trait in Chinese Arbas Cashmere goat. Genes & Genomics, 2020, 42(8): 893-899. doi:10.1007/s13258-020-00937-5. doi: 10.1007/s13258-020-00937-5
[16]	E G X, ZHOU D K, YANG B G, DUAN X H, NA R S, HAN Y G, ZENG Y. Association analysis of sixty-seven single nucleotide polymorphisms with litter size in Dazu Black goats. Animal Genetics, 2020, 51(1): 151-152. doi:10.1111/age.12879. doi: 10.1111/age.12879
[17]	WANG J J, ZHANG T, CHEN Q M, ZHANG R Q, LI L, CHENG S F, SHEN W, LEI C Z. Genomic signatures of selection associated with litter size trait in Jining gray goat. Frontiers in Genetics, 2020, 11: 286. doi:10.3389/fgene.2020.00286. doi: 10.3389/fgene.2020.00286
[18]	WANG K, LIU X F, QI T, HUI Y Q, YAN H L, QU L, LAN X Y, PAN C Y. Whole-genome sequencing to identify candidate genes for litter size and to uncover the variant function in goats (Capra hircus). Genomics, 2021, 113(1): 142-150. doi:10.1016/j.ygeno.2020.11.024. doi: 10.1016/j.ygeno.2020.11.024
[19]	ZI X D, MU X K, LU J Y, MA L, WANG Y. Polymorphisms of growth hormone(GH) and insulin-like growth factor I(IGF-I) genes in prolific Lezhi Black Goat: Possible association with litter size. Journal of Southwest University for Nationalities (Natural Science Edition), 2014, 40(3): 344-349.
[20]	LÜ M J, LI H, ZI X D. Assessment of estrous synchronization protocols and pregnancy specific protein B concentration for the prediction of kidding rate in Lezhi black goats. Small Ruminant Research, 2021, 195: 106299. doi:10.1016/j.smallrumres.2020.106299. doi: 10.1016/j.smallrumres.2020.106299
[21]	YANG C X, ZI X D, WANG Y, YANG D Q, MA L, LU J Y, NIU H R, XIAO X. Cloning and mRNA expression levels of GDF9, BMP15, and BMPR1B genes in prolific and non-prolific goat breeds. Molecular Reproduction and Development, 2012, 79(1): 2. doi:10.1002/mrd.21386. doi: 10.1002/mrd.21386
[22]	ZI X D, LU J Y, MA L. Identification and comparative analysis of the ovarian microRNAs of prolific and non-prolific goats during the follicular phase using high-throughput sequencing. Scientific Reports, 2017, 7: 1921. doi:10.1038/s41598-017-02225-x. doi: 10.1038/s41598-017-02225-x
[23]	ZI X D, LU J Y, ZHOU H, MA L, XIA W, XIONG X R, LAN D L, WU X H. Comparative analysis of ovarian transcriptomes between prolific and non-prolific goat breeds via high-throughput sequencing. Reproduction in Domestic Animals, 2018, 53(2): 344-351. doi:10.1111/rda.13111. doi: 10.1111/rda.13111 pmid: 29134700
[24]	LI H, DURBIN R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics, 2009, 25(14): 1754- 1760. doi:10.1093/bioinformatics/btp324. doi: 10.1093/bioinformatics/btp324 pmid: 19451168
[25]	BICKHART D M, ROSEN B D, KOREN S, SAYRE B L, HASTIE A R, CHAN S, LEE J, LAM E T, LIACHKO I, SULLIVAN S T, BURTON J N, HUSON H J, NYSTROM J C, KELLEY C M, HUTCHISON J L, ZHOU Y, SUN J J, CRISÀ A, PONCE DE LEÓN F A, SCHWARTZ J C, HAMMOND J A, WALDBIESER G C, SCHROEDER S G, LIU G E, DUNHAM M J, SHENDURE J, SONSTEGARD T S, PHILLIPPY A M, VAN TASSELL C P, SMITH T P L. Single-molecule sequencing and chromatin conformation capture enable de novo reference assembly of the domestic goat genome. Nature Genetics, 2017, 49(4): 643-650. doi:10.1038/ng.3802. doi: 10.1038/ng.3802 pmid: 28263316
[26]	LI H, HANDSAKER B, WYSOKER A, FENNELL T, RUAN J, HOMER N, MARTH G, ABECASIS G, DURBIN R. 1000 genome project data processing subgroup. The sequence alignment/ map format and SAMtools. Microbiology Spectrum, 2009, 25(16): 2078-2079. doi:10.1093/bioinformatics/btp352. doi: 10.1093/bioinformatics/btp352
[27]	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. Cell Reports, 2010, 20(9): 1297-1303. doi:10.1101/gr.107524.110. doi: 10.1101/gr.107524.110
[28]	YANG H, WANG K. Genomic variant annotation and prioritization with ANNOVAR and wANNOVAR. Nature Protocols, 2015, 10(10): 1556-1566. doi:10.1038/nprot.2015.105. doi: 10.1038/nprot.2015.105 pmid: 26379229
[29]	RAUDVERE U, KOLBERG L, KUZMIN I, ARAK T, ADLER P, PETERSON H, VILO J. G: Profiler: A web server for functional enrichment analysis and conversions of gene lists (2019 update). Nucleic Acids Research, 2019, 47(W1): W191-W198. doi:10.1093/nar/gkz369. doi: 10.1093/nar/gkz369
[30]	BU D C, LUO H T, HUO P P, WANG Z H, ZHANG S, HE Z H, WU Y, ZHAO L H, LIU J J, GUO J C, FANG S S, CAO W C, YI L, ZHAO Y, KONG L. KOBAS-i: intelligent prioritization and exploratory visualization of biological functions for gene enrichment analysis. Nucleic Acids Research, 2021, 49(W1): W317-W325. doi:10.1093/nar/gkab447. doi: 10.1093/nar/gkab447 pmid: 34086934
[31]	MOKHTARI M S, ASADI FOZI M, GUTIERREZ J P, NOTTER D R. Genetic and phenotypic aspects of early reproductive performance in Raeini Cashmere goats. Tropical Animal Health and Production, 2019, 51(8): 2175-2180. doi:10.1007/s11250-019-01915-0. doi: 10.1007/s11250-019-01915-0 pmid: 31104225
[32]	ĐURIČIĆ D, BENIĆ M, ŽAJA I Ž, VALPOTIĆ H, SAMARDŽIJA M. Influence of season, rainfall and air temperature on the reproductive efficiency in Romanov sheep in Croatia. International Journal of Biometeorology, 2019, 63(6): 817-824. doi:10.1007/s00484-019-01696-z. doi: 10.1007/s00484-019-01696-z pmid: 30790044
[33]	ASTUTI D A, KHOTIJAH L, MAIDIN M S, NUGROHO P. Reproductive profile of etawah crossbred does fed Flushing diet containing different kinds of plant oil and animal fat. Pakistan Journal of Biological Sciences, 2020, 23(5): 650-657. doi:10.3923/pjbs.2020.650.657. doi: 10.3923/pjbs.2020.650.657
[34]	MCCARTHY M I, ABECASIS G R, CARDON L R, GOLDSTEIN D B, LITTLE J, IOANNIDIS J P A, HIRSCHHORN J N. Genome-wide association studies for complex traits: consensus, uncertainty and challenges. Nature Reviews Genetics, 2008, 9(5): 356-369. doi:10.1038/nrg2344. doi: 10.1038/nrg2344 pmid: 18398418
[35]	HONG E P, PARK J W. Sample size and statistical power calculation in genetic association studies. Genomics & Informatics, 2012, 10(2): 117-122. doi:10.5808/gi.2012.10.2.117. doi: 10.5808/gi.2012.10.2.117
[36]	LIU N, CUI W B, CHEN M Y, ZHANG X L, SONG X Y, PAN C Y. A 21-bp indel within the LLGL1 gene is significantly associated with litter size in goat. Animal Biotechnology, 2021, 32(2): 213-218. doi:10.1080/10495398.2019.1677682. doi: 10.1080/10495398.2019.1677682
[37]	JIANG E H, KANG Z H, WANG X Y, LIU Y, LIU X F, WANG Z, LI X C, LAN X Y. Detection of insertions/deletions (InDels) within the goat Runx2 gene and their association with litter size and growth traits. Animal Biotechnology, 2021, 32(2): 169-177. doi:10.1080/10495398.2019.1671858. doi: 10.1080/10495398.2019.1671858
[38]	BALBACH M, FUSHIMI M, HUGGINS D J, STEEGBORN C, MEINKE P T, LEVIN L R, BUCK J. Optimization of lead compounds into on-demand, nonhormonal contraceptives: Leveraging a public- private drug discovery institute collaboration. Biology of Reproduction, 2020, 103(2): 176-182. doi:10.1093/biolre/ioaa052. doi: 10.1093/biolre/ioaa052
[39]	CHEN H, CHAN H C. Amplification of FSH signalling by CFTR and nuclear soluble adenylyl cyclase in the ovary. Clinical and Experimental Pharmacology & Physiology, 2017, 44(Suppl 1): 78-85. doi:10.1111/1440-1681.12756. doi: 10.1111/1440-1681.12756
[40]	JAYARAJAN V, APPUKUTTAN A, ASLAM M, REUSCH P, REGITZ-ZAGROSEK V, LADILOV Y. Regulation of AMPK activity by type 10 adenylyl cyclase: Contribution to the mitochondrial biology, cellular redox and energy homeostasis. Cellular and Molecular Life Sciences, 2019, 76(24): 4945-4959. doi:10.1007/s00018-019-03152-y. doi: 10.1007/s00018-019-03152-y pmid: 31172217
[41]	WANG C, LI S J, LI C, FENG Y P, PENG X L, GONG Y Z. Molecular cloning, expression profile, polymorphism and the genetic effects of the dopamine D1 receptor gene on duck reproductive traits. Molecular Biology Reports, 2012, 39(9): 9239-9246. doi:10.1007/s11033-012-1797-3. doi: 10.1007/s11033-012-1797-3 pmid: 22740132
[42]	LIU Z, YANG N, YAN Y, LI G, LIU A, WU G, SUN C. Genome-wide association analysis of egg production performance in chickens across the whole laying period. BMC Genetics, 2019, 20(1): 67. doi:10.1186/s12863-019-0771-7. doi: 10.1186/s12863-019-0771-7 pmid: 31412760
[43]	BARONCHELLI S, VILLA N, REDAELLI S, LISSONI S, SACCHERI F, PANZERI E, CONCONI D, BENTIVEGNA A, CROSTI F, SALA E, BERTOLA F, MAROZZI A, PEDICINI A, VENTRUTO M, POLICE M A, DALPRÀ L. Investigating the role of X chromosome breakpoints in premature ovarian failure. Molecular Cytogenetics, 2012, 5(1): 32. doi:10.1186/1755-8166-5-32. doi: 10.1186/1755-8166-5-32 pmid: 22794123
[44]	FESTA A, UMANO G R, MIRAGLIA DEL GIUDICE E, GRANDONE A. Genetic evaluation of patients with delayed puberty and congenital hypogonadotropic hypogonadism: Is it worthy of consideration? Frontiers in Endocrinology, 2020, 11: 253. doi:10.3389/fendo.2020.00253. doi: 10.3389/fendo.2020.00253 pmid: 32508745
[45]	LI J, LIU J, CAMPANILE G, PLASTOW G, ZHANG C, WANG Z, CASSANDRO M, GASPARRINI B, SALZANO A, HUA G, LIANG A, YANG L. Novel insights into the genetic basis of buffalo reproductive performance. BMC Genomics, 2018, 19(1): 814. doi:10.1186/s12864-018-5208-6. doi: 10.1186/s12864-018-5208-6 pmid: 30419816
[46]	NALLASAMY S, KAYA OKUR H S, BHURKE A, DAVILA J, LI Q X, YOUNG S L, TAYLOR R N, BAGCHI M K, BAGCHI I C. Msx homeobox genes act downstream of BMP2 to regulate endometrial decidualization in mice and in humans. Endocrinology, 2019, 160(7): 1631-1644. doi:10.1210/en.2019-00131. doi: 10.1210/en.2019-00131 pmid: 31125045
[47]	LIU Z K, WANG R C, HAN B C, YANG Y, PENG J P. A novel role of IGFBP₇ in mouse uterus: regulating uterine receptivity through Th1/Th2 lymphocyte balance and decidualization. PLoS ONE, 2012, 7(9): e45224. doi:10.1371/journal.pone.0045224. doi: 10.1371/journal.pone.0045224
[48]	GERHART J, SCHEINFELD V L, MILITO T, PFAUTZ J, NEELY C, FISHER-VANCE D, SUTTER K, CRAWFORD M, KNUDSEN K, GEORGE-WEINSTEIN M. Myo/Nog cell regulation of bone morphogenetic protein signaling in the blastocyst is essential for normal morphogenesis and striated muscle lineage specification. Developmental Biology, 2011, 359(1): 12-25. doi:10.1016/j.ydbio.2011.08.007. doi: S0012-1606(11)01194-8 pmid: 21884693
[49]	NYEGAARD M, OVERGAARD M T, SU Y Q, HAMILTON A E, KWINTKIEWICZ J, HSIEH M, NAYAK N R, CONTI M, CONOVER C A, GIUDICE L C. Lack of functional pregnancy- associated plasma protein-A (PAPPA) compromises mouse ovarian steroidogenesis and female Fertility1. Biology of Reproduction, 2010, 82(6): 1129-1138. doi:10.1095/biolreprod.109.079517. doi: 10.1095/biolreprod.109.079517
[50]	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. doi:10.1007/s10815-019-01489-8. doi: 10.1007/s10815-019-01489-8
[51]	YU M, WANG J, LIU S, WANG X Q, YAN Q. Novel function of pregnancy-associated plasma protein A: promotes endometrium receptivity by up-regulating N-fucosylation. Scientific Reports, 2017, 7: 5315. doi:10.1038/s41598-017-04735-0. doi: 10.1038/s41598-017-04735-0 pmid: 28706275
[52]	WON J, DE EVSIKOVA C M, SMITH R S, HICKS W L, EDWARDS M M, LONGO-GUESS C, LI T S, NAGGERT J K, NISHINA P M. NPHP₄ is necessary for normal photoreceptor ribbon synapse maintenance and outer segment formation, and for sperm development. Human Molecular Genetics, 2010, 20(3): 482-496. doi:10.1093/hmg/ddq494. doi: 10.1093/hmg/ddq494
[53]	MAO S, WU F, CAO X, HE M, LIU N, WU H, YANG Z, DING Q, WANG X. Tdrp deficiency contributes to low sperm motility and is a potential risk factor for male infertility. American Journal of Translational Research, 2016, 8(1): 177-187. pmid: 27069551
[54]	FARACH M C, TANG J P, DECKER G L, CARSON D D. Heparin/heparan sulfate is involved in attachment and spreading of mouse embryos in vitro. Developmental Biology, 1987, 123(2): 401-410. doi:10.1016/0012-1606(87)90398-8. doi: 10.1016/0012-1606(87)90398-8
[55]	潘阳阳, 王萌, 芮弦, 王立斌, 何翃闳, 王靖雷, 马睿, 徐庚全, 崔燕, 樊江峰, 余四九. IGF-1调控RBM3表达抑制低温应激诱导牦牛卵丘细胞凋亡. 中国农业科学, 2020, 53(11): 2285-2296.
	PAN Y Y, WANG M, RUI X, WANG L B, HE H H, WANG J L, MA R, XU G Q, CUI Y, FAN J F, YU S J. RNA-binding motif protein 3(RBM3) expression is regulated by insulin-like growth factor(IGF-1) for protecting yak(Bos grunniens) cumulus cells from apoptosis during hypothermia stress. Scientia Agricultura Sinica, 2020, 53(11): 2285-2296. (in Chinese)
[56]	THOMAS N, VENKATACHALAPATHY T, ARAVINDAKSHAN T, RAGHAVAN K C. Molecular cloning, SNP detection and association analysis of 5' flanking region of the goat IGF₁ gene with prolificacy. Animal Reproduction Science, 2016, 167: 8-15. doi:10.1016/j.anireprosci.2016.01.016. doi: 10.1016/j.anireprosci.2016.01.016
[57]	CHENG Y Y, LIU S C, WANG G, WEI W Z, HUANG S, YANG R, GENG H W, LI H Y, SONG J, SUN L D, YU H, HAO L L. Porcine IGF₁ synonymous mutation alter gene expression and protein binding affinity with IGF1R. International Journal of Biological Macromolecules, 2018, 116: 23-30. doi:10.1016/j.ijbiomac.2018.05.022. doi: 10.1016/j.ijbiomac.2018.05.022
[58]	INAGAKI H, OTA S, NISHIZAWA H, MIYAMURA H, NAKAHIRA K, SUZUKI M, NISHIYAMA S, KATO T, YANAGIHARA I, KURAHASHI H. Obstetric complication-associated ANXA5 promoter polymorphisms may affect gene expression via DNA secondary structures. Journal of Human Genetics, 2019, 64(5): 459-466. doi:10.1038/s10038-019-0578-4. doi: 10.1038/s10038-019-0578-4
[59]	ARANDA F, UDRY S, PERÉS WINGEYER S, AMSHOFF L C, BOGDANOVA N, WIEACKER P, LATINO J O, MARKOFF A, LARRAÑAGA G. Maternal carriers of the ANXA5 M2 haplotype are exposed to a greater risk for placenta-mediated pregnancy complications. Journal of Assisted Reproduction and Genetics, 2018, 35(5): 921-928. doi:10.1007/s10815-018-1142-4. doi: 10.1007/s10815-018-1142-4 pmid: 29497952
[60]	DRYLLIS G, GIANNOPOULOS A, ZOI C, POULIAKIS A, LOGOTHETIS E, VOULGARELIS M, ZOI K, KOUSKOUNI E, DINOU A, STAVROPOULOS-GIOKAS C, KREATSAS G, KONSTANTOPOULOS K, POLITOU M. Correlation of single nucleotide polymorphisms in the promoter region of the ANXA5 (annexin A5) gene with recurrent miscarriages in women of Greek origin. The Journal of Maternal-Fetal & Neonatal Medicine, 2020, 33(9): 1538-1543. doi:10.1080/14767058.2018.1521799. doi: 10.1080/14767058.2018.1521799
[61]	DI GERLANDO R, MASTRANGELO S, MOSCARELLI A, TOLONE M, SUTERA A M, PORTOLANO B, SARDINA M T. Genomic structural diversity in local goats: Analysis of copy-number variations. Animals, 2020, 10(6): E1040. doi:10.3390/ani10061040. doi: 10.3390/ani10061040
[62]	HOHOS N M, ELLIOTT E M, GIORNAZI A, SILVA E, RICE J D, SKAZNIK-WIKIEL M E. High-fat diet induces an ovulatory defect associated with dysregulated endothelin-2 in mice. Reproduction (Cambridge, England), 2021, 161(3): 307-317. doi:10.1530/rep-20- 0290. doi: 10.1530/rep-20- 0290
[63]	YANG Y, GUO T, LIU R, KE H, XU W, ZHAO S, QIN Y. FANCL gene mutations in premature ovarian insufficiency. Human Mutation, 2020, 41(5): 1033-1041. doi:10.1002/humu.23997. doi: 10.1002/humu.23997
[64]	YANG Y, ZHAO S, QIN Y. Response to “Should FANCL heterozygous pathogenic variants be considered as potentially causative of primary ovarian insufficiency? ”. Human Mutation, 2020, 41(9): 1700-1701. doi:10.1002/humu.24073. doi: 10.1002/humu.24073
[65]	FERGANI C, NAVARRO V M. Expanding the role of tachykinins in the neuroendocrine control of reproduction. Reproduction (Cambridge, England), 2016, 153(1): R1-R14. doi:10.1530/rep-16-0378. doi: 10.1530/rep-16-0378
[66]	LEÓN S, FERGANI C, TALBI R, SIMAVLI S, MAGUIRE C A, GERUTSHANG A, NAVARRO V M. Characterization of the role of NKA in the control of puberty onset and gonadotropin release in the female mouse. Endocrinology, 2019, 160(10): 2453-2463. doi:10.1210/en.2019-00195. doi: 10.1210/en.2019-00195 pmid: 31504389
[67]	LEÓN S, FERGANI C, TALBI R, MAGUIRE C A, GERUTSHANG A, SEMINARA S B, NAVARRO V M. Tachykinin signaling is required for induction of the preovulatory luteinizing hormone surge and normal luteinizing hormone pulses. Neuroendocrinology, 2021, 111(6): 542-554. doi:10.1159/000509222. doi: 10.1159/000509222
[68]	GUO J Z, TAO H X, LI P F, LI L, ZHONG T, WANG L J, MA J Y, CHEN X Y, SONG T Z, ZHANG H P. Whole-genome sequencing reveals selection signatures associated with important traits in six goat breeds. Scientific Reports, 2018, 8: 10405. doi:10.1038/s41598-018-28719-w. doi: 10.1038/s41598-018-28719-w pmid: 29991772
[69]	PASTEL E, POINTUD J C, LOUBEAU G, DANI C, SLIM K, MARTIN G, VOLAT F, SAHUT-BARNOLA I, VAL P, MARTINEZ A, LEFRANÇOIS-MARTINEZ A M. Aldose reductases influence prostaglandin F2α levels and adipocyte differentiation in male mouse and human species. Endocrinology, 2015, 156(5): 1671-1684. doi:10.1210/en.2014-1750. doi: 10.1210/en.2014-1750 pmid: 25730106
[70]	ZHANG Q, PEI L G, LIU M, LV F, CHEN G H, WANG H.Reduced testicular steroidogenesis in rat offspring by prenatal nicotine exposure: Epigenetic programming and heritability via nAChR/ HDAC4. Food and Chemical Toxicology, 2020, 135: 111057. doi:10.1016/j.fct.2019.111057. doi: 10.1016/j.fct.2019.111057
[71]	OLABARRIETA E, TOTORIKAGUENA L, AGIRREGOITIA N, AGIRREGOITIA E. Implication of mu opioid receptor in the in vitro maturation of oocytes and its effects on subsequent fertilization and embryo development in mice. Molecular Reproduction and Development, 2019, 86(9): 1236-1244. doi:10.1002/mrd.23248. doi: 10.1002/mrd.23248
[72]	OLABARRIETA E, TOTORIKAGUENA L, ROMERO- AGUIRREGOMEZCORTA J, AGIRREGOITIA N, AGIRREGOITIA E. Mu opioid receptor expression and localisation in murine spermatozoa and its role in IVF. Reproduction Fertility and Development, 2020, 32(4): 349-354. doi:10.1071/rd19176. doi: 10.1071/RD19176 pmid: 31718767
[73]	WATSON L N, MOTTERSHEAD D G, DUNNING K R, ROBKER R L, GILCHRIST R B, RUSSELL D L. Heparan sulfate proteoglycans regulate responses to oocyte paracrine signals in ovarian follicle morphogenesis. Endocrinology, 2012, 153(9): 4544-4555. doi:10.1210/en.2012-1181. doi: 10.1210/en.2012-1181 pmid: 22759380
[74]	TIWARI M, PRASAD S, SHRIVASTAV T G, CHAUBE S K. Calcium signaling during meiotic cell cycle regulation and apoptosis in mammalian oocytes. Journal of Cellular Physiology, 2017, 232(5): 976-981. doi:10.1002/jcp.25670. doi: 10.1002/jcp.25670 pmid: 27791263
[75]	STEWART T A, DAVIS F M. An element for development: Calcium signaling in mammalian reproduction and development. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 2019, 1866(7): 1230-1238. doi:10.1016/j.bbamcr.2019.02.016. doi: 10.1016/j.bbamcr.2019.02.016
[76]	LU N S, LI M J, LEI H L, JIANG X Y, TU W L, LU Y, XIA D. Butyric acid regulates progesterone and estradiol secretion via cAMP signaling pathway in porcine granulosa cells. The Journal of Steroid Biochemistry and Molecular Biology, 2017, 172: 89-97. doi:10.1016/j.jsbmb.2017.06.004. doi: 10.1016/j.jsbmb.2017.06.004
[77]	JOZKOWIAK M, HUTCHINGS G, JANKOWSKI M, KULCENTY K, MOZDZIAK P, KEMPISTY B, SPACZYNSKI R Z, PIOTROWSKA- KEMPISTY H. The stemness of human ovarian granulosa cells and the role of resveratrol in the differentiation of MSCs-A review based on cellular and molecular knowledge. Cells, 2020, 9(6): E1418. doi:10.3390/cells9061418. doi: 10.3390/cells9061418
[78]	ZHANG T, CHEN L, HAN K P, ZHANG X Q, ZHANG G X, DAI G J, WANG J Y, XIE K Z. Transcriptome analysis of ovary in relatively greater and lesser egg producing Jinghai Yellow Chicken. Animal Reproduction Science, 2019, 208: 106114. doi:10.1016/j.anireprosci.2019.106114. doi: 10.1016/j.anireprosci.2019.106114
[79]	CHEN X, SUN X, CHIMBAKA I M, QIN N, XU X, LISWANISO S, XU R, GONZALEZ J M. Transcriptome analysis of ovarian follicles reveals potential pivotal genes associated with increased and decreased rates of chicken egg production. Frontiers in Genetics, 2021, 12: 622751. doi:10.3389/fgene.2021.622751. doi: 10.3389/fgene.2021.622751
[80]	XU R Y, PAN L Q, YANG Y Y, ZHOU Y Y. Characterizing transcriptome in female scallop Chlamys farreri provides new insights into the molecular mechanisms of reproductive regulation during ovarian development and spawn. Gene, 2020, 758: 144967. doi:10.1016/j.gene.2020.144967. doi: 10.1016/j.gene.2020.144967
[81]	HUANG D X, ZHANG B, HAN T, LIU G B, CHEN X, ZHAO Z H, FENG J Q, YANG J W, WANG T M. Genome-wide prediction and comparative transcriptomic analysis reveals the G protein-coupled receptors involved in gonadal development of Apostichopus japonicus. Genomics, 2021, 113(1): 967-978. doi:10.1016/j.ygeno.2020.10.030. doi: 10.1016/j.ygeno.2020.10.030
[82]	NADERI N, HOUSE J D. Recent developments in folate nutrition// Advances in Food and Nutrition Research. Amsterdam: Elsevier, 2018: 195-213. doi:10.1016/bs.afnr.2017.12.006. doi: 10.1016/bs.afnr.2017.12.006
[83]	BROWN L L, COHEN B E, EDWARDS E, GUSTIN C E, NOREEN Z. Physiological need for calcium, iron, and folic acid for women of various subpopulations during pregnancy and beyond. Journal of Womens Health (Larchmt), 2021, 30(2): 207-211. doi:10.1089/jwh.2020.8873. doi: 10.1089/jwh.2020.8873

山羊编号 Symbol	胎产羔数（只） Litter size
山羊编号 Symbol	头胎First birth	二胎Second pregnancy
HF1	4	5
HF2	4	4
HF3	5	4
HF4	4	6
HF5	5	4
HF6	4	4
LF1	1	1
LF2	1	1
LF3	1	1
LF4	1	1
LF5	1	1
LF6	1	1

位点Locus	引物序列Primer sequence (5′→3′)	产物大小Product size（bp）	退火温度Tm (℃)
PRLHR（G529A）	F: GGCGTAGGAGGGGTTGGATA	1524	59.8
PRLHR（G529A）	R: CAACCCCAACCAGCCATTT
DRD1（A281T）	F:ACCGCATCCATCCTCAACCT	473	60.0
DRD1（A281T）	R: ATCACCGACAGAGTCTTCAG
MSX2（G770T）	F：GCAAAACCTATGCTGCCCTC	350	60.0
MSX2（G770T）	R：GGCTTGGGTGTCTCCAGTCA
ADCY10（A4662G）	F：CTACCCAAGGCTTTACCATC	242	60.0
ADCY10（A4662G）	R：CCACACCCAGTGAAATCCAA

项目 Category	SNPs数 Number of SNPs
项目 Category	低繁组LF	高繁组HF
基因上游1 kb区域Upstream	50798	44949
使基因获得终止密码子的变异Stop gain	323	300
使基因失去终止密码子的变异Stop loss	39	31
同义变异Synonymous	35520	31260
非同义变异Non-synonymous	24706	21834
变异位于内含子区域Intronic	2473891	2145836
变异位于剪接位点Splicing	237	214
基因下游1 kb区域Downstream	50349	44201
基因上游1 kb区域/基因下游1 kb区域Upstream/downstream	828	720
变异位于基因间区Intergenic	6262768	5450243
变异位于5’ UTR UTR5	16072	14378
变异位于3’ UTR UTR3	20376	17451
转换Transitions	6290461	5466615
颠换Transversions	2645446	2304802
转换与颠换的比率 Transitions/Transversions	2.377	2.371
SNP总个数Total SNP	8935907	7771417

GO条目 GO Term	GO分类号 GO ID	矫正P值 Corrected P-value	基因名 Symbols
繁殖过程 Reproductive process	GO:0022414	2.02563E-05	ADCY10、AKR1B3、DRD1、HDAC4、HS6ST1、IGFBP7、MSX2、NOG、NPHP4、OPRM1、PAPPA、PRLHR、TDRP、ANXA5、EDNRA、FANCL、IGF1、SPINK1、TAC1
繁殖 Reproduction	GO:0000003	2.04405E-05	ADCY10、AKR1B3、DRD1、HDAC4、HS6ST1、IGFBP7、MSX2、NOG、NPHP4、OPRM1、PAPPA、PRLHR、TDRP、ANXA5、EDNRA、FANCL、IGF1、SPINK1、TAC1
胚胎发育Embryo development	GO:0009790	0.003942756	EDNRA、HS6ST1、IGF1、MSX2、NOG、TDGF1、XYLT1
生殖过程调控 Regulation of reproductive process	GO:2000241	0.009505443	HDAC4、MSX2、ANXA5、IGF1、SPINK1、TAC1