植物SNP数据库及转化CAPS的方法

单核苷酸多态性(SNP)在植物基因组中广泛存在,基于SNP的分子标记也正越来越广泛地应用到植物基因定位、图位克隆及分子标记辅助育种等方面。模式植物拟南芥和水稻的全基因组序列测定已经完成,拟南芥有两种生态型完成了全序列测定,水稻有两个品种完成了全序列测定。许多植物有来自不同品种或不同组织器官或生长发育阶段的大量的EST序列。这些序列是植物SNP开发的重要资源。利用生物信息学手段对全基因组序列或EST序列进行分析已经形成了许多SNP位点数据库,这些数据库的建立为基于SNP的基因功能研究及分子标记开发提供了宝贵的资源。本文对植物SNP位点开发涉及的数据库资源及已经形成的SNP位点数据库进行了总结,并讨论了将SNP位点转化成CAPS或dCAPS标记的方法和相应的工具软件。     

利用基因组简约法开发烟草SNP标记及遗传作图

提出了一种基于基因组简约法开发SNP标记的方法, 即利用特定限制性内切酶酶切降低基因组复杂度, 利用高通量测序平台对酶切位点周围的目标片段进行富集测序, 设计一个生物信息学流程进行序列分析和SNP鉴定。以烤烟DH群体为例, 通过基因组简约法收集烟草基因组代表性片段和高通量测序产生11.4 Gb数据, 经生物信息学分析获得了1015个高质量SNP位点。以SSR标记为骨架, 绘制包括SNP标记在内、标记总数为1307的烤烟遗传连锁图。最后利用该遗传图谱和普通烟草2个祖先种的基因组序列, 分析烟草24个连锁群(染色体)之间的同源关系, 发现了大量染色体之间的重组或交换事件以及部分染色体之间的共线性。     

基于高通量测序开发玉米高效KASP分子标记

SNP (Single Nucleotide Polymorphism)在基因组中数量多、分布广,适用于大规模、自动化基因型检测。本研究利用205份不同来源的玉米自交系全基因组重测序数据鉴定出一系列多态性高的二态性SNP位点并开发出700个KASP分子标记。其中, 202个在46个玉米代表系中得到验证的KASP标记进一步用于系统进化树构建及群体结构分析。结果显示,开发成功的KASP标记在染色体上分布均匀,平均PIC为0.463,平均MAF为0.451。基于KASP标记位点和总SNP位点的聚类分析结果高度吻合。KASP标记位点与总SNP位点的遗传距离相似性系数高达89.5%,能成功区分玉米的杂种优势群。该KASP标记可在玉米种质资源分析、连锁群构建以及杂种优势群划分等方面发挥重要作用。     

亚洲棉EST-SNP的挖掘及其在陆地棉中的验证

【目的】随着不同棉种序列数据库的逐步完善以及高通量测序技术的发展,棉花单核苷酸多态性(Single nucleotide polymorphism,SNP)标记开发可利用的公共数据资源逐步增加。【方法】本研究基于陆地棉祖先基因组的现代种亚洲棉表达序列标签(Expressed sequence tag,EST)数据库,利用CAP3对亚洲棉EST数据库进行拼接。拼接获得7 187个重叠群(Contig),再利用Quality SNP软件进行SNP位点分析。【结果】在807条含有4条以上EST序列的Contig中查找到2 690个SNP位点。通过筛选次要等位基因频率大于30%的位点,获得953个可靠度较高的候选SNP,通过电子筛选,最终获得可用于陆地棉分析的SNP 149个,利用位点特异性聚合酶链式反应以及酶切扩增多态序列验证了EST-SNP的准确性。【结论】本研究证实基于亚洲棉EST数据库挖掘用于陆地棉研究的EST-SNP切实可行,并有望将EST-SNP用于陆地棉遗传图谱构建、重要性状的基因定位以及分子标记辅助育种。     

基于SLAF-seq技术的树莓SNP位点开发

为分析树莓群体的遗传进化关系,开发特异性SNP标记。选用23 个栽培种树莓以用SLAF-seq技术测序,从树莓全基因组范围开发SNP位点,以黑树莓基因组为参考基因组,通过生物信息学分析进行电子酶切预测,筛选特异长度的DNA片断,构建SLAF-seq 文库。通过高通量测序的方式获得海量序列标签,利用软件分析比对,获得多态性SLAF标签,进而开发大量特异性SNP位点。对照水稻的测序数据与参考基因组比对结果,双端比对效率为95.60%,酶切效率为93.52%,说明SLAF建库正常。通过测序共产生59.93 Mb的读长数据,测序质量值Q30 平均为95.07%,所有样品GC含量均值为39.16%,GC含量普遍不高,说明达到测序要求。本研究共获得425402 个SLAF标签,其中多态性的SLAF标签共有121610 个。获得749811 个有效单核苷酸多态(SNP),利用这些SNP分析了23 个树莓种质的群体结构与系统发生树。利用简化基因组测序技术SLAF-seq 可以高效地、低成本地开发出大量的可用于群体遗传结构分析的SNP标记。     

基于转录组数据的三雌蕊小麦中SNP/InDel位点的挖掘

为了进一步定位Pis1基因,加快小麦的分子标记辅助育种工作,获得高质、高产、稳产的小麦品种。以川麦28(CM28)与其三雌蕊近等基因系CM28TP为研究材料,对CM28和CM28TP幼穗的3个阶段(幼穗长度为0.2～0.5 cm, 0.5～0.7 cm, 0.7～1.0 cm)进行转录组测序,然后通过GO数据库对变异位点所在的基因进行分类分析,并选取4个位于Pis1基因附近的SNP标记进行验证。结果表明,CM28TP和CM28幼穗3个阶段共有的SNP/InDel位点为5 310个,其中SNP位点5 024个,InDel位点286个。SNP位点中转换类型(63.33%)多于颠换类型(31.28%),两者的比值达到了2.02;InDel位点中插入类型(152个)多于缺失类型(134个);SNP/InDel位点在A基因组上分布最多、其次是B基因组、D基因组上最少。对SNP/InDel所在的基因进行GO分类注释表明,生物学进程中基因的占比最高,其次是细胞组分和分子功能。SNP/InDel位点对蛋白质功能的影响预测表明,有36个变异位点会严重影响蛋白质功能,中度影响蛋白质功能的位点有1 279个。从Pis1基因的定位区间附近筛选了4个SNP位点进行PCR扩增和测序验证,发现这4个位点与RNA-Seq分析结果一致,这表明挖掘出的SNP/InDel位点是准确的。本研究丰富了小麦中的SNP/InDel标记,为小麦高密度遗传图谱构建、基因定位和分子标记辅助选择育种奠定了基础,同时开发出的4个位于Pis1基因附近的SNP标记,为图位克隆该基因提供了可能。     

基于单拷贝SNP标记的棉花杂交种纯度高通量检测技术

利用有代表性的材料进行SNP位点的全基因组扫描分析与综合评估,基于KASP技术开发1套适用于我国棉花杂交种纯度高通量检测的核心SNP组合。从63K的棉花全基因组芯片中筛选获得具有单拷贝特性的SNP标记分别为5474个(中棉所TM-1基因组版本)和1850个(南京农大TM-1基因组版本)。根据芯片扫描分析结果,权衡考虑位点多态性、分型效果、纯合率与杂合率等因素,最终从每条染色体上优选1个杂交种杂合率高且分型效果理想的核心SNP位点,合计26个。采用KrakenTM软件将SNP位点转化成KASP标记,利用SNPline平台进行SNP分型检测,实现了对大量样品的高通量基因分型,尤其适用于品种纯度快速检测,为SNP标记技术在棉花品种鉴定及指纹数据库构建等方面的应用奠定基础。     

基于名优谷子品种晋谷21全基因组重测序的分子标记开发

小米因其营养丰富日益受到重视, 而小米的品质是民众选择小米时最为关注的指标。晋谷21米质优异, 但由于缺少基因组信息, 严重阻碍了其优异米质形成机制的研究。本研究利用高通量测序技术, 对晋谷21全基因组进行重测序, 获得了14.95 Gb高质量测序数据。进一步将其与豫谷1号参考基因组比较, 发掘了169 037个InDel位点和1 167 555个SNP位点, 其中长度在13~50 bp之间适于琼脂糖凝胶电泳检测的InDel位点为14 578个。选择其中1个SNP位点和68个InDel位点验证, 表明利用二代测序技术开发的InDel和SNP标记真实可靠。基于名优谷子晋谷21重测序数据开发的InDel和SNP分子标记具有通用性, 可用于其他谷子、狗尾草和谷莠子等种质资源的相关研究。同时, 开发了一个晋谷21特异的InDel标记2G5501976, 利用该标记即可快速鉴定待测材料是否为晋谷21及其衍生品种。本研究初步揭示了晋谷21的基因组特征, 不仅为深入解析其优异米质形成的分子机制奠定了基础, 而且为相关分子标记辅助育种、遗传分析和基因克隆提供了分子标记资源。     

基于重测序的芥蓝(Brassica alboglabra)全基因组InDel标记开发

碱基插入/缺失(InDel)在基因组中的分布密度仅次于SNP且易于基因型分型,成为分子标记开发的主要来源。为了开发芥蓝品种间有多态性的分子标记,本研究利用2份芥蓝自交系重测序数据鉴定的InDel位点,在全基因组范围内设计了367对InDel候选标记。通过PCR检测比较8个芥蓝自交系的多态性,发现284对标记在至少2个芥蓝自交系间有多态性,阳性率为77.4%。本研究利用重测序技术开发芥蓝品种间InDel标记效率较高,为种质资源分析、基因定位、分子标记辅助育种等提供了便捷工具。     

基于二代测序数据开发以93-11为亲本的水稻SNP-dCAPS标记的研究实例

作为第一个全基因组序列被测定的籼稻材料,93-11,已成为开展水稻分子遗传、功能基因定位和克隆等研究的最重要亲本之一。公共数据库公布的93-11序列为开发水稻SNP标记奠定了重要基础,但其序列中存在的部分错误,也给相应研究工作带来困扰。本研究通过双重参考日本晴序列和93-11序列,进行候选SNP位点碱基多重比对,建立了一个推测、甄别93-11公共数据库误差序列,开发SNP标记的实践方法。基于本研究建立的方法,针对一个涉及稻瘟病抗性材料P400突变基因所定位区间,结合dCAPS策略,展示了高效开发SNP-dCAPS标记的实例。研究结果表明,本研究建立的方法对利用93-11为亲本,开展水稻功能基因分离等工作,有积极的借鉴意义。     

Molecular markers in Brassica oilseed breeding: current status and future possibilities

As PCR techniques have developed over the last 15 years, a wealth of new DNA marker technologies have arisen which have enabled the generation of high‐density molecular maps for all the major Brassica crop species. Molecular markers have also been heavily used in analyses of genetic diversity in Brassica crops. The majority of the work utilizing molecular markers in Brassica oilseed breeding has to date been based on genetic mapping using various DNA marker systems in segregating populations generated for specific investigations of particular traits of interest. For numerous qualitative traits, traditional mapping approaches have led to the development of marker‐assisted selection strategies in oilseed Brassica breeding, and in some cases to map‐based cloning of the responsible genes. For quantitative traits, however, it has become apparent that traditional mapping of quantitative trait loci (QTL) is often not sufficient to develop effective markers for trait introgression or for identification of the genes responsible. In this case, allele‐trait association studies in non‐structured genetic populations represent an interesting new approach, provided the degree of gametic phase disequilibrium between the QTL and the marker loci is sufficient. Because Brassica species represent the closest crop plant relatives to the model plant Arabidopsis thaliana, significant progress will be achieved in the coming years through integration of candidate gene approaches in crop brassicas, using the detailed information now available for the Arabidopsis genome. Integration of information from the model plant with the increasing supply of data from physical mapping and sequencing of the diploid Brassica genomes will undoubtedly give great insight into the genetics underlying both simple and complex traits in oilseed rape. This review describes the current use of available genetic marker technologies in oilseed rape breeding and provides an outlook for use of new technologies, including single‐nucleotide polymorphism markers, candidate gene approaches and allele‐trait association studies.     

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).     

棉花生殖器官发育的分子生物学研究进展

 棉花生殖器官发育与棉花的经济价值紧密相关,因此一直以来都是研究热点。随着棉花三系育种体系的应用推广,棉纤维发育相关基因的克隆、棉花生殖器官高效表达启动子的克隆和应用,棉花生殖器官发育的分子生物学研究的重要性也日益凸显。为方便相关研究人员了解该领域的最新进展,就国内外在棉花核质互作雄性不育、核不育、棉纤维发育以及棉花生殖器官特异型启动子等方面取得的分子生物学研究进展作了详细介绍,并对以上各方面的研究趋势进行了展望。     

Molecular markers: actual and potential contributions to wheat genome characterization and breeding

The demands for increasing global crop production have prompted the development of new approaches relying on molecular marker technologies to investigate and improve the plant genome. The merits of molecular markers make them valuable tools in a range of research areas. This review describes novel approaches based on modern molecular marker technologies for characterization and utilization of genetic variation for wheat improvement. Large-scale genome characterization by DNA-fingerprinting has revealed no declining trends in the molecular genetic diversity in wheat as a consequence of modern intensive breeding thus opposing the genetic ‘erosion’ hypothesis. A great number of important major genes and quantitative trait loci have been mapped with molecular markers. Marker-assisted selection based on a tight linkage between a marker allele and a gene(s) governing a qualitative or quantitative trait is gaining considerable importance as it facilitates and accelerates cultivar improvement through precise transfer of chromosome regions carrying the gene of interest. The implementations of molecular markers in wheat genotyping, mapping and breeding complemented by specific approaches associated with the complex polyploid nature of common wheat are analyzed and presented.     

构建数量性状基因图谱的统计方法

随着分子生物学研究的迅速发展和人类基因组统计的深入，数量遗传学在数量性状基因（ＱＴＬ）的定位方面正在经历着一场深刻的变化。人类和动、植物基因组计划的早期成果之一，就是建立人类以及实验、家养动物和栽培植物的遗传连锁图、利用这些连锁图，可以对许多ＱＴＬ进行单个分离测定和定位。许多复杂疾病和具有重要农 生物学意义的性状都属于数量性状。在ＱＴＬ定位的研究中，统计分析起着很重要的作用。近年来有许多新的统计方     

大豆数量性状定位的研究进展

大豆的许多重要农艺性状和经济性状是受多基因控制的数量性状。针对大豆产量性状、种子品质性状和重要病害的抗性等，综述了近年来大豆数量性状基因座位(quantitative trait locus, QTL)定位研究的进展,并讨论了目前大豆QTL定位研究存在的问题及解决途径。     

用(培矮64s/Nipponbare)F2群体对水稻产量构成性状的QTL定位分析

用水稻测序品种培矮64s和Nipponbare为亲本构建的含137个SSRs标记的连锁遗传图谱和（培矮64s/Nipponbare）F₂群体的180个单株，对水稻的单株有效穗数、穗粒数、穗实粒数、结实率、穗着粒密度、千粒重等6个产量构成性状进行了QTL定位分析。共检测到6个性状的22个QTLs，分布在第1、2、4、5、6、9、10、11、12等9条染色体的14个区域,表型贡献率5.0%-19.3%;相关性较强的性状之间具有较多共同或紧密连锁的QTLs;集中分布的QTLs之间既有同向连锁.对不同水稻群体定位的同源QTL进行了比较,对QTL在染色体上的集中分布,以及用QTL定位结果和生物信息学方法相结合预测基因的英勇等进行了探讨。     

Research Progress on the Mendelian Genetic Analysis and Molecular Mapping of Morphological Qualitative Traits in Cotton

Genetic analysis and molecular mapping of qualitative traits are important tools in cotton breeding. Research on cotton qualitative traits has been reported since the early 1900s. Rapid advances in cotton genomics and molecular mapping over the past several years have facilitated the mapping and cloning of important qualitative trait genes in cotton. Although most of the 101 genes and alleles related to cotton morphological qualitative traits have not been mapped to linkage groups or chromosomes, several genes have been cloned and identified via map-based cloning. This paper describes research progress on the genetic and linkage analysis, molecular mapping, and candidate gene cloning of eight major types of morphological qualitative traits: plant color, leaf color, leaf shape, bract, flower, nectary, gland, and fiber characteristics. The presented information lays a foundation for studying the molecular basis of qualitative traits, favorable gene-based transfer, and gene pyramiding in cotton.     

作物产量性状QTL定位的研究现状及应用前景

作物的许多农艺性状和经济性状是数量性状。研究作物数量性状遗传对农作物育种具有十分重要的意义。本文综述了数量性状基因座QTL（quantitative trait locus）定位的原理和常用方法及分子标记在水稻、小麦、玉米、棉花、大豆、番茄、大麦和油菜等重要作物产量性状基因定位中的研究现状,并对目前产量性状QTL定位存在的问题和发展前景进行了探讨     

Construction of a high-density genetic map using genotyping by sequencing (GBS) for quantitative trait loci (QTL) analysis of three plant morphological traits in upland cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis> L.)

In recent years, the production costs of cotton (Gossypium hirsutum L.) in China have continued to rise, and this has been accompanied by relatively low productivity, diminished enthusiasm of Chinese farmers for planting cotton, and the difficulty caused by high subsidies as well as the high degree of mechanized harvesting for competing crops like grains. Therefore, it is urgent to improve the level of mechanization and the scale of cotton production in China. Morphological traits play an important role in the mechanized harvesting of cotton. Plant height (PH), height of the first fruiting branch node (HFFBN), and the number of vegetative shoot (NOVS) are key cotton morphological traits that influence mechanical harvesting. The genetic basis of PH, HFFBN, and NOVS were examined in the Z571 and CCRI 49 parents as well as 188 individuals comprising the F₂ mapping population. This F₂ population was examined using genotyping by sequencing (GBS) with 5571 high-density polymorphism single nucleotide polymorphism (SNP) markers to construct a genetic linkage map comprised of 3187 polymorphic markers. The genetic map spanned 3828.551 cM, with an average distance of 0.687 cM between markers. The complete interval mapping method identified 17 quantitative trait loci (QTL) for PH, HFFBN, and NOVS located on chromosomes 3, 4, 5, 7, 9, 17, 19, 23, and 25. Our study provides an efficient approach for fast detection of QTL underlying complex trait variation with high accuracy, thus providing preliminary information that can improve the efficiency of subsequent machine cotton picking through breeding and molecular marker-assisted selection methods.