水稻抗白叶枯病基因Xa-4的PCR标记研究

根据与水稻抗白叶枯病基因Ｘａ－４紧密连锁的分子标记Ｍ５５的序列设计引物,通过对国际水稻研究所育成的抗白叶枯病近等基因系和基因累加系的叶片ＤＮＡ、半粒种子提取物及Ｘａ－４基因的杂合体ＤＮＡ的ＰＣＲ特异扩增,初步建立了Ｘａ－４的ＰＣＲ标记体系。进而用该标记体系对我国籼型杂交水稻常用的亲本材料进行分析,揭示出了Ｘａ－４在这些材料中的分布情况。     

水稻抗白叶枯病基因Xα—4的PCR标记研究

根据与水稻抗白叶枯病基因Ｘα－４紧密连锁的分子标记Ｍ５５的序列设计引物,通过对国际水稻研究所育成的抗白叶枯病近等基因系和基因累加系的叶片ＤＮＡ。半粒种子提取物及Ｘα－４基因的杂合体ＤＮＡ的ＰＣＲ特异扩增。初步建立了Ｘα－４的ＰＣＲ标记体系。揭示出了Ｘα－４在这些材料中的分布情况。     

利用微卫星标记鉴定水稻的稻瘟病抗性

应用水稻稻瘟病抗性基因Pid(t)紧密连锁的微卫星标记RM262对含有该抗病基因的品种地谷与感病品种江南香糯和8987的杂交F2群体进行遗传分析和抗性鉴定,结果表明,RM262的PCR扩增物在抗、感品种之间的多态性较好;在2个F2群体中,RM262和抗病基因间的重组率分别为5.74%和8.17%,应用该标记的抗性纯合和杂合带型选择抗性植株,其准确率可达98%以上。此外,还就分子标记辅助育种进行了讨论。     

籼稻品种地谷抗稻瘟病基因的遗传

籼稻地谷是我国杂效裟育种上重要的稻瘟病抗源之一。利用我国稻区的稻瘟病菌系ＺＢ１３和ＺＢ１５对地谷与感病品种江南香糯的杂效Ｆ１、Ｆ２和Ｂ１Ｆ１群体,以及地谷与感病品种丽江新团黑谷、冈４６Ｂ和８９８７的Ｆ２群体进行接种鉴定,根据抗病性的分离确认地谷对ＺＢ１３和ＺＢ１５的抗性受显性基因控制。利用菌系ＺＢ１３接种地谷和１０个具有已知抗病基因的鉴别品种及其杂交Ｆ１和Ｆ２群体,进一步证明了地谷的抗温性受１对显     

一个新的水稻迟熟性基因的遗传分析和分子标记定位

中籼迟熟水稻品系８９８７含未知的长生育期基因,在杂交水稻育种中有重要的利用价值,应用该品系与４个不同生态类型的水稻品种杂交,对其Ｆ１和Ｆ２群体进行生育期调查和遗传分析,确认８９８７的长生育期受１对隐性主效基因控制。以（８９８７Ｘ地谷）Ｆ２群体为基础,应用ＲＦＬＰ和微卫星标记结合群分法,发现第７染色体的ＲＦＬＰ标记Ｃ２１３与该基因连锁;进一步应用Ｆ２分离群体将该基因定位于第７染色体上,暂定名为ｌｆ－３。此基因的发现和定位将有助于分子标记辅助选择和杂交水稻的改良。     

利用分子标记定位农垦58S的光敏核不育基因

对农垦５８Ｓ（Ｏｒｙｚａｓａｔｉｖａｓｐ．ｊａｐｏｎｉｃａ）／大黑矮生标记基因系ＦＬ２组合组建可育集团和不育集团,并以亲本为对照进行了ＲＦＬＰ、ＲＡＰＤ和双引物ＲＡＰＤ分析,结果第１２染色体上的一个单拷贝标记Ｇ２１４０与光敏核不育基因连锁遗传,二者间的遗传图距为１４．１ｃＭ（ｃｅｎｔｉｍｏｒｇａｎ）。在筛选过的１０４０个随机单引物和１９０个双引物中,仅引物ＯＰＡＵ１０扩增出与光敏核不育基因连锁的１．５ｋｂＤＮＡ片段,回收、克隆该ＤＮＡ片段并制备探针,将其转换成共显性的ＲＦＬＰ标记并命名为ＯＰＡＵ１０１５００。分离群体连锁分析表明该标记与标记Ｇ２１４０紧密连锁,将农垦５８Ｓ的一对光敏核不育基因定位于第１２染色体上。     

分子标记技术在林木常规育种中的应用及其问题

分子生物学技术在植物和动物常规育种中的应用主要包括两个方面,即分子标记和基因工程。分子标记,或称ＤＮＡ标记,是基因型在ＤＮＡ水平上的表现形式。目前,已产生了多种分子标记技术,在方法上可以分为三类［１７］：（１）非ＰＣＲ类,如ＲＦＬＰ（Ｒｅｓｔｒｉｃｔｉｏｎｆｒａｇｍｅｎｔｌｅｎｇｔｈｐｏｌｙｍｏｒｐｈｉｓｍ）和ＶＮＴＲｓ（Ｖａｒｉａｂｌｅｎｕｍｂｅｒｏｆｔａｎｄｅｍｒｅｐｅａｔｓ）。（２）以...     

稻瘟病抗病基因Pi15的精细定位

稻瘟病抗病基因Pi15曾被作者鉴定为与已知抗病基因Pii具有连锁关系,但是,Pii基因究竟位于染色体6还是9上存在争议。为了确定Pi15基因的染色体位置,利用分子标记在由15个抗病个体和141个感病个体组成的F_2群体中,通过混合群体分离法(BSA)与隐性群体分析法(RCA)相结合的手段,对目标基因进行了连锁分析。首先,从染色体6和9分别选择10个微卫星标记进行了分析,结果表明,只有位于染色体9的RM316与目标基因连锁,重组率为(19.1±3.7)％。为了进一步确定这种连锁关系,从染色体9选择了4个序列标定位点(STS)标记进行分析,结果表明,只有G103与目标基因连锁,重组率为(5.7±2.1)％。为了获得与目标基因更加紧密连锁的分子标记,对目标基囚进行了RAPD分析。在筛选、分析了1000个随机引物之后,从中获得了3个目标基因紧密连锁的分子标记BAPi15_(486)、BAPi15_(782)、BAPi15_(844)。它们与目标基因的重组率分别为0.35％、0.35％和1.1％。这些紧密连锁的分子标记可作为分子标记辅助基因聚合和克隆的出发点。     

对高梁BTAM428×ICS-12B杂交F3代及部分抗感品种的SCAR分析

用与抗蚜基因紧密连锁的OPN-0727、OPN-08373RAPD标记,转化得到的SCAR标记对F3代及部分抗感品种进行SCAR检测.将F2代抗感群体留种种于田间,取在孕穗期F3植株叶片提取总DNA进行SCAR分析,得到了F3抗感个体的特异性谱带,同时在部分抗性品种中也得到了OPN-07727、OPN-08373二条特异谱带,而感性品种中则未见,从而为分子标记辅助育种提供了可靠的依据.     

水稻细菌性条斑病和白叶枯病抗性遗传研究

分析了Ｈａｓｈｉｋａｌｍｉ,Ｄｕｌａｒ和９０ＩＲＢＢＮ４４三个抗源品种对水稻细菌性条斑病Ｓ－１０３菌株和白叶枯病Ｐ１菌系的抗性遗传。结果表明,Ｈａｓｈｉｋａｌｍｉ和Ｄｕｌａｒ对Ｓ－１０３的抗性均由２对隐性基因所控制,９０ＩＲＢＢＮ４４则带有１对隐性抗性基因。经等位性测定表明,Ｈａｓｈｉｋａｌｍｉ和Ｄｕｌａｒ的２对基因中至少有１对是等位的,但它们与９０ＩＲＢＢＮ４４的１对基因均不等位。３个抗源品种对Ｐ１的抗性都受１对隐性基因控制,该基因与ｘａ－５等位。连锁遗传分析表明,Ｈａｓｈｉｋａｌｍｉ和Ｄｕｌａｒ对Ｓ－１０３的２对抗细条病基因中的１对与ｘａ－５相连锁,而９０ＩＲＢＢＮ４４的１对抗性基因与ｘａ－５呈独立遗传。本文还就开展水稻抗细菌性条斑病育种进行了讨论。     

药用野生稻转育后代一个抗白叶枯病新基因的定位

从药用野生稻渗入后代选育的水稻株系B5表现为高抗褐飞虱、白背飞虱和白叶枯病。对B5与籼稻品种明恢63杂交组合的187个重组自交系(RILs)进行了抗白叶枯病接种鉴定,采用分离集团分析法(Bulked Segregant Analysis,BSA),在第1染色体上筛选到与水稻抗白叶枯病基因相连锁RFLP分子标记。利用RILs抗病性表现型鉴定资料和构建的分子标记连锁图谱,将抗白叶枯病基因定位在第1染色体短臂的C904和R596之间,这两个分子标记间遗传距离为1．3cM。该基因对RILs群体抗病性变异的贡献率为52．96％,是一效应值较大的主效基因。这一抗白叶枯病基因不同于已报道的抗白叶枯病基因的位点,因此将其命名为Xa29(t)。     

High-resolution genetic mapping of rice bacterial blight resistance gene Xa23

Bacterial blight (BB) caused by Xanthomonas oryzae pv. oryzae (Xoo) is the most devastating bacterial disease of rice (Oryza sativa L.), a staple food crop that feeds half of the world’s population. In management of this disease, the most economical and effective approach is cultivating resistant varieties. Due to rapid change of pathogenicity in the pathogen, it is necessary to identify and characterize more host resistance genes for breeding new resistant varieties. We have previously identified the BB resistance (R) gene Xa23 that confers the broadest resistance to Xoo strains isolated from different rice-growing regions and preliminarily mapped the gene within a 1.7 cm region on the long arm of rice chromosome 11. Here, we report fine genetic mapping and in silico analysis of putative candidate genes of Xa23. Based on F₂ mapping populations derived from crosses between Xa23-containing rice line CBB23 and susceptible varieties JG30 or IR24, six new STS markers Lj36, Lj46, Lj138, Lj74, A83B4, and Lj13 were developed. Linkage analysis revealed that the new markers were co-segregated with or closely linked to the Xa23 locus. Consequently, the Xa23 gene was mapped within a 0.4 cm region between markers Lj138 and A83B4, in which the co-segregating marker Lj74 was identified. The corresponding physical distance between Lj138 and A83B4 on Nipponbare genome is 49.8 kb. Six Xa23 candidate genes have been annotated, including four candidate genes encoding hypothetical proteins and the other two encoding a putative ADP-ribosylation factor protein and a putative PPR protein. These results will facilitate marker-assisted selection of Xa23 in rice breeding and molecular cloning of this valuable R gene.     

Marker assisted selection of bacterial blight resistance genes in rice

Bacterial leaf blight caused by Xanthomonas oryzae pv. oryzae is one of the most important diseases affecting rice production in Asia. We were interested in surveying rice genotypes that are popularly used in the Indian breeding program for conferring resistance to bacterial blight, using 11 STMS and 6 STS markers. The basis of selection of these DNA markers was their close linkage to xa5, xa13, and Xa21 genes and their positions on the rice genetic map relative to bacterial blight resistance genes. Eight lines were found to contain the xa5 gene while two lines contained Xa21 gene and none of the lines contained the xa13 gene with the exception of its near-isogenic line. Using the polymorphic markers obtained in the initial survey, marker-assisted selection was performed in the F3 population of a cross between IR-64 and IET-14444 to detect lines containing multiple resistance genes. Of the 59 progeny lines analyzed, eight lines contained both the resistance genes, xa5 and Xa4.     

云南野生稻中的抗白叶枯病基因Xa21的鉴定

水稻白叶枯病是水稻生产上的主要细菌病害之一。从野生稻中发掘优异的水稻白叶枯病抗性材料,可以拓宽栽培稻抗白叶枯病遗传基础。经过温室接菌鉴定和PCR标记分析,对云南野生稻进行Xa21基因的检测鉴定。温室接菌鉴定表明,云南野生稻对广谱致病小种PX099及云南强致病菌Y8具有较好的抗性能力,特别是疣粒野生稻对致病菌株达到免疫程度;PCR标记分析表明,云南野生稻不含有Xa21基因,但含有与Xa21基因某些区域同源的片段。本研究结果为寻找新的抗源材料及快速发掘利用云南野生稻中的抗白叶枯病基因提供理论依据。     

DNA markers and marker-assisted breeding for durable resistance to bacterial blight disease in rice

Bacterial leaf blight caused by the bacterial pathogen Xanthomonas oryzae pv oryzae (Xoo) limits rice yield in all major rice-growing regions of the world, especially in irrigated lowland and rainfed conditions where predisposition factors favor disease development to epidemic proportions. Since bacterial pathogens are difficult to manage, development of host plant resistance is the most effective means of disease management. As many as 24 major genes conferring resistance to various races of the pathogen have been identified and utilized in rice breeding programs. However, large-scale and long-term cultivation of varieties carrying a single gene for resistance resulted in a significant shift in pathogen race frequency with consequent breakdown of resistance in these cultivars. To combat the problem of resistance breakdown, pyramiding of resistance genes into different cultivars is being carried out. Pyramiding of resistance genes is now possible with molecular markers that are developed for individual genes. This review discusses the various bacterial blight resistance genes identified and their corresponding molecular markers developed for breeding durable resistance into modern rice cultivars.     

Molecular breeding: marker-assisted selection combined with biolistic transformation for blast and bacterial blight resistance in Indica rice (cv. CO39)

Based on blast pathogen population dynamics and lineage exclusion assays, we found that the major blast resistance genes Pi-1 and Piz-5 confer resistance against most Magnaporthe grisea lineages. Near-isogenic rice lines C101LAC and C101A51 carrying these two major genes for blast resistance in the background of a most blast-susceptible genotype were used for developing the pyramids. The closely linked RFLP marker RZ536 and NBS-LRR r10 marker for Pi-1 and a PCR-based SAP marker RG64 for Piz-5 were used to identify the genes in the parents and in marker-assisted breeding of the pyramided populations. To achieve multiple resistance against blast and blight in this cultivar, these blast-resistant pyramids were transformed with the cloned bacterial blight resistance gene Xa21 known to confer resistance to all races of Xanthomonas oryzae pv. oryzae (Xoo). Bioassays with six independent transformants showed that transgenic CO39 plants were resistant to both pathogens, M. grisea and Xoo. We report here the stacking of three major genes (Pi-1 + Piz-5 + Xa21) into rice using two different approaches of molecular breeding: marker-assisted selection (MAS) and genetic transformation.     

Introduction of a rice blight resistance gene,Xa21, into five Chinese rice varieties through anAgrobacterium-mediated system

A cloned gene, Xa21 was transferred into five widely-used Chinese rice varieties through an Agrobacterium-mediated system, and over 110 independent transgenic lines were obtained. PCR and Southern analysis of transgenic plants revealed the integration of the whole Xa21 gene into the host genomes. The integrated Xa21 gene was stably inherited, and segregated in a 3 : 1 ratio in the selfed T1 generation when one copy of the gene was integrated in the transfor-mants. Inoculation tests displayed that transgenic T0 plants and Xa21 PCR-positive T1 plants were highly resistant to bacterial blight disease. The selected Xa21 homozygous resistant transgenic lines with desirable qualities may be propagated as new varieties or utilized in hybrid rice breeding.     

Chromosome landing at the bacterial blight resistance gene<Emphasis Type="Italic"> Xa4</Emphasis> locus using a deep coverage rice BAC library

Xa4 is a dominantly inherited rice gene that confers resistance to Philippine race 1 of the bacterial blight pathogen Xanthomonas oryzae pv. oryzae in rice. In order to isolate the gene by positional cloning, a bacterial artificial chromosome (BAC) library was constructed from genomic DNA isolated from an Xa4-harboring accession, IRBB56. The library contains 55,296 clones with an average insert size of 132 kb, providing 14 rice genome equivalents. Three DNA markers closely linked to Xa4 were used to screen the library. The marker RS13, a resistance gene analogue that co-segregates with Xa4, identified 18 clones, of which four and six, respectively, were simultaneously detected by the other two markers, G181 and L1044. Fingerprinting and Southern analysis indicated that these clones overlapped and define an interval spanning 420 kb. In an F2 population derived from an indica variety, IR24, and its Xa4-containing near isogenic line (NIL), IRBB4, the susceptible plants were screened in order to map the Xa4 gene genetically and physically. Out of 24 insert ends isolated from the BACs in the contig, three revealed polymorphisms between IR24 and IRBB4. Two insert ends, 56M22F and 26D24R, flanked Xa4 on each side. Based on the overlap of the BACs, six overlapping clones were considered to include the Xa4 allele, one of which, 106P13, was chosen for further investigation.     

Introduction of a rice blight resistance gene, Xa21, into five Chinese rice varieties through an Agrobacterium-mediated system

A cloned gene, Xa21 was transferred into five widely-used Chinese rice varieties through an Agrobacterium-mediated system, and over 110 independent transgenic lines were obtained. PCR and Southern analysis of transgenic plants revealed the integration of the whole Xa21 gene into the host genomes. The integrated Xa21 gene was stably inherited, and segregated in a 3∶1 ratio in the selfed T1 generation when one copy of the gene was integrated in the transformants. Inoculation tests displayed that transgenic T0 plants and Xa21 PCR-positive T1 plants were highly resistant to bacterial blight disease. The selected Xa21 homozygous resistant transgenic lines with desirable qualities may be propagated as new varieties or utilized in hybrid rice breeding.     

农杆菌介导将白叶枯病抗性基因Xa21导入水稻获得转基因植株

利用农杆菌介导的高效遗传转化系统,将白叶枯病抗性基因Xa21转入黄淮稻区主栽品种豫粳6号的胚性愈伤组织,获得转基因植株,GUS染色和PCR分析证明Xa21基因已整合到水稻基因组中,其自交T1代植株经GUS染色和白叶枯病接种鉴定呈现3：1分离,研究为培育抗白叶枯病水稻品种奠定了基础。