水稻InDel和SSR标记多态性的比较分析

为评价插入缺失（InDel）标记在水稻分子育种中的应用价值,本研究用日本晴和9311序列筛选得到遍布每条染色体的20对InDel标记和53对SSR标记,分析46份粳稻和47份籼稻的遗传多样性。研究表明这些InDel标记在籼粳亚种间具有很高的多态性,亚种内多态性较低,平均多态性水平显著小于SSR标记,InDel标记具有数量多,扩增产物稳定和易于检测等优点,将InDel标记应用到遗传图谱构建、基因定位分析和标记辅助选择中可以加速研究进程。     

水稻多蘖矮杆突变体htd7(t)的遗传分析与基因定位

分蘖是水稻最重要的农艺性状之一,其决定水稻的最终产量。多蘖矮杆突变体htd7(t)是粳稻品种‘日本晴’经350 Gy 的60Co-γ射线辐射处理后产生的突变体。为了克隆HTD7(t)基因,将htd7(t)与‘9311’配制正反杂交组合进行遗传分析发现,htd7(t)多蘖矮杆性状是受1 对隐性核基因控制。利用SSR分子标记将HTD7(t)初步定位在第11 染色体分子标记RM21 与RM254 之间,遗传距离分别为5.6 cM和3.2 cM。利用已经公布的水稻基因组数据,在该基因附近新发展了13 对InDel 标记,对HTD7(t)进行精细定位。根据定位结果构建覆盖HTD7(t)基因的BAC 重叠群,最终将HTD7(t)定位在InDel11-3 和InDel11-5之间的64.8 kb的物理距离内。     

籼粳稻基因组295个InDel标记的开发

插入/缺失(InDel)分子标记具有使用简单,结果清晰可靠的优点。本研究通过比对粳稻品种日本晴和籼稻品种93-11的基因组序列,在全基因组范围内设计了634对InDel候选标记,通过PCR检测比较2种粳稻(日本晴和台中65)和2种籼稻(93-11和黄华占)的多态性,发现295对标记在2种籼稻间及2种粳稻间均带型一致,而在籼、粳亚种间有多态性,因此这套295对标记可以在涉及籼粳亚种的基因定位和分子育种中应用。     

Molecular mapping of S-c, an F1 pollen sterility gene in cultivated rice

Hybrids between indica and japonica rice varieties usually show partial sterility, and are a major limiting factor in the utilization of heterosis at subspecific level. When studying male-gamete (pollen) abortion, a possibly important cause for sterility, six loci (S-a, S-b, S-c, S-d, S-e and S-f) for F₁ pollen sterility were identified. Here we report genetic and linkage analysis of S-c locus using molecular markers in a cross between Taichung 65, a japonica variety carrying allele S-c ^j, and its isogenic line TISL5, carrying alleleS-c ^j. Our results show that pollen sterility occurring in the hybrids is controlled by one locus. We used 208 RFLP markers, as well as 500 RAPD primers, to survey the polymorphism between Taichung 65 and TISL5. Six RFLP markers located on a small region of chromosome 3, detected different RFLP patterns. Co-segregation analysis of fertility and RFLP patterns with 123 F₂ plants confirmed that the markers RG227, RG391, R1420 were completely linked with the S-c locus. The genetic distances between the markers C730, RG166 and RG369 and the S-c locus were 0.5 cM, 3.4 cM, and 3.4 cM respectively. Distorted F₂ ratios were also observed for these 4 RFLP markers in the cross. This result suggests that the `one locus sporo-gametophytic' model could explain F₁ hybrid pollen sterility in cultivated rice. RG227, the completely linked marker, has been converted to STS marker for marker-assisted selection. This revised version was published online in August 2006 with corrections to the Cover Date.     

Localization of a Dwarfing Mutation in Upland Cotton Using InDel Markers Based on Genome Re-Sequencing Data

[Objective] InDel markers that were developed by comparing whole genome re-sequencing data were used to map the AS98 gene of a dwarf mutant of upland cotton (Gossypium hirsutum L.). [Method] An F2 segregation population was constructed from an upland-cotton dwarf mutant (AS98) and its wild type (LHF10), and InDel markers were developed based on genome re-sequencing data from the two parents. The InDel markers and the F2 generation were then used to map the AS98 gene of the dwarf mutant. [Result] A total of 114 InDel markers located in a previous mapping region were screened and used to genotype 223 F2 individuals. Ultimately, phenotypic data and 20 polymorphic primer pairs were used to construct a genetic linkage map. Molecular marker analysis mapped AS98 within a 6.9 cM distance of the marker InDel-50 on chromosome D12. [Conclusion] This study has demonstrated the feasibility of using InDel markers developed from re-sequencing data for gene localization in isolated populations derived from upland cotton dwarf materials. The resulting gene map provides a basis for future fine mapping and molecular-assisted selection breeding.     

水稻功能性插入缺失标记（InDel）的筛选与应用

开展水稻品种纯度和真实性鉴定对水稻遗传育种和种子生产有着重要的意义。以245个长江中下游主推水稻品种为研究材料,从643个插入缺失（InDel）位点中筛选出124个功能性多态性位点,建立了InDel高效检测体系。进一步利用InDel标记vf0121641804和SSR标记RM7120分析水稻样品的纯度,同时选用覆盖12条染色体的24对InDel引物和48对SSR标准引物分析水稻品种真实性,结果表明,采用InDel标记检测水稻品种纯度和真实性的结果与采用SSR标记检测结果一致,说明水稻功能性InDel标记也可用于水稻品种纯度和品种真实性的鉴定。在此基础上,鉴定了245个水稻品种124个InDel标记的基因型遗传多样性并进行了聚类分析,结果表明,水稻材料间存在明显的群体结构,基本反映了不同品种间的亲缘关系。水稻功能性InDel标记的筛选与应用,不仅为品种纯度和真实性鉴定、遗传多样性分析提供了新方法;而且可用于分子辅助育种,提高强优势组合的预见性。     

构建水稻优良恢复系背景的重叠片段代换系及其效应分析

通过回交程序结合分子标记辅助选择构建了一套以优良籼稻恢复系9311为背景、导入片段来源于粳稻日本晴的代换系群体。该套群体由125个代换系组成,每系含有单一或少量导入染色体片段,导入片段间相互重叠或衔接能覆盖粳稻全基因组。代换系的平均背景回复率为98.4%,导入片段平均长度为20.9 cM,纯合和杂合导入片段分别占水稻基因组的1.4%和0.2%。利用该群体,两年共检测到31个QTL影响水稻穗重、穗长、结实率和秃顶等性状;导入片段QTL对穗重和结实率均起减效作用。该套重叠片段代换系将为重要性状的基因定位、功能鉴定以及籼粳杂交育种研究提供极有价值的遗传材料。     

TRAP molecular markers as a system for saturation of the genetic map of durum wheat

Target region amplification polymorphism (TRAP) is a relatively new PCR-based technique that detects large numbers of loci in a single reaction without extensive pre-PCR processing of samples. The aim of this study was to integrate TRAP markers in an EST-derived SSR linkage map of a RIL mapping population from the cross of the durum wheat cultivars Ciccio and Svevo, for a more general purpose of establishing a high-throughput system for genetic map saturation. Primer combinations producing PCR products with at least 4–5 polymorphic bands were selected and analyzed across the mapping population. The PCR reactions produced a total of 2,881 fragments with an average of 52 peaks per reaction. A total of 142 new TRAP markers were mapped and found to be randomly distributed in the genome. The total length of the map was 2,043.0 cM, with an average chromosome length of 145.9 cM. Homoeologous group one had the highest number of TRAP markers (38 loci) and the longest map length (407.9 cM) for a total of 87 markers, while the homoeologous group five had the lowest TRAP marker number (5 loci) and the shortest map length (232.5 cM). The distribution of markers among the seven homoeologous groups was random. The results indicate that TRAP is highly efficient in genetic mapping, generating a large number of markers scattered across the genome. This closes many existing gaps in marker coverage and may join otherwise separate linkage groups.     

东乡野生稻与日本晴多态性标记的开发

东乡野生稻是分布于全球最北端的一种普通野生稻,与亚洲栽培稻基因组差异较大,目前缺少覆盖其全基因组的分子标记。本文以东乡野生稻和日本晴为材料,通过筛选已有的1017个标记,并利用东乡野生稻基因组重测序信息设计的217个InDel标记,共检测出203个标记在东乡野生稻与日本晴间呈现多态性。这些标记均匀分布于12条染色体,平均间隔1.9 Mb,基本覆盖东乡野生稻全基因组区域。通过对籼粳亚种的检验分析,发现该套多态性分子标记在东乡野生稻与粳稻杂交后代群体基因型分析上具有较高的应用价值。本研究结果为发掘东乡野生稻的有利基因以及分子标记辅助选择育种提供了有力的工具。     

水稻垩白基因Chalk5功能标记的开发与应用

水稻垩白是衡量水稻外观品质优劣的重要指标之一,降低稻米的垩白是培育优质水稻品种的前提。Chalk5是一个能显著降低稻米垩白从而提高稻米外观品质的主效基因。为提高垩白基因Chalk5在育种中的选择效率,在前人克隆的基础上,根据其启动子区域存在的2个功能突变位点,分别设计等位基因特异PCR(ARMS-PCR)引物,最终筛选出2对Chalk5基因的功能标记Chalk12、NChalk12和Chalk22、NChalk22,通过PCR产物测序的方法对这2对标记的检查结果进行了验证,说明Chalk12、NChalk12和Chalk22、NChalk22可以准确鉴定出Chalk5的不同基因型。利用该标记对不同来源的43份籼稻资源、江苏2007-2013年审定的65份粳稻品种和太湖流域179份粳稻地方资源进行了Chalk5基因型检测,筛选到携带chalk5低垩白率等位基因的籼稻资源20份,太湖流域粳稻地方资源仅8份,其余259份粳型资源中均不携带chalk5低垩白率等位基因,这也说明chalk5主要分布在籼型水稻资源中,在粳型资源中分布很少。     

用SSR标记提高水稻分子连锁图谱密度

本课题组利用圭630／台湾粳的DH群体，建立了一个水稻RFLP连锁图。该图谱含175个标记，总长1224．6cM，相邻标记间平均距离为7．0cM。但该图标记分布不够均匀，存在较多空白区，且在第4和第8染色体上分别存在一个断点。本研究试图在原有RFLP图谱的基础上，添加一些SSR标记，以使该图谱标记更加密集和均匀，并消除两个断点。共筛选了361对RM引物，获得了183对多态标记，在12条染色体上共整合了57个RM标记，染色体连锁图总长度为1811．2cM，比原图谱增长了47．9％，相邻标记间平均距离为7．8cM，与原图谱相近。不过，两条染色体上的两个断点仍无法消除，暗示这两个断点区域多态性较低或重组率较高。     

A linkage map for flowering dogwood (<Emphasis Type="Italic">Cornus florida</Emphasis> L.) based on microsatellite markers

A genetic linkage map of flowering dogwood (Cornus florida L.) was constructed using 94 individuals derived from a cross of two F₁ trees designated 97-6 and 97-7, which were originally from a cross between ‘Appalachian Spring’ and ‘Cherokee Brave’. Out of approximately 800 SSR loci examined, 271 were polymorphic between ‘Appalachian Spring’ and ‘Cherokee Brave’, but were monomorphic between 97-6 and 97-7. These 271 segregating markers were used to build a linkage map for flowering dogwood. Eleven linkage groups were obtained with a log-of-odds (LOD) value of 6.0 using JoinMap® 4.0 software, which matches the chromosome number of flowering dogwood haploid genome. This linkage map consisted of 255 SSR loci, spanned a total of 1,175 centimorgans (cM) with an average internal distance of 4.6 cM. Several larger gaps and slight clustering of markers were present on this linkage map. This is the first linkage map of flowering dogwood and will be a fundamental tool for new gene identification and marker-assisted selection in our flowering dogwood breeding program.     

籼稻背景的单片段代换系群体的构建

以国内、外12个水稻品种（包括8个籼稻品种和4个粳稻品种）为供体亲本，利用回交和微卫星标记辅助选择相结合的方法构建了一个以籼稻品种“华粳籼74”为遗传背景的单片段代换系（SSSL）群体。该群体由260个不同的SSSL组成，其代换片段分布在水稻的12条染色体上，长度分布于0.2～109.9 cM，平均长度为19.9 cM；代换片段的总长度为5 166.0 cM，相当于水稻基因组总长度的3.4倍，代换片段在水稻基因组上的覆盖率为83.8%。这些SSSL的代换片段包含了水稻基因组丰富的等位基因变异，将在基因（QTL）的定位、克隆、功能分析及水稻分子育种中具有重要的利用价值。     

水稻亚种间优质米偏籼组合选育研究

在１６个亚种间杂交组合中,有６个偏籼组合的米质达到部优二级标准。选择优质米偏籼组合时,应抓好亲本选择：（１）选用优质米籼型（或偏籼型）不育系作母本;（２）重视双亲的垩白度和透明度的选择,最好两个性状在双亲中都属于部优级标准;（３）选用双亲性状互补,特别是对米质起重要作用的性状,不要选用都是非部优级标准的双产性状。     

用Rim2超级家族分子指纹鉴别杂交水稻及预测杂种优势

选用5对Rim2引物对21份籼稻和20份粳稻材料进行了DNA指纹扩增,共扩增出38条谱带,其中多态性谱带26条,多态性水平为69.64%;粳稻平均多态性水平为56.67%,明显高于籼稻。根据Nei’s遗传距离计算获得的聚类树状图表明,参试21份籼稻材料聚为6组,20份粳稻材料聚为7组。亲缘关系分析表明,Rim2分子指纹可以区分多数的籼稻或粳稻材料,且显示出材料的遗传特征。考察部分籼稻和粳稻杂交组合的产量对照优势与双亲间遗传距离的关系,发现杂交粳稻的遗传距离与杂交优势表现出相关性,而杂交籼稻无此相关性。     

甬优籼粳杂交稻花后干物质积累模型与特征分析

以甬优籼粳杂交稻甬优1538和甬优7176为试材,常规粳稻宁粳3号和武运粳24,杂交籼稻扬两优6号和两优培九为对照,研究甬优籼粳杂交稻花后干物质积累特征及比较不同类型品种花后干物质积累特征差异。结果表明: (1)两年中甬优籼粳杂交稻的平均产量为11.5 t hm^-2 (11.3~11.7 t hm^-2),较常规粳稻和杂交籼稻分别高7.8%和10.4% (两年平均值)。甬优籼粳杂交稻抽穗至成熟期的干物质积累量为8.9 t hm^-2,较常规粳稻和杂交籼稻分别高19.1%和26.9%(两年平均值)。(2)不同类型品种花后干物质积累量与花后天数(抽穗当天为0 d)均可用Richards方程拟合(R²均大于0.990);各品种花后干物重积累速率均呈先平缓增加后下降的趋势,花后最大干物重积累速率和平均干物重积累速率呈杂交籼稻>常规粳稻>籼粳杂交稻,籼粳杂交稻达最大干物重积累速率的时间大致在花后42~44 d,常规粳稻和杂交籼稻则在花后26~28 d;籼粳杂交稻在花后渐增期天数和干物质积累量显著高于常规粳稻和杂交籼稻,渐增期干物重积累速率以杂交籼稻最高。常规粳稻在花后快增期和花后缓增期天数和干物质积累量均显著高于籼粳杂交稻和杂交籼稻,快增期和缓增期干物重积累速率则以杂交籼稻最高。本研究表明甬优籼粳杂交稻花后较强的干物质积累优势主要体现在花后渐增期,而花后渐增期较强的干物质积累能力主要在于其较长的渐增期持续天数。     

Development and Application of Cotton InDel Markers Based on High Throughput Sequencing

[Objective] Dimorphic InDel markers can be used for cotton variety identification and purity detection, to improve the accuracy and efficiency of cotton seed testing, and to play a role in molecular breeding of cotton. [Method] Based on the whole genome sequencing of 121 cotton varieties from different sources, the InDel markers with high polymorphism were developed according to polymorphism information content(PIC) and were applied in the genetic distance analysis and cluster analysis by using 66 cotton varieties in China. [Result] Totally 10 967 InDel were identified based on the next generation sequencing data of 121 cotton varieties. Among the 85 pairs of InDel primers synthesized, 64 were selected including 35 from At group and 29 from Dt group. The minimum average allelic frequency(MAF) of At and Dt chromosomes were 0.45 and 0.32, respectively, while the PIC were 0.49 and 0.40, respectively. The genetic distances of the 66 cotton varieties ranged from 0.04 to 0.65 centimorgan (cM), with an average of 0.39 cM. The two varieties with the largest genetic distance were Simian 3 and CCRI 36, and the two varieties with the smallest genetic distance were Xumian 18 and Xuza 3. [Conclusion] The 64 cotton dimorphic InDel markers can effectively reveal the relationships among varieties based on the genetic distance, and distinguish cotton varieties from different sources, which has certain theoretical significance and application value.     

Construction of an integrated genetic map based on maternal and paternal lineages of tea (Camellia sinensis)

Genetic study on important traits of tea is difficult because of its self-incompatibility in nature. Moreover, development of a new variety usually needs more than 20 years, since it takes many years from seedling to matured plants for trait investigation. Genetic map is an essential tool for genetic study and breeding. In this study, we have developed an integrated genetic map of tea (Camellia sinensis) using a segregating F1 population derived from a cross between two commercial cultivars (‘TTES 19’ and ‘TTES 8’). A total of 574 polymorphic markers (including SSR, CAPS, STS, AFLP, ISSR and RAPD), 69 markers with highly significant levels of segregation distortion (P < 0.001) (12.0 %) were excluded from further analyses. Of the 505 mapped markers, there were 265 paternal markers (52.5 %), 163 maternal markers (32.3 %), 65 doubly heterozygous dominant markers (12.9 %), and 12 co-dominant markers (2.4 %). The co-dominant markers and doubly heterozygous dominant markers were used as bridge loci for the integration of the paternal and maternal maps. The integrated map comprised 367 linked markers, including 36 SSR, 3 CAPS, 1 STS, 250 AFLP, 13 ISSR and 64 RAPD that were assigned to 18 linkage groups. The linkage groups represented a total map length of 4482.9 cM with a map density of 12.2 cM. This genetic map has the highest genetic coverage so far, which could be applied to comparative mapping, QTL mapping and marker assisted selection in the future.     

Construction of a high-density SSR marker-based linkage map of zoysiagrass (<Emphasis Type="Italic">Zoysia japonica</Emphasis> Steud.)

A consensus genetic linkage map with 447 SSR markers was constructed for zoysiagrass (Zoysia japonica Steud.), using 86 F₁ individuals from the cross ‘Muroran 2’ × ‘Tawarayama Kita 1’. The consensus map identified 22 linkage groups and had a total length of 2,009.9 cM, with an average map density of 4.8 cM. When compared with a previous AFLP-SSR linkage map, the SSR markers from each linkage group mapped to similar positions in both maps. Eight pairs of linkage groups from the AFLP-SSR map were joined into eight new groups in the current map. This zoysiagrass consensus map contained 35 SSR markers exhibiting high homology with rice genomic sequences from known chromosomal locations. This allowed synteny to be identified between Zoysiagrass linkage groups 2, 3, 9, 19 and rice chromosomes 3, 12, 2, 7 respectively. These results provide important comparative genomics information and the new map is now available for quantitative trait locus analysis, marker-assisted selection and breeding for important traits in zoysiagrass.     

Genetic analysis and fine mapping of a semi-dwarf gene in a centromeric region in rice (Oryza sativa L.)

Superior plant architecture is a key means of enhancing yield potential in high yielding varieties. A newly identified recessive gene, named sd-c, controls plant height and tiller number. Genetic analysis of an F₂ population from a cross between the semi-dwarf mutant and japonica cv. Houshengheng showed that the sd-c locus was flanked by SSR markers RM27877 and RM277 on chromosome 12. Thirty nine InDel markers were developed in the region and the sd-c gene was further mapped to a 1 cM centromeric region between InDel markers C11 and C12. These sequenced markers can be used to distinguish wild type and mutants and thus can be used in marker-assisted selection. The sd-c mutant decreases culm length by about 26% and doubles the tiller number without changing seed weight. Until now only sd-1 has been used in indica rice breeding programs. The sd-c mutant seems to have no undesirable pleiotropic effects and is therefore a potential genetic resource for breeding semi-dwarf indica rice cultivars.