MITE类转座子mPing在水稻不同亚种间的差异分析

mPing是水稻中第一个被鉴定出的有活性的MITE类转座子,为了探索mPing在水稻粳稻品种日本晴和籼稻品种93-11基因组中的分布差异,本研究首先运用Southern杂交的方法初步检测m Ping在两个亚种中拷贝数的差异,然后通过同源性探寻方法发现,m Ping在水稻亚种日本晴和93-11基因组中拷贝数分别为52和14,并且日本晴基因组中的m Ping均为m Ping-1,93-11中m Ping-1的拷贝数为3,m Ping-2的拷贝数为11。通过分析m Ping上下游5 kb侧翼序列发现m Ping在日本晴和93-11中分别与23和3个已知基因相关联。本研究为阐明以m Ping的分布多样性为主要原因的粳稻和籼稻之间的遗传差异提供初步理论基础。     

利用两个测序水稻品种构建微卫星连锁图谱

利用已完成基因组测序的两个水稻品种日本晴和931l的数据库成功开发出水稻微卫星新标记,并利用由90个单株组成的日本晴×9311 F2作图群体,构建了一张包含152个SSR标记位点、覆盖基因组总长度2 455.7 cM的连锁图谱,有46个SSR新标记为自主开发,该图谱标记间的平均遗传距离为16.16 cM;并将未能在Temnykh等人(2001)构建的图谱上定位的微卫星标记RM345和RM494定位在第6染色体上.通过与Temnykh等人(2001)和兰涛等人(2003)所构建的图谱从作图群体的类型和大小、标记的类型和数量、标记在染色体上的线性排列顺序等几个方面进行比较,所绘制的图谱其标记在染色体线性排列上与Temnykh等人绘制的图谱具有很高的一致性,达93.81%.     

70个水稻微卫星标记染色体位置的更正

微卫星标记(SSR)因其操作简单和稳定可靠的特点而成为一种重要的分子标记,被广泛应用于遗传作图和种质鉴定等方面。但其在染色体上位置的正确性将直接影响到基因定位的正确性和后续研究的方向。利用美国国家生物信息技术中心(NCBI)网站的Blast程序,将2740个SSR标记的前后引物序列与水稻粳稻品种日本晴基因组进行比对,共发现70个标记位于另一条染色体,对这70个标记重新锚定的染色体进行了更正。这将有助于今后水稻分子标记遗传连锁图的正确构建。     

水稻日本晴与广陆矮4号杂交F2群体SSR标记偏分离原因探析

以全基因组测序已经完成的材料粳稻日本晴和完成了第4染色体全序列测序的籼稻广陆矮4号的杂交F2作为构图群体,共90个单株,构建了一张含148个微卫星标记的水稻分子遗传图谱。该F2群体显著偏分离非常高,发现有49个分子标记表现偏分离（P〈0．05）,占总标记数的33．11％,这些偏分离标记中有36个偏向广陆4号,13个偏向杂合体,没有偏向日本晴的偏分离标记。讨论了配子体基因和孢子体基因导致偏分离的原因,通过已经定位的配子体基因和杂种不育基因分布在偏分离集中的区域来进一步说明配子体基因和杂种不育基因确实是导致偏分离形成的原因,而且还通过未定位的标记分析了偏分离的原因。     

水稻“三明”显性核不育基因的定位

"三明显性核不育水稻"突变体是由福建省三明市农业科学研究所于2001年在杂交组合"SE21S/Basmati370"的F2代群体中发现的。其不育性受1个显性基因控制(将该基因命名为SMS)。经过多代回交,该显性不育基因已导入籼稻品种佳福占的遗传背景中(将该不育材料称为佳不育)。为了定位SMS,文章将佳不育与粳稻品种日本晴杂交,并将F1与佳福占测交,构建了一个作图群体。利用SSR和INDEL标记,通过混合分离分析和连锁分析,将SMS定位于第8号染色体上两个INDEL标记ZM30和ZM9之间,约99 kb的区间内。该结果为克隆SMS奠定了基础。     

利用染色体片段置换系定位水稻落粒性主效QTL

水稻落粒性是与其生产密切相关的重要性状之一。以7个染色体片段置换系为材料,采用重叠群代换作图法对控制落粒性的2个主效QTL进行定位。结果表明,104个SSR标记在亲本间具有多态性,多态率为68.0%;4个置换系的落粒性与亲本日本晴的落粒性相似,表现难落粒。3个置换系与亲本93-11的落粒性相似,表现易落粒;7个染色体片段置换系在第1和第6染色体上检出7个置换片段,其长度分别为23.6、16.5、6.6、9.9、10.4、20.2和7.1 cM;qSH-1-1被定位在第1染色体RM472-RM1387之间,遗传距离约为6.6 cM。qSH-6-1为新发现的落粒性主效QTL,被定位在第6染色体RM6782-RM3430之间,遗传距离约为4.2 cM。利用染色体片段置换系能准确地定位水稻落粒性QTL,qSH-1-1与qSH-6-1的鉴定和初步定位为其进一步的精细定位、图位克隆及分子标记辅助选择奠定了基础。     

利用染色体片段置换系定位水稻落粒性主效QTL

水稻落粒性是与其生产密切相关的重要性状之一。以7个染色体片段置换系为材料, 采用重叠群代换作图法对控制落粒性的2个主效QTL进行定位。结果表明, 104个SSR标记在亲本间具有多态性, 多态率为68.0%; 4个置换系的落粒性与亲本日本晴的落粒性相似, 表现难落粒。3个置换系与亲本93-11的落粒性相似, 表现易落粒; 7个染色体片段置换系在第1和第6染色体上检出7个置换片段, 其长度分别为23.6、16.5、 6.6、 9.9、 10.4、 20.2和7.1 cM; qSH-1-1被定位在第1染色体RM472－RM1387之间, 遗传距离约为6.6 cM。qSH-6-1为新发现的落粒性主效QTL, 被定位在第6染色体RM6782－RM3430之间,遗传距离约为4.2 cM。利用染色体片段置换系能准确地定位水稻落粒性QTL, qSH-1-1与qSH-6-1的鉴定和初步定位为其进一步的精细定位、图位克隆及分子标记辅助选择奠定了基础。     

大粒裸燕麦(Avena nuda L.)遗传连锁图谱的构建

以“元莜麦”和“555”杂交得到的281个F2单株为作图群体,利用20对AFLP引物、3对SSR引物和1个穗型性状构建了一张大粒裸燕麦遗传连锁图。该图谱全长1544.8cM,包含19个连锁群,其上分布有92个AFLP标记、3个SSR标记和1个穗型形态标记,不同连锁群标记数为2-14个,长度在23.7-276.3cM之间,平均长度为81.3cM,标记间平均距离为20.1cM。穗型标记分离比符合3：1,11个AFLP标记表现为偏分离,偏分离比为11.5%。该图谱符合遗传连锁框架图的要求,为今后大粒裸燕麦的QTL定位、分子标记辅助育种和比较基因组学等研究奠定基础。     

水稻矮秆突变体dtl1的分离鉴定及其突变基因的精细定位

文章通过对所构建的水稻突变体库进行大规模筛选,获得一个稳定遗传的矮秆突变体,与野生型日本晴相比,该突变体表现为植株矮化、叶片卷曲、分蘖减少和不育等性状,命名为dtl1(dwarf and twist leaf 1)。dtl1属于nl型矮秆,激素检测表明,矮秆性状与赤霉素和油菜素内酯无关。遗传分析显示,突变性状受单一隐性核基因控制。利用dtl1与籼稻品种Taichung Native 1杂交构建F2群体,将该突变基因DTL1定位于水稻第10染色体长臂2个SSR标记RM25923和RM6673之间约70.4 kb区域内,并与InDel标记Z10-29共分离,在该区域预测有13个候选基因,但未见调控水稻株高相关基因的报道,因此,认为DTL1基因是一个新的控制水稻株高的基因。     

水稻粒长基因GL3的遗传分析和分子标记定位

为了解析水稻粒长的遗传机制,以大粒水稻品种‘80018-TR161-2-1’和小粒水稻品种‘日本小黑稻’及其F2代200个株系和F2：3家系为材料,分析水稻粒长的遗传学性状。结果表明,谷粒长度的分离比在F2及F2：3家系中都表现为3：1,长粒性状受1对隐性核基因控制,命名为GL3。用简单重复序列（simple sequence repeat,ssR）分子标记结合群体分组混合分析的方法,将此种基因定位在水稻第3号染色体上SSR标记PSM379和RM16之间,它们的遗传距离分别为4．0cM和11．2cM。     

Genome-wide Investigation of Intron Length Polymorphisms and Their Potential as Molecular Markers in Rice (Oryza sativa L.)

Intron length polymorphisms (ILPs) have been used as geneticmarkers in some studies. However, a systematic investigationand large-scale exploitation of ILP markers has not been reported.In this study, we performed a genome-wide search of ILPs betweentwo subspecies (indica and japonica) in rice using the draftgenomic sequences of cultivars 93-11 (indica) and Nipponbare(japonica) and 32 127 full-length cDNA sequences of Nipponbareobtained from public databases. We identified 13 308 putativeILPs. Based on these putative ILPs, we developed 5811 candidateILP markers via electronic-PCR with primers designed in flankingexons. We further conducted experiment to verify the candidateILP markers. Out of 215 candidate ILP markers tested on 93-11,Nipponbare and their hybrid, we successfully exploited 173 codominantILP markers. Further analyses on 10 rice accessions showed thatthese ILP markers were widely applicable and most (71.1%) exhibitedsubspecies specificity. This feature suggests that ILPs wouldbe useful for the studies of genome evolution and inter-subspeciesheterosis and for cross-subspecies marker-assisted selectionin rice. In addition, by testing 51 pairs of the ILP primerson five Gramineae plants and three dicot plants, we found anotherdesirable characteristic of rice ILP markers that they havehigh transferability to other plants.     

Exploring the genetic characteristics of 93-11 and Nipponbare recombination inbred lines based on the GoldenGate SNP assay

Understanding genetic characteristics in rice populations will facilitate exploring evolutionary mechanisms and gene cloning. Numerous molecular markers have been utilized in linkage map construction and quantitative trait locus (QTL) mappings. However, segregation-distorted markers were rarely considered, which prevented understanding genetic characteristics in many populations. In this study, we designed a 384-marker GoldenGate SNP array to genotype 283 recombination inbred lines (RILs) derived from 93-11 and Nipponbare Oryza sativa crosses. Using 294 markers that were highly polymorphic between parents, a linkage map with a total genetic distance of 1,583.2 cM was constructed, including 231 segregation-distorted markers. This linkage map was consistent with maps generated by other methods in previous studies. In total, 85 significant quantitative trait loci (QTLs) with phenotypic variation explained (PVE) values≥5% were identified. Among them, 34 QTLs were overlapped with reported genes/QTLs relevant to corresponding traits, and 17 QTLs were overlapped with reported sterility-related genes/QTLs. Our study provides evidence that segregation-distorted markers can be used in linkage map construction and QTL mapping. Moreover, genetic information resulting from this study will help us to understand recombination events and segregation distortion. Furthermore, this study will facilitate gene cloning and understanding mechanism of inter-subspecies hybrid sterility and correlations with important agronomic traits in rice.     

Development of genome-wide DNA polymorphism database for map-based cloning of rice genes

DNA polymorphism is the basis to develop molecular markers that are widely used in genetic mapping today. A genome-wide rice (Oryza sativa) DNA polymorphism database has been constructed in this work using the genomes of Nipponbare, a cultivar of japonica, and 93-11, a cultivar of indica. This database contains 1,703,176 single nucleotide polymorphisms (SNPs) and 479,406 Insertion/Deletions (InDels), approximately one SNP every 268 bp and one InDel every 953 bp in rice genome. Both SNPs and InDels in the database were experimentally validated. Of 109 randomly selected SNPs, 107 SNPs (98.2%) are accurate. PCR analysis indicated that 90% (97 of 108) of InDels in the database could be used as molecular markers, and 68% to 89% of the 97 InDel markers have polymorphisms between other indica cultivars (Guang-lu-ai 4 and Long-te-pu B) and japonica cultivars (Zhong-hua 11 and 9522). This suggests that this database can be used not only for Nipponbare and 93-11, but also for other japonica and indica cultivars. While validating InDel polymorphisms in the database, a set of InDel markers with each chromosome 3 to 5 marker was developed. These markers are inexpensive and easy to use, and can be used for any combination of japonica and indica cultivars used in this work. This rice DNA polymorphism database will be a valuable resource and important tool for map-based cloning of rice gene, as well as in other various research on rice (http://shenghuan.shnu.edu.cn/ricemarker).     

Analysis of Segregation Distortion of Molecular Markers in F2 Population of Rice

A genetic linkage map comprising 148 SSR markers loci was constructed using an F₂ population consisting of 90 lines derived from a sub-specific cross between a japonica variety Nipponbare and an indica variety Guangluai-4. The F₂ population showed high significantly distorted segregations. Among these SSR markers, 49 markers (33.11%) showed the genetics distortion(P<0.05). Of them, 36 markers deviated toward male parent indica GuangLuAi-4 and 13 markers toward heterozygote, but none toward the female parent Nipponbare. It was found that the segregation distortion might be caused by gametophyte and zygote. Since most gametophyte loci and sterility loci were mapped in segregation distortion regions, it indicated that the segregation distortion may be caused by these gametophyte loci and sterility loci. Finally, this research also analyzed the skewed segregation of some markers, which had not been mapped on chromosome.     

Identification and mapping of Pi41, a major gene conferring resistance to rice blast in the Oryza sativa subsp. indica reference cultivar, 93-11

The Oryza sativa subsp. indica reference cultivar (cv.), 93-11 is completely resistant to many Chinese isolates of the rice blast fungus. Resistance segregated in a 3:1 (resistance/susceptible) ratio in an F₂ population from the cross between 93-11 and the japonica reference cv. Nipponbare, when challenged with two independent blast isolates. The chromosomal location of this monogenic resistance was mapped to a region of the long arm of chromosome 12 by bulk segregant analysis, using 180 evenly distributed SSR markers. Five additional SSR loci and nine newly developed PCR-based markers allowed the target region to be reduced to ca. 1.8 cM, equivalent in Nipponbare to about 800 kb. In the reference sequence of Nipponbare, this region includes an NBS-LRR cluster of four genes. The known blast resistance gene Pi-GD-3 also maps in this region, but the 93-11 resistance was distinguishable from Pi-GD-3 on the basis of race specificity. We have therefore named the 93-11 resistance Pi41. Seven markers completely linked to Pi41 will facilitate both marker-assisted breeding and gene isolation cloning.