应用SSR分子标记鉴定超级杂交水稻组合及其纯度

 应用SSR分子标记技术对超级杂交稻5个组合（HYS 1/R105、培矮64S/E32、两优培九、88S/0293、J23A/Q611）及其9个亲本进行了鉴定。用144对SSR引物进行筛选，有47对能够在实验材料中显示较好的多态性，其中，RM337与RM154呈现丰富多态性，可鉴别供试组合并分别与其亲本区分开。对于水稻的每一条染色体，各筛选出两条产生多态性的引物，共24对，并提供一组作为鉴定参考的图谱；通过杂种表现为父母本互补带型的特点，找到在杂交稻组合及其亲本间具有多态性的引物，筛选出5对引物分别作为鉴定上述5个超级杂交稻组合的特异引物，进而针对杂交稻不同的纯度问题设计鉴定方法。     

Identification and Purity Test of Super Hybrid Rice with SSR Molecular Markers

Five super hybrid rice combinations, i.e. HYS-1/R105, Pei‘ai 64S/E32, Liangyoupeijiu (Pei‘ai 64S/9311), 88S/0293, and J23A/Q611, and their parental lines were tested by means of SSR analysis. A total of 144 SSR primer pairs distributed on 12 rice chromosomes were used, out of which 47 detected polymorphism among the tested rice lines. Among all these primers, RM337 and RM154 produced polymorphic patterns in four or more of the tested experimental materials respectively, and they could distinguish among most rice genotypes tested. Twenty-four primer pairs, two on each rice chromosome, were selected to make a reference SSR marker-based fingerprinting for the rice lines. For most of the primer pairs, F1 hybrids mainly showed complementary pattern of both parents, which could be very useful to distinguish the F1 from its parental lines. In addition, 5 primer pairs were selected as special primer pairs for five hybrid rice combinations respectively. By combining the rapid, simple method on DNA extraction, it is suggested that SSR technique has wide prospective in variety authentication and purity identification.     

Genetic Diversity of Tropical Hybrid Rice Germplasm Measured by Molecular Markers

Investigation of genetic diversity and relationships among breeding lines is of great importance to facilitate parent selection in hybrid rice breeding programs.In this study,we characterized 168 hybrid rice parents from International Rice Research Institute with 207 simple sequence repeat (SSR) and 353 single nucleotide polymorphism (SNP) markers.A total of 1 267 SSR and 706 SNP alleles were detected with the averages of 6.1 (SSR) and 2.0 (SNP) alleles per locus respectively across all lines.Based on the genetic distances estimated from the SSR and SNP markers separately and combined,the unrooted neighbor-joining cluster and STRUCTURE analyses consistently separated the 168 hybrid rice parents into two major groups:B-line and R-line,which is consistent with known parent pedigree information.The genetic distance matrices derived from the SSR and SNP genotyping were highly correlated (r=0.81,P < 0.001),indicating that both of the SSR and SNP markers have distinguishable power to detect polymorphism and are appropriate for genetic diversity analysis among tropical hybrid rice parents.A subset of 60 SSR markers were also chosen by the Core Hunter with 368 alleles,and the cluster analysis based on the total and subset of SSR markers highly corresponded at r=0.91 (P < 0.001),suggesting that fewer SSR markers can be used to classify and evaluate genetic diversity among parental lines.     

利用RAPD和微卫星标记对协优杂交稻及其亲本进行区别与鉴定

选用400个随机寡核苷酸和40个荧光标记的微卫星引物对,对我国主栽的协优杂交稻组合及其亲本进行多态性分析。RAPD分析显示,400个随机寡核苷酸引物中,有364个能够在供试基因型材料中扩增出肉眼可见的DNA条带,其中28个显示扩增的多态性,这中间9个引物产生的13条多态性条带具有清晰、易分辨的特点,可以在供试的水稻组合与亲本之间,以及杂交组合之间进行区别和鉴定。微卫星分析指出,40对荧光标记引物中,13对引物产生的31条标记带,能够在供试基因型材料中显示稳定的多态性,且杂种表现为双亲的互补型。与RAPD相比,微卫星分析具有多态性强、精确性高和重复性好的特点。     

我国主要杂交水稻组合及其亲本SSR标记和纯度鉴定

选用分布于水稻12条染色体上的26对SSR引物对我国9个主要的杂交水稻组合及其亲本进行了SSR标记分析,21对多态性引物共扩增出62条条带,平均2.95条,能够有效地区分所有恢复系和大部分不育系。杂交种条带均为父母本的互补型,很适合做杂交种纯度鉴定。用引物RM17对杂交稻组合汕优63和两优培九进行了100粒单种子SSR鉴定,所测纯度分别为96.0%和98.0%,与田间纯度96.2%和97.7%非常接近,显示出SSR技术在品种认证和纯度鉴定中有广阔的前景。     

利用微卫星标记鉴定北方杂交粳稻种子纯度的研究

利用48对SSR引物对7个北方杂交粳稻组合的杂种F1与其父母本间的多态性进行了筛选，每个组合获得4-11对多态性好的特异引物，利用特异性引物可将杂交种与其相应的不育系、保持系和恢复系分开，说明SSR标记可应用于北方杂交梗稻种子纯度鉴定，具有较高的实用价值。     

Development and Application of SCAR Markers for Discriminating Cytoplasmic Male Sterile Lines from Their Cognate Maintainer Lines in Indica Rice

The DNA fragments about 1 600 bp were amplified using random amplified polymorphism DNA (RAPD) primer OPA12 with the templates of mitochondrial DNA of Zhenshan 97A and Zhenshan 97B,and were sequenced.The nucleotide sequences and lengths of the fragments from Zhenshan 97A and Zhenshan 97B showed no difference.The precise length of the fragment was 1 588 bp.Sequence characterized amplification region (SCAR) primers were then developed to discriminate the cytoplasmic male sterile (CMS) lines and their maintainer lines.A specific 1 588 bp fragment could be amplified with SCAR primers,CHI19F2/CHI19R2 and CHI20F3/CHI23R3,in the mitochondrial DNA of Zhenshan 97A,but not Zhenshan 97B.Furthermore,the specific fragment could be also amplified from the total DNA from green leaf tissues of Zhenshan 97A with SCAR primers,but not Zhenshan 97B.With the corresponding primers,the specific fragment could also be amplified from the total DNA of green leaves of other two CMS lines with wild abortive type cytoplasm (CMS-WA),namely Zhenpin A and Tianfeng A,but not in their maintainer lines.Moreover,using total DNA as template,each of the four pairs of SCAR primers could also be used to amplify the 1 588 bp fragment in CMS-ID (Indonesia paddy type) line Ⅱ-32A,but not in II-32B,and the specific fragment was amplified from the DNA of both F1 and F2 seedlings of Shanyou 63.The results of detecting the genetic purity of a man-made mixture of the seeds of Zhenshan 97A using CHI20F3/CHI23R3 were completely consistent with the phenotypes.Taken together,these results indicated that the specific 1 588 bp-fragment amplified by CHI20F3/CHI23R3 was the unique amplification products of CMS mitochondrial DNA,and could be used to distinguish CMS-WA and CMS-ID lines from their corresponding maintainer lines at the seedling stage.     

稻米淀粉理化特性相关的水稻淀粉分支酶基因标记开发

 基于籼稻93 11和粳稻日本晴基因组间的序列差异,成功发展了水稻淀粉分支酶基因(Sbe)9个分子标记。利用这些标记对102份非糯材料进行了基因型检测,并分析了Sbe1、Sbe3基因标记基因型多态性对稻米淀粉理化特性的影响。Sbe1、Sbe3基因标记多态性位点对揭示非糯材料间稻米淀粉理化特性的差异不显著,说明淀粉分支酶基因这些等位性变异位点对非糯稻米的蒸煮食味品质影响不显著;在鉴定籼稻品种时,有6个标记的鉴定准确率达80%以上。还对标记开发的意义和分子标记辅助选择育种进行了探讨。     

SCAR Markers Assisted Selection for a Bentazon Susceptible Lethality Gene (ben) in Rice

In progenies resulting from crosses involving rice cultivar Norin 8m susceptible to bentazon as the donor of ben gene, SCARs tightly linked to ben were utilized for selection of ben. The homozygous and heterozygous genotypes with ben could be identified with the SCARs. The molecular markers offer a powerful tool for indirect selection of ben and can accelerate the introgression of ben into current rice cultivars.     

应用微卫星标记评估我国水稻主栽品种的一致性

为较准确地评估我国水稻主栽品种的一致性,挑选５个微卫星标记,对４１个有2~7个来源的水稻品种进行了微卫星标记检测。结果表明,１８个（４３．９０％）品种存在同一品种名称不同来源间的差异。进一步选择１１个代表性水稻品种或杂交稻组合作较大样本分析（５０个单株）,它们在５个微卫星标记上的不一致率为０．０％～１０．０％,平均为１．６７％,每个微卫星标记的异质率变化幅度为０．３６％～５．４５％。１１个品种中有６个（５４．５５％）在所有位点上完全一致,不一致率通常都不超过５％ 。因此,在构建水稻品种微卫星数据库时,必须慎重选择取样来源。当鉴别特定材料时,则应相应增加标记数。     

利用微卫星DNA标记进行杂交水稻种子纯度鉴定的研究

利用微卫星分子标记，对湖南目前推广的6个杂交稻组合及其亲本之间的多态性进行了研究。在已筛选的178对引物中，有52对能在一个或多个组合中显示稳定的多态性，杂种表现为父母本互补带型。不同组合间多态性的比例具有差异。用表现多态性的两对引物分别对掺假的威优46和金优207进行纯度鉴定，都能有效地将掺入组合中的假种子区分开。该方法具有准确可靠、多态性高、稳定性好、易于操作的特点。     

SCAR 标记对水稻苯达松敏感致死基因的辅助选育

 利用与苯达松敏感致死基因（ben）紧密连锁的SCAR标记，对田间以农林8号m为ben基因供体转育的后代进行了PCR检测。利用该标记不仅能鉴别出转育的材料是否含有ben基因，而且能区别它是含ben基因的纯合体还是杂合体，从而有效地指导田间选育，加速育种进程。     

SCAR Markers Assisted Selection for a Bentazon Susceptible Lethality Gene （ben） in Rice

In progenies resulting from crosses involving rice cultivar Norin 8m susceptible to bentazon as the donor of ben gene, SCARs tightly linked to ben were utilized for selection of ben. The homozygous and heterozygous genotypes with ben could be identified with the SCARs. The molecular markers offer a powerful tool for indirect selection of ben and can accelerate the introgression of ben into current rice cultivars.     

八个抗稻瘟病基因在华南籼型杂交水稻中的分布

【目的】已克隆的稻瘟病抗性基因Pi1、Pik-p、Pik-h、Pi2、Pi9、Piz-t、Pita、Pii对不同稻区的稻瘟病菌表现较广谱的抗性,被广泛应用于水稻抗瘟性育种。为了明确上述抗性基因在华南稻区杂交稻组合中的分布及其组合的抗病有效性,【方法】利用上述8个抗病基因的功能标记,对华南328个杂交稻组合进行了抗瘟基因型分子检测。【结果】抗性基因Pita和Pii分布频率最高,在测试组合中检出率分别为84.76%与67.68%;其次是Pi2与Pik-p,分别为22.87%与13.72%;检出频率较低的是Pi1、Piz-t和Pik-h,分别为5.18%、3.35%与2.13%,检测的品种都不携带抗性基因Pi9。在单个杂交稻组合中,检出的抗瘟基因数量最多是4个。抗病性评价结果表明,杂交稻组合中检出的抗病基因数量越多,其表现为抗病品种的频率就越高;含4个抗病基因的杂交稻组合中,抗病品种所占比率达91.67%。含有不同抗瘟基因的组合表现出不同水平的抗瘟性,其中Pi2与Pi1对华南稻区稻瘟病的抗病性贡献最大,其他抗病基因的贡献大小依次是Pik-h、Pik-p、Pita、Pii与Piz-t。【结论】本研究为华南稻区杂交稻抗病品种基因型的合理布局以及抗瘟基因的应用提供了科学依据。     

鉴定水稻品种的微卫星标记筛选

 选用58个水稻微卫星标记，对目前应用较广的28份杂交水稻亲本材料进行多态性分析，并比较了其中14个标记应用3.0%琼脂糖凝胶电泳和6.0%非变性聚丙烯酰胺凝胶电泳的检测结果。研究表明，所应用的标记可将所有供试水稻材料区分开，不同标记的多态性检测能力差异较大；两种检测方法所得结果高度一致，但非变性聚丙烯酰胺凝胶电泳分辨率较高、带型清晰，能更准确反映水稻材料间的差异。对于一般水稻材料而言，选用12个标记可将不同材料区别开的几率大于99.9%，但要区分遗传相似性较高的材料，则可能需挑选高多态性标记和（或）增加检测的标记数。     

SSR分子标记技术在杂交水稻种子纯度鉴定中的应用

SSR标记由于具有数量丰富、多态性高、遗传上呈共显性、实验操作简单、结果稳定可靠、引物序列易交流等优点，已成为杂交水稻种子纯度鉴定的一种理想分子标记。概述了SSR分子标记技术应用于杂交水稻种子纯度鉴定的原理、技术要点及其研究进展，并就该技术产业化应重点考虑的问题进行了讨论。     

分子标记在杂交粳稻育种上的应用现状及展望

分子标记的应用为杂交粳稻育种提供了新的技术手段,分子标记技术与传统育种技术相结合,可大大提高育种效率。本文综述了分子标记在杂交粳稻种质资源遗传多样性分析及核心种质构建、分子标记辅助选择育种、杂种优势预测以及DNA指纹图谱构建等方面的研究和应用现状,探讨了分子标记在杂交粳稻育种上应用存在的主要问题,并对其应用前景进行了展望。     

利用SSR分子标记快速鉴定杂交水稻种子纯度技术体系的优化

针对目前SSR分子标记技术快速鉴定杂交水稻种子纯度方法商业化应用的限制因素,从DNA提取、PCR反应体系和程序及凝胶电泳等方面进行了技术体系的优化。原方法经优化后,DNA提取时间从每次5h以上减少到2.5min,PCR反应过程从200min缩短至65min,改聚丙烯酰胺凝胶电泳为琼脂糖凝胶电泳,电泳时间由3.0-3.5h缩短为1h,且通过减少dNTP、Taq酶的用量,对琼脂糖凝胶反复利用,大大降低了成本,减少了废弃物的污染,从而建立了准确、简单、快速、成本低廉的快速鉴定杂交稻种子纯度的技术体系。     

利用SSR标记构建湖南省审杂交水稻品种DNA指纹图谱

筛选出48对SSR引物对湖南省审定的77个杂交水稻品种进行了DNA分子指纹图谱构建和品种鉴定,成功建立了品种指纹图谱数据库,可有效进行杂交水稻品种鉴定.     

杂交稻与常规稻对涝渍环境适应能力的比较研究

利用形态和产量性状指标,比较分析了杂交稻和常规稻在分蘖期和孕穗期对淹涝胁迫环境适应能力的差异性。在深水涝渍条件下,杂交稻生长发育普遍快于常规稻,单蔸苗数显著高于常规稻,有效穗相对受害率低于常规稻,因而单蔸产量显著高于常规稻。分蘖期遭受淹涝胁迫杂交稻的忍耐力优于常规稻,淹涝后的活苗率显著高于常规稻;而且,受淹涝后的杂交稻比常规稻能更快地恢复生长,成熟时主要农艺性状表现是成穗率极显著高于常规稻,每穗实粒数和结实率的相对受害率极显著低于常规稻,单蔸产量的受害程度低于常规稻。孕穗期淹涝胁迫后,杂交稻和常规稻之间产量及相关经济性状的差异均未达到显著水平。