紫花苜蓿QTL与全基因组选择研究进展及其应用

四倍体紫花苜蓿是重要的豆科牧草之一,由于其复杂的遗传背景与二倍体作物相比遗传作图与重要性状数量性状位点(quantitative trait locus,QTL)定位研究相对滞后。然而,二倍体苜蓿的相关研究起步较早,已经建立了高密度遗传图谱和物理图谱,这些研究为四倍体苜蓿遗传作图与QTL定位奠定了基础。随着第三代分子标记与测序技术的快速发展,极大地促进了四倍体苜蓿的高密度遗传图谱构建与QTL定位研究,并借助分子标记辅助育种技术对提高苜蓿选育效率,加速育种进程具有重要意义。本文对苜蓿遗传图谱构建与QTL定位研究及发展趋势进行了总结,并对苜蓿关联作图与全基因组选择的研究进展及应用前景加以概述,旨在为读者就相关研究领域有较全面的了解。     

绵羊基因组研究进展

过去几年中,家畜基因组计划取得了巨大进展。已经构建了猪、鸡、牛、绵羊和马的遗传图谱,其遗传标记间距在5-20cM。这些图谱对于家畜中与重要经济性状相关的基因或遗传标记的鉴定非常重要。文章从绵羊的遗传图谱、物理图谱、重要经济性状基因及QTL定位等方面对绵羊基因组的研究进展作了简要阐述。     

Exclusion of the swine leukocyte antigens as candidate region and reduction of the position interval for the Sus scrofa chromosome 7 QTL affecting growth and fatness

Pig chromosome 7 (SSC 7) has been shown to be rich in QTL affecting performance and quality traits. Most studies mapped the QTL close to the swine leukocyte antigens (SLA), which has a large effect on adaptability and natural selection. Previous comparative mapping studies suggested that the 15-cM region limited by markers LRA1 (mapped at 55 cM) and S0102 (mapped at 70 cM) contains hundreds of genes. To decrease the number of candidate genes, we improved the mapping resolution with a genetic chromosome dissection through a backcross recombinant progeny test program between Meishan (MS) and European (EU; i.e., Large White or Landrace) breeds. Three first-generation backcross--(EU x MS) x EU--and two second-generation backcross--([EU x MS] x EU) x EU--sires carrying a recombination in the QTL mapping interval were progeny-tested (i.e., measured for a total of 44 growth, fatness, carcass and meat quality traits). Progeny family size varied from 29 to 119 pigs. Animals were genotyped for markers covering the region of interest. Progeny-test results allowed the QTL interval to be decreased from 15 to 20 cM down to 10 cM, and even less than 6 cM if we assumed that the EU pigs used in this study share only one QTL allele. Except for a putative QTL affecting some carcass composition traits, the SLA is excluded as a candidate region, suggesting that it might be possible to apply a marker-assisted selection strategy for this QTL, while controlling SLA allele diversity. The strong QTL effects remaining in animals with only 12.5% (issued from first-generation backcross boars) and 6.25% (issued from second-generation back-cross boars) Meishan genetic background shows that epistatic interactions are likely to be limited. Finally, the QTL does not have strong effects on meat quality traits.     

奶牛生长性状研究进展与展望

近年来,生长性状在奶牛育种中备受关注,随着对奶牛生长性状研究的日益深入,其与重要经济性状间的相关性逐渐凸显出来,对奶牛的育种工作也变得越来越重要。本文就奶牛生长性状定义、遗传参数、与经济性状的关联、生长性状QTL定位、GWAS分析等方面进行了综述,并明确了生长性状在奶牛未来育种中的地位。     

家畜全基因组关联分析研究进展

全基因组关联分析（genome-wide association study,GWAS）是一种研究经济性状候选基因的分析方法。近年来,随着家畜全基因组测序的完成,大量的单核苷酸多态性（single nucleotide polymorphisms,SNPs）被标识,GWAS也越来越多地应用于家畜重要性状的研究领域中,在动物遗传育种中,通过对家畜基因组进行GWAS分析研究,找到控制家畜主要经济性状的重要SNPs,从而挖掘重要经济性状的候选基因。作者详细综述了GWAS的分析方法及其在重要家畜育种中的研究进展。GWAS分析方法包括基因组控制法（genommic control）、分层分析法（stratification analysis）、主成分分析法（principal components analysis,PAC）和混合线性模型分析法（mixed-linear-model association,MLMA）,通路分析方法包括非核算法（基因功能富集分析（gene set enrichment analysis,GSEA）和分层贝叶斯优取（hierarchical Bayes prioritization,HBP））和核算法。依据不同的目标性状选择合理的分析方法,提高GWAS分析结果的准确性,为进一步利用GWAS分析各种性状的遗传基础提供合理的借鉴。     

Building phenotype networks to improve QTL detection: a comparative analysis of fatty acid and fat traits in pigs

Models in QTL mapping can be improved by considering all potential variables, i.e. we can use remaining traits other than the trait under study as potential predictors. QTL mapping is often conducted by correcting for a few fixed effects or covariates (e.g. sex, age), although many traits with potential causal relationships between them are recorded. In this work, we evaluate by simulation several procedures to identify optimum models in QTL scans: forward selection, undirected dependency graph and QTL-directed dependency graph (QDG). The latter, QDG, performed better in terms of power and false discovery rate and was applied to fatty acid (FA) composition and fat deposition traits in two pig F2 crosses from China and Spain. Compared with the typical QTL mapping, QDG approach revealed several new QTL. To the contrary, several FA QTL on chromosome 4 (e.g. Palmitic, C16:0; Stearic, C18:0) detected by typical mapping vanished after adjusting for phenotypic covariates in QDG mapping. This suggests that the QTL detected in typical mapping could be indirect. When a QTL is supported by both approaches, there is an increased confidence that the QTL have a primary effect on the corresponding trait. An example is a QTL for C16:1 on chromosome 8. In conclusion, mapping QTL based on causal phenotypic networks can increase power and help to make more biologically sound hypothesis on the genetic architecture of complex traits.     

四倍体紫花苜蓿遗传图谱的初步构建

分别以早熟低产和晚熟高产苜蓿单株为父母本,通过人工杂交构建了四倍体紫花苜蓿(Medicago Sativa)F1遗传作图群体,采用单因子变量分析法,以降落式PCR和常规PCR结合的反应程序,建立了适宜于紫花苜蓿的分子标记扩增体系;应用130对SSR引物进行筛选,获得60对引物在父母本间存在多态性而被用于绘制遗传连锁图。采用PAGE电泳分析,对作图群体进行基因型分析。通过TetraploidMap软件对60个SSR标记进行连锁作图分析,有44个标记可以定位在8个连锁群上,占总标记数的33.8%,覆盖遗传距离979 cM,两标记间平均图距为22.25 cM,初步构建了四倍体紫花苜蓿遗传图谱的框架图,还需要进一步添加标记数量增大其饱和度,为重要性状的QTL定位奠定基础。     

小反刍动物基因定位及其应用的回顾与展望

小反刍动物即绵羊和山羊是重要的可再生资源,对21世纪农业及世界经济的可持续发展具有重要意义.绵羊的中等分辨率和山羊的低分辨率连锁图谱已经构建.绵羊和山羊与牛在核型、带型及微卫星标记等方面存在广泛的保守性,这对于比较基因定位是非常有用的.已在绵羊和山羊中发现了一些主要基因和QTL关联,其中遗传印记和性别决定基因属首次在家畜中发现.探讨了基因图谱和遗传标记在标记辅助选择/导入(MAS/MAI)、亲子鉴定及DNA系谱记录等方面的应用.     

Detection and characterization of quantitative trait loci for meat quality traits in pigs.

In an experimental cross between Meishan and Dutch Large White and Landrace lines, 785 F2 animals with carcass information and their parents were typed for molecular markers covering the entire porcine genome. Linkage was studied between these markers and eight meat quality traits. Quantitative trait locus analyses were performed using interval mapping by regression under two genetic models: 1) the line-cross approach, where the founder lines were assumed to be fixed for different QTL alleles and 2) a half-sib model where a unique allele substitution effect was fitted within each of the 38 half-sib families. The line-cross approach included tests for genomic imprinting and sex-specific QTL effects. In total, three genome-wide significant and 26 suggestive QTL were detected. The significant QTL on chromosomes 3, 4, and 13, affecting meat color, were only detected under the half-sib model. Failure of the line-cross approach to detect the meat color QTL suggests that the founder lines have similar allele frequencies for these QTL. This study provides information on new QTL affecting meat quality traits. It also shows the benefit of analyzing experimental data under different genetic and statistical models.     

Genetic and genomic analyses for predicted methane‐related traits in Japanese Black steers

The objectives of this study were to estimate genetic parameters and to perform a genome‐wide association study (GWAS) for predicted methane‐related traits in Japanese Black steers. The methane production and yield traits were predicted using on‐farm measurable traits, such as dry matter intake and average daily gain. A total of 4,578 Japanese Black steers, which were progenies of 362 sires genotyped with imputed 551,995 single nucleotide polymorphisms (SNPs), had phenotypes of predicted methane‐related traits during the total fattening period (52 weeks). For the estimation of genetic parameters, the estimated heritabilities were moderate (ranged from 0.57 to 0.60). In addition, the estimated genetic correlations of methane production traits with most of carcass traits and feed‐efficiency traits were unfavorable, but those of methane yield traits were favorable or low. For the GWAS, no genome‐wide significant SNP was detected, but a total of four quantitative trait locus (QTL) regions that explained more than 5.0% of genetic variance were localized on the genome, and some candidate genes associated with growth and feed‐efficiency traits were located on the regions. Our results suggest that the predicted methane‐related traits are heritable and some QTL regions for the traits are localized on the genome in Japanese Black steers.     

Genetic control of immune responsiveness: a review of its use as a tool for selection for disease resistance

Disease resistance and immune responsiveness have been traits generally ignored by animal breeders. Recent advances in immunology and molecular biology have opened new avenues towards our understanding of genetic control of these traits. The major histocompatibility gene complex (MHC) appears to play a central role in all immune functions and disease resistance. The need to understand the relationship between immune responsiveness, disease resistance and production traits is discussed in this review. Antagonistic relationships might prevent simultaneous improvement of all of these traits by conventional breeding methods. It is suggested that genetic engineering methods may allow the simultaneous improvement of disease resistance and production traits in domestic animals. Genes of the MHC will be especially good candidates for genetic engineering experiments to improve domestic species.     

Effects of quantitative trait loci on chromosomes 1, 2, 4, and 7 on growth, carcass, and meat quality traits in backcross Meishan x Large White pigs

The aim of this work was to estimate whether genetic dissection of QTL on chromosomes 1, 2, 4, and 7, detected in an F2 Meishan x Large White population, can be achieved with a recombinant back-cross progeny test approach. For this purpose, a first generation of backcross (BC1) was produced by using frozen semen of F1 Large White x Meishan boars with Large White females. Four BC1 boars were selected because of their heterozygosity for at least 1 of the 4 regions. The BC1 boars were crossed with Large White sows, and the resulting BC2 offspring were measured for several growth and body composition traits. Contrary to the F2 animals, BC2 animals were also measured for meat quality traits in adductor, gluteus superficialis (GS), longissimus dorsi, and biceps femoris (BF) muscles. Each BC1 boar was tested for a total of 39 traits and for the 4 regions with statistical interval mapping analyses. The QTL effects obtained in BC1 families showed some differences compared with those described in F1 families. However, we confirmed QTL effects for growth in the SW1301-SW2512 markers interval on chromosome 1 and also for body composition in the SW1828-SW2512 markers interval on chromosome 1, in the SW2443-SWR783 markers interval on chromosome 2, and in the SW1369-SW632 markers interval on chromosome 7. In addition, we detected new QTL for growth traits on chromosome 2 and for meat quality traits on chromosomes 1 and 2. Growth of animals from weaning to the end of the test was influenced by the IGF2 gene region on chromosome 2. Concerning meat quality, ultimate pH of adductor, longissimus dorsi, and BF were affected by the interval delimited by UMNP3000 and SW2512 markers on chromosome 1, and a* of GS, L* of BF, and water-holding capacity of GS were affected by QTL located between marker loci SW2443 and SWR783 on chromosome 2. Recombinant progeny testing appeared to be a suitable strategy for the genetic dissection of the QTL investigated.     

畜禽生长发育相关性状的全基因组关联分析研究进展

全基因组关联分析(GWAS)是近年兴起的用于分析复杂性状的重要研究方法。高通量测序技术的成熟发展使得基于全基因组测序技术和基因芯片技术的GWAS解析畜禽复杂性状成为可能,GWAS的运用对畜禽经济性状相关的SNP、QTL和候选功能基因研究起到关键作用。本文主要对GWAS的基本原理和方法、优劣势以及GWAS在畜禽生长发育相关性状中的应用现状进行综述,并对GWAS在今后畜禽育种中的应用前景进行展望,以期为GWAS在畜禽育种中的深入研究提供参考。     

Whole-genome association analyses for lifetime reproductive traits in the pig

Profits for commercial pork producers vary in part because of sow productivity or sow productive life (SPL) and replacement costs. During the last decade, culling rates of sows have increased to more than 50% in the United States. Both SPL and culling rates are influenced by genetic and nongenetic factors. A whole-genome association study was conducted for pig lifetime reproductive traits, including lifetime total number born (LTNB), lifetime number born alive (LNBA), removal parity, and the ratio between lifetime nonproductive days and herd life. The proportion of phenotypic variance explained by markers was 0.15 for LTNB and LNBA, 0.12 for removal parity, and 0.06 for the ratio between lifetime nonproductive days and herd life. Several informative QTL regions (e.g., 14 QTL regions for LTNB) and genes within the regions (e.g., SLC22A18 on SSC2 for LTNB) were associated with lifetime reproductive traits in this study. Genes associated with LTNB and LNBA were similar, reflecting the high genetic correlation (0.99 ± 0.003) between these traits. Functional annotation revealed that many genes at the associated regions are expressed in reproductive tissues. For instance, the SLC22A18 gene on SSC2 associated with LTNB has been shown to be expressed in the placenta of mice. Many of the QTL regions showing associations coincided with previously identified QTL for fat deposition. This reinforces the role of fat regulation for lifetime reproductive traits. Overall, this whole-genome association study provides a list of genomic locations and markers associated with pig lifetime reproductive traits that could be considered for SPL in future studies.     

Identification of quantitative trait loci affecting corpora lutea and number of teats in a Meishan x Duroc F2 resource population

Understanding of the genetic control of female reproductive performance in pigs would offer the opportunity to utilize natural variation and improve selective breeding programs through marker-assisted selection. The Chinese Meishan is one of the most prolific pig breeds known, farrowing 3 to 5 more viable piglets per litter than Western breeds. This difference in prolificacy is attributed to the Meishan's superior prenatal survival. Our study utilized a 3-generation resource population, in which the founder grandparental animals were purebred Meishan and Duroc pigs, in a genome scan for QTL. Grandparent, F1, and F2 animals were genotyped for 180 microsatellite markers. Reproductive traits, including number of corpora lutea (number of animals = 234), number of fetuses per animal (n = 226), number of teats (n = 801), and total number born (n = 288), were recorded for F2 females. Genome-wide significance level thresholds of 1, 5, and 10% were calculated using a permutation approach. We identified 9 QTL for 3 traits at a 10% genome-wise significance level. Parametric interval mapping analysis indicated evidence of a 1% genome-wise significant QTL for corpora lutea on SSC 3. Nonparametric interval mapping for number of teats found 4 significant QTL on chromosomes SSC3 (P < 0.01), SSC7 (P < 0.01), SSC8 (P < 0.01), and SSC12 (P < 0.05). Partial imprinting of a QTL affecting teat number (P < 0.10) was detected on SSC8. Using the likelihood-ratio test for a categorical trait, 2 QTL for pin nipples were detected on SSC2 and SSC16 (P < 0.01). Fine mapping of the QTL regions will be required for their application to introgression programs and gene cloning.     

Quantitative trait loci for behavioural traits in chickens

The detection of quantitative trait loci (QTL) of behavioural traits has mainly been focussed on mouse and rat. With the rapid development of molecular genetics and the statistical tools, QTL mapping for behavioural traits in farm animals is developing. In chicken, a total of 30 QTL involved in pecking-related traits, open-field behaviour, tonic immobility, response to novel objects, and response to a restraint test were detected in different studies. In the search for a useful early predictor for feather pecking (FP) behaviour in adult laying hens, the following was found: FP in young animals is not a predictor for FP in adult animals, however, open-field behaviour in young animals is genetically correlated with FP in adult hens. Before the implementation of FP behaviour or open-field behaviour in breeding programmes, it is essential to know more about the correlation between these behavioural traits and also their relationship with production traits. Nevertheless, with the QTL for behavioural traits and the chicken genome sequence in progress, a better understanding of the underlying genetic mechanisms of behavioural traits will be feasible.     

基于单倍型肉牛屠宰性状全基因组关联分析研究

单倍型标记与数量性状基因座(quantitative trait loci，QTL)之间具有较强的连锁不平衡(linkage disequilibrium，LD)关系，在基因定位和因果突变鉴定方面具有较高的应用价值。为了评估单倍型标记在基因组研究中的作用，本研究在华西牛资源群体中，选取该群体于2008—2021年间屠宰的共计1 478头平均月龄为24个月的个体进行研究，其中公牛1 333头，母牛145头。利用770K高密度芯片数据，基于LD阈值(r²>0.3)及固定单核苷酸多态(single nucleotide polymorphism,SNP)个数(5个连续SNP)两种方法进行单倍型构建，分别采用单位点SNP标记和两种单倍型标记共3种标记，基于GCTA的混合线性模型(mixed linear model,MLM)，开展宰前活重(LW)和屠宰率(DP)等屠宰性状的全基因组关联分析(genome-wide association study，GWAS)，定位影响屠宰性状的显著(P<0.05) SNPs、单倍型块和候选基因，同时比较3种标记的GWAS结果，评估3种标记的优劣。结果显示，3种标记在全基因组范围内共找到16个的显著SNPs及单倍型区域，主要分布于1、5、6、14、16、17和28号染色体上，同时鉴定到FAM184B、PPM1K、LCORL、RIMS2等10个与屠宰性状相关的候选基因，其中，基于SNP标记方法鉴定到的3个候选基因，在利用基于单倍型标记的方法中也鉴定到，且单倍型鉴定到的显著性位点或区域大多位于基因内部。在两种单倍型构建方法中，与基于固定SNP个数构建单倍型进行GWAS相比，基于LD阈值的构建方法鉴定到了更多候选基因。本研究结果表明，以单倍型开展GWAS可以综合考虑SNP位点间连锁关系，能较好地揭示复杂性状的遗传结构。     

标记辅助选择在猪育种中应用效果的研究

本研究针对猪育种中重点考虑的窝产活仔猪数（NBA）、达100 kg体重日增重（ADG）、饲料利用率（FCR）、达100 kg体重的背膘厚（BF）、肌内脂肪含量（IMF） 5个性状,利用连锁平衡（linkage equilibrium,LE）、连锁不平衡（linkage disequilibrium,LD）标记和直接标记（direct marker,DR）3种类型的分子遗传标记,设计了3个规模不同的基础群,母猪数分别为100、200、300头,公猪数都为10头,基础群个体间无亲缘关系,育种群实施闭锁繁育。用Monte Carlo方法模拟了MAS的5个世代选择试验。育种值估计采用标准BLUP（Standard BLUP,SBLUP）模型（此育种值作为对照）、QBLUP模型（使用DR标记）、MBLUP模型（使用LD和LE标记）。结果表明,利用DR标记在各种情况下都比利用LD和LE标记获得的选择效率高;5个性状中,MAS对低遗传力、限性性状NBA的选择效率最高;当性状的QTL方差占遗传方差基本相同时,中等遗传力性状FCR的选择效率比高遗传力性状BF的更高;当性状的遗传力差异不大时,QTL方差占遗传方差比例大的性状FCR的选择效率比QTL方差占遗传方差比例小的性状ADG的更高。当利用QBLUP模型时,MAS对NBA的选择效率最高,ADG的选择效率最低。     

Accounting for heterogeneity of variances to improve the precision of QTL mapping in dairy cattle

The principle of interval mapping for quantitative trait loci (QTL) was originally developed for the analysis of single backcross data but it has been increasingly applied to more complicated experimental designs and data structures. It is important to study whether accounting for the heterogeneity of variance would improve the precision of QTL mapping based on data of multiple populations or families. This study compared homogeneous and heterogeneous maximum likelihood approaches for QTL mapping. The data consisted of 433 sons from six sire families with 69 microsatellite markers distributed over 12 chromosomes. The results of this study indicate that the heterogeneous approach generally produced a smaller residual variance and thus provided a better fit to the data than the homogeneous approach, meaning that the heterogeneous approach offers better precision in estimating both positions and effects of QTL. The results further showed that accounting for the heterogeneity of residual variance led to different statistical inferences from ignoring the heterogeneity of variance in QTL mapping. The heterogeneous approach is useful for QTL mapping based on the joint data of diverse reference populations or heteroscedastic data obtained from crossing animals with different genetic backgrounds.     

蒺藜苜蓿、天蓝苜蓿、金花菜基因组SNP穿梭标记开发

蒺藜苜蓿是继拟南芥和水稻之后又一个进行全基因组测序的植物,利用蒺藜苜蓿的基因组序列,开发出可以在其他豆科植物上应用的分子标记,即穿梭标记,已成为缺乏基因组信息或基因组复杂的豆科植物基因组学及分子遗传学研究的有效手段。天蓝苜蓿和金花菜是我国最重要的两种一年生苜蓿,由于缺乏有效的分子标记,这两种苜蓿在基因组水平上的研究很少。SLAF-seq是近年来开发出的一种简化基因组测序技术,具有高通量、准确性、成本低、周期短的优点,已在众多物种的全基因组SNP标记开发上得到应用。本研究通过SLAF-seq技术对12份蒺藜苜蓿、天蓝苜蓿和金花菜材料进行简化基因组测序,共得到28.04×10⁶个读长的测序数据,276432个高质量的SLAF标签,其中58748个SLAF标签为多态性标签,平均测序深度为17.44。在58748个多态性SLAF标签中,共检测出次要基因型频率(MAF)大于0.05的SNP标记189133个。本研究开发出的SNP标记可用于一年生苜蓿的遗传多样性、遗传图谱构建和重要农艺性状的QTL定位等的研究,其种间穿梭的特性可为苜蓿属种间基因组排列顺序、系统进化关系、比较图谱构建等方面的研究提供帮助。