数量性状基因定位研究中若干常见问题的分析与解答

QTL作图是基因精细定位、克隆以及有效开展分子育种的基础,在利用QTL作图开展数量性状基因定位研究的过程中经常会碰到一些问题,与统计方法有关的一些问题包括LOD的统计学意义是什么？检测QTL的可信度和LOD临界值的关系是什么？如何评价不同的QTL作图方法？提高QTL检测效率的途径有哪些？与遗传参数估计有关的一些问题包括QTL的贡献率是如何计算出来的？如何确定QTL有利等位基因的来源？选择基因型分析的有效性如何？复合性状是否适宜于QTL作图？与作图群体及遗传图谱有关的一些问题包括QTL作图群体中表型数据是否要求服从正态分布？加密标记是否可以显著提高QTL检测功效？缺失分子标记对QTL作图有什么影响？奇异分离标记对QTL作图有什么影响？文章试图结合笔者多年研究工作对这12个有共性的常见问题做出分析和解答,以供科研工作者参考。     

分子选择

选择是指在一个群体中选择符合需要的基因型，它是育种实践中最重要的环节之一。在传统育种中。选择的依据通常是表现型，从表现型推断基因型。表型选择对寡基因控制的质量性状较为有效，而对多基因控制的数量性状则效率不高。随着现代分子生物学的迅速发展，分子选择技术为实现对基因型的直接选择提供了可能。本文概述了分子选择的原理、方法及应用，讨论了分子选择存在的问题和发展前景。     

西藏三联小穗小麦中三联小穗性状控制基因的SSR分子标记定位

西藏三联小穗小麦是中国西藏地区一种独特的小麦地方品种,拥有特殊的三联小穗性状,超多的小穗数和小花数。分子定位控制三联小穗基因的基因座,发掘与之紧密连锁的分子标记,可为小麦高产育种提供分子标记辅助选择工具。本研究利用西藏三联小穗小麦的衍生系TTSW-5与普通穗型小麦,间3和川麦55,分别构建F2群体,成熟后进行穗部性状的表型分析和SSR基因型鉴定。性状表型遗传分析表明,西藏三联小穗小麦的三联小穗性状由两个独立遗传的隐性基因控制;通过SSR标记鉴定来自TTSW-5/间3组合的F2群体,在2A染色体上检测到1个与三联小穗性状相关的QTL,定位于SSR标记Xgwm275和Xgwm122之间,两标记间的遗传距离为6.6cM,该QTL的LOD值为6.19,可解释的表型变异值为33.1%,初步命名为qTS2A-1。我们推测qTS2A-1可能是控制三联小穗性状相关的主效QTL,SSR标记Xgwm275和Xgwm122可能可用于三联小穗性状的辅助选择。     

数量性状基因作图精度的主要影响因子

采用计算机模拟数据和数量性状基因作图的双侧标记基因型均值回归方法，研究了分子标记密度、性状遗传力和样本容量3因素对F2群体数量性状基因图谱构建的影响。结果表明：（1）在QTL被发现的能力上，适当大(标记间距15cM)的标记密度较有利于QTL的检测，过大或过小均不利；随着遗传力提高和样本容量变大发现QTL的能力提高，但     

基于株平均值的胚乳性状QTL作图的极大似然方法

根据三倍体胚乳性状的数量遗传模型,发展出一种新的专用于胚乳性状数量基因座位（QTL）区间作图的统计方法。该方法以分离群体中各植株的分子标记基因型以及植株上若干粒种子胚乳性状的平均值为数据模式,采用基于平均值混合分布理论的极大似然方法进行QTL分析。QTL效应估计通过EM算法实现。由于该方法利用标记基因型内QTL基因型的混合分布特性,因此,它比同样基于株平均值的最小平方QTL分析方法以及迭代重新加权最小平方QTL分析方法具有更高的统计功效和精确度。方法的可行性和有效性通过计算机模拟数据分析得到了进一步验证。     

水稻细菌性条斑病抗性QTL qB1sr5a的精细定位

分别以高抗细菌性条斑病的品种Acc8558和高感的品种H359为供体和受体亲本,通过回交和分子标记辅助选择,育成了只渗入5号染色体短臂上单个供体亲本细条病抗性QTL（qBlsr5a）的近等基因系H359-BLSR5a。将该近等基因系与H359杂交,建立了一个大的F2群体。采用选择极端表型个体并验证其后代（F2：3）株系的方法,在F2群体中鉴定出目标QTL为抗病纯合基因型的个体。通过对这些个体进行分子标记基因型检测和连锁分析,将qBlsr5a定位在SSR标记RM153和RM159之间,大约2.4cM或290kb的范围内。     

数量性状基因的完备区间作图方法

结合分子标记和表型数据的QTL作图已成为数量性状遗传分析的常规方法。复合区间作图是近10多年来广泛应用的一种QTL定位方法,但它在算法上有一些缺陷,致使QTL效应可能会被侧连标记区间之外的标记变量吸收,同时不同的背景标记选择方法对作图结果的影响较大,并且难以推广到上位型互作QTL的定位。针对这些问题,笔者提出完备区间作图方法。本文介绍了该方法的遗传和统计原理,并通过一个大麦加倍单倍体群体说明其在定位加性QTL和加性×加性互作QTL中的应用。完备区间作图包含两个步骤：首先利用所有标记的信息,通过逐步回归选择重要的标记变量并估计其效应;然后利用逐步回归得到的线性模型校正表型数据,通过一维扫描定位加(显)性效应QTL,通过二维扫描定位上位型互作QTL。这种作图策略简化了复合区间作图中控制背景遗传变异的过程,提高了对QTL的检测功效。     

抗青枯病兼大果和高出仁率的花生新种质创制

青枯病是影响花生产量和品质的重要土传性细菌病害,百果重和出仁率是与花生产量相关的重要性状。本研究利用远杂9102和徐州68-4杂交构建的RIL群体,在B02染色体上定位到青枯病抗性主效QTL qBWRB02。结合前期对百果重和出仁率QTL的定位结果发现,所涉及的3个性状的主效QTL分布在不同的染色体上。以RIL群体基因型数据和多个环境的青枯病抗性、百果重和出仁率表型数据为基础,利用与主效QTL紧密连锁分子标记筛选出6份聚合抗青枯病、荚果大、出仁率高3种优良性状的新种质,可以作为育种中间材料或亲本培育高产抗病新品种。本研究利用分子标记辅助选择和表型鉴定相结合有效筛选抗病高产种质,为未来花生育种提供了新思路。     

分子标记技术在甜菜育种中的应用

分子标记技术与常规育种技术相互紧密结合能显著提高育种效率。为了更好地阐明分子标记在甜菜育种中的作用,总结了国内外分子标记在甜菜亲缘关系及遗传多样性研究、遗传连锁图谱构建、数量性状基因定位（QTL）、分子标记辅助选择育种、杂种优势及种质鉴定中的研究现状和存在的问题。指出建立相应的高效分子标记辅助选育体系,创造出高产、优质、多抗或具广谱抗性的甜菜种质或品种是甜菜分子育种的研究方向。当前甜菜种质资源鉴定的关键任务是大力开发新型的分子标记进行甜菜种质资源遗传分析,绘制指纹图谱、进一步构建甜菜种质分子身份证。今后应加强对甜菜重要农艺性状基因进行精细定位,充分发掘QTL的信息,构建更为饱和的分子标记连锁图谱。     

利用重组自交系定位水稻种子耐低温发芽QTL

具有强耐低温发芽直播稻的种子可以克服低温胁迫导致的发育迟缓,保证幼苗旺盛生长。水稻种子耐低温发芽是多基因控制的复杂数量性状,遗传力低,导致育种家对该性状直接选择的效率很低。耐低温发芽QTL定位研究,有助于开展分子标记辅助选择,提高选择效率。本研究发现,江苏省优质粳稻品种‘南粳46’与云南地方品种‘扎西玛’低温发芽率存在极显著差异。利用‘扎西玛’/‘南粳46’RIL群体定位了3个控制低温发芽的QTL (q LTG-2, qLTG-4和qLTG-7),分别位于第2、4和7染色体上。3个QTL分别可以解释7.69%、8.75%和22.93%表型变异。其中,qLTG-2所在的染色体区域未见报道QTL,是新的QTL位点。该结果为耐低温发芽分子标记辅助选择育种提供了材料基础和分子标记。     

Present and future of quantitative trait locus analysis in plant breeding

The joint analysis of genotype marker segregation and phenotypic values of individuals or lines enables the detection and location of loci affecting quantitative traits (QTL). The availability of DNA markers and powerful biometric methods has led to considerable progress in QTL mapping in plants. The most obvious applications of QTL analysis seem to be marker‐assisted selection (MAS) in breeding and pre‐breeding and QTL cloning. However, other areas are envisaged where QTL analysis can contribute decisively. These are: the understanding of complex traits such as plant‐pathogen interaction; plant genomics, connecting proteins and regulatory elements of known functions to QTL by candidate gene analysis; and germplasm enhancement through a characterization that allows its efficient utilization. The success in all these applications depends primarily on the reliability and accuracy of the QTL analysis itself. Therefore, the discussion of its limitations will constitute an important part of this review.     

Marker-assisted selection of Verticillium wilt resistance in progeny populations of upland cotton derived from mass selection-mass crossing

Two progeny populations of upland cotton derived from mass selection-mass crossing, M₃S₂F₅ and its family lines M₃S₂F_5:6, were generated from 17 hybrid cotton lines derived from regional trials conducted in the Yellow River basin and Yangtze River basin in China. These populations were used to verify 39 reported molecular markers that were related to quantitative trait loci (QTLs) for Verticillium wilt resistance of cotton. Only 12 of 39 markers were polymorphic; 19 had no polymorphisms, and amplification failed for eight markers. The differences in disease grades of aa/AA genotype individuals for five markers, BNL3241, NAU1225, NAU1230, JESPR153, and BNL3031, reached either significant or highly significant levels in at least one population. These markers can thus be effectively used for marker-assisted selection (MAS) of the target trait. Especially for JESPR153 and BNL3031, the differences in disease grades of aa/AA genotype individuals both reached either significant or highly significant levels in the two populations. These two markers should be given preferential consideration when undertaking MAS. The two flanking markers were more effective than the single flanking marker for MAS of single-loci QTL. The selection effect will be greatly enhanced through a reasonable allocation of marker combinations for multi-locus QTL polymerization. When using multi-locus markers for multi-locus QTL-assisted polymerization breeding, the selection effect can be improved progressively by increasing the number of polymerization markers. The possible interaction of different QTLs or genetic backgrounds does not influence the selection effect. A combination of resistant genotypes and disease grade performance enabled final selection of three individuals resistant to Verticillium wilt.     

分子标记辅助选择技术在水稻育种上的应用

分子标记辅助选择技术给水稻育种提供了新的途径,与传统育种技术相结合,可大大提高育种效率,缩短育种周期。因此,加强分子标记辅助选择技术在水稻育种上的应用研究具有重要的实践意义。在此,综述了分子标记的特点与类型及分子标记辅助选择原理,着重介绍了分子标记辅助选择在水稻育种上的利用现状,主要包括在回交育种、基因聚合、数量性状改良等方面的应用进展。同时讨论了该技术存在的问题,并展望了其应用前景。     

An SSR-based molecular genetic map of cassava

Summary Microsatellites or simple sequence repeats (SSR) are the markers of choice for molecular genetic mapping and marker-assisted selection in many crop species. A microsatellite-based linkage map of cassava was drawn using SSR markers and a F₂ population consisting of 268 individuals. The F₂ population was derived from selfing the genotype K150, an early yielding genotype from an F₁ progeny from a cross between two non-inbred elite cassava varieties, TMS 30572 and CM 2177-2 from IITA and CIAT respectively. A set of 472 SSR markers, previously developed from cassava genomic and cDNA libraries, were screened for polymorphism in K150 and its parents TMS 30572 and CM 2177-2. One hundred and twenty two polymorphic SSR markers were identified and utilized for linkage analysis. The map has 100 markers spanning 1236.7 cM, distributed on 22 linkage groups with an average marker distance of 17.92 cM. Marker density across the genome was uniform. This is the first SSR based linkage map of cassava and represents an important step towards quantitative trait loci mapping and genetic analysis of complex traits in M. esculenta species in national research program and other institutes with minimal laboratory facilities. SSR markers reduce the time and cost of mapping quantitative trait loci (QTL) controlling traits of agronomic interest, and are of potential use for marker-assisted selection (MAS).     

Common bean breeding for resistance against biotic and abiotic stresses: From classical to MAS breeding

Summary Breeding for resistance to biotic and abiotic stresses of global importance in common bean is reviewed with emphasis on development and application of marker-assisted selection (MAS). The implementation and adoption of MAS in breeding for disease resistance is advanced compared to the implementation of MAS for insect and abiotic stress resistance. Highlighted examples of breeding in common bean using molecular markers reveal the role and success of MAS in gene pyramiding, rapidly deploying resistance genes via marker-assisted backcrossing, enabling simpler detection and selection of resistance genes in absence of the pathogen, and contributing to simplified breeding of complex traits by detection and indirect selection of quantitative trait loci (QTL) with major effects. The current status of MAS in breeding for resistance to angular leaf spot, anthracnose, Bean common mosaic and Bean common mosaic necrosis viruses, Beet curly top virus, Bean golden yellow mosaic virus, common bacterial blight, halo bacterial blight, rust, root rots, and white mold is reviewed in detail. Cumulative mapping of disease resistance traits has revealed new resistance gene clusters while adding to others, and reinforces the co-location of QTL conditioning resistance with specific resistance genes and defense-related genes. Breeding for resistance to insect pests is updated for bean pod weevil (Apion), bruchid seed weevils, leafhopper, thrips, bean fly, and whitefly, including the use of arcelin proteins as selectable markers for resistance to bruchid seed weevils. Breeding for resistance to abiotic stresses concentrates on drought, low soil phosphorus, and improved symbiotic nitrogen fixation. The combination of root growth and morphology traits, phosphorus uptake mechanisms, root acid exudation, and other traits in alleviating phosphorus deficiency, and identification of numerous QTL of relatively minor effect associated with each trait, reveals the complexity to be addressed in breeding for abiotic stress resistance in common bean.     

An introduction to markers, quantitative trait loci (QTL) mapping and marker-assisted selection for crop improvement: The basic concepts

Recognizing the enormous potential of DNA markers in plant breeding, many agricultural research centers and plant breeding institutes have adopted the capacity for marker development and marker-assisted selection (MAS). However, due to rapid developments in marker technology, statistical methodology for identifying quantitative trait loci (QTLs) and the jargon used by molecular biologists, the utility of DNA markers in plant breeding may not be clearly understood by non-molecular biologists. This review provides an introduction to DNA markers and the concept of polymorphism, linkage analysis and map construction, the principles of QTL analysis and how markers may be applied in breeding programs using MAS. This review has been specifically written for readers who have only a basic knowledge of molecular biology and/or plant genetics. Its format is therefore ideal for conventional plant breeders, physiologists, pathologists, other plant scientists and students.     

Crop breeding for salt tolerance in the era of molecular markers and marker‐assisted selection

Crop salt tolerance (ST) is a complex trait affected by numerous genetic and non‐genetic factors, and its improvement via conventional breeding has been slow. Recent advancements in biotechnology have led to the development of more efficient selection tools to substitute phenotype‐based selection systems. Molecular markers associated with genes or quantitative trait loci (QTLs) affecting important traits are identified, which could be used as indirect selection criteria to improve breeding efficiency via marker‐assisted selection (MAS). While the use of MAS for manipulating simple traits has been streamlined in many plant breeding programmes, MAS for improving complex traits seems to be at infancy stage. Numerous QTLs have been reported for ST in different crop species; however, few commercial cultivars or breeding lines with improved ST have been developed via MAS. We review genes and QTLs identified with positive effects on ST in different plant species and discuss the prospects for developing crop ST via MAS. With the current advances in marker technology and a better handling of genotype by environment interaction effects, the utility of MAS for breeding for ST will gain momentum.     

作物磷效率相关性状的QTL分析研究进展

土壤缺磷是作物生产的主要限制因素之一。利用遗传育种的途径选育磷高效的作物新品种,结合传统的改土施肥方法,是经济、环保地解决土壤缺磷问题的有效措施。数量性状座位(QTL)的定位分析为磷效率的数量性状遗传学研究和磷高效品种的选育提供了有效的手段和途径。本文对近年来利用分子标记对磷效率及其相关性状进行数量性状QTL定位分析的研究成果进行了综述,讨论了作物磷效率遗传改良存在的问题,展望了该领域的发展前景。     

Family-based QTL mapping of heat stress tolerance in primitive tetraploid wheat (Triticum turgidum L.)

Identification of quantitative trait loci (QTL) and markers associated with heat and drought tolerance is warranted for marker-assisted selection in wheat (Triticum aestivum L.) breeding programs in areas prone to these abiotic stresses. Our study used a family-based mapping approach in which 19 families consisting of 384 individuals were developed by three-way crosses involving the heat tolerant, tetraploid cultivated emmer (Triticum turgidum L. var dicoccum) genotype IG45069 and ten heat susceptible tetraploid genotypes, IG44999, IG44961, IG45413, IG83047, IG45441, IG127682, IG45448, IG110572, IG88723 and IG54073, in order to detect QTL and markers associated with heat tolerance. The 384 individuals were phenotyped for physiological traits associated with heat tolerance and genotyped by SSR markers. The QTL associated with heat stress tolerance, as measured by chlorophyll content, flag leaf temperature depression (FLTD) and individual kernel weight (IKW) were mapped on chromosomes 1B (QChlc.tamu-1B), 2B (QFlt.tamu-2B), and 5A (QIkw.tamu-5A), respectively, using linkage analysis. Alleles from IG45069 possessed the highest associations with the phenotypic data for the studied traits. This study demonstrates that a family-based mapping approach can be utilized in rapid detection of QTL associated with heat tolerance in wheat based on linkage and association analyses.