103个杂交稻亲本白叶枯病和稻瘟病抗性基因型检测

白叶枯病和稻瘟病是我国水稻生产的主要病害,选育抗病品种是一种经济有效的防治手段。明确抗性基因在水稻品种资源中的分布,将有助于应用分子标记辅助选择培育抗性品种。本研究针对13个重要抗病基因,包括8个白叶枯病抗性基因(Xa1、Xa4、xa5、Xa7、xa13、Xa21、Xa23和Xa26)和5个稻瘟病抗性基因(Pib、Pi2、Pi9、Pi25和Pita),应用这些基因的基因标记或连锁标记共29个,检测103份杂交稻亲本,初步确定了其等位基因分布和利用情况,为水稻基础材料的筛选及应用提供参考。     

利用功能标记鉴定普通野生稻中的白叶枯病抗性基因

 以9个菲律宾白叶枯病菌小种对供试的9份普通野生稻（Oryza rufipogon Griff.）及1份高感白叶枯病材料IR24进行抗性鉴定,发现IR24对所有的菌株都表现为高感, 6份野生稻材料对9个菌株表现全抗,占参试野生稻总数的67%。取自广西玉林的1份材料只感PXO280(P8),海南万宁的1份材料感PXO71(P4),广州高州的1份材料对PXO79、PXO99和PXO339感病,而这几份材料对其余菌株都表现为抗病,说明每份材料至少含有1个抗性基因。利用已克隆的白叶枯病抗性基因xa5、xa13、Xa21和Xa27的功能标记检测,结果表明9份供试普通野生稻中都不含抗性基因xa5、Xa21;5份为显性Xa13纯合体,4份为隐性抗病xa13杂合体;5份为抗病显性Xa27纯合体,3份为隐性xa27纯合体,1份材料中xa27和Xa27都不存在。     

四川主要水稻恢复系抗白叶枯病基因的分子检测

用分别与水稻抗白叶枯病基因Xa4、Xa7、Xa21和Xa23紧密连锁的PCR标记MP12、M3、pTA248和RM206对四川省新选育的以及主要应用的39份恢复系进行了分子检测，结果表明，这些恢复系均不含有Xa7（M3）和Xa21（pTA248）抗性等位点，17份携有榭（MP12）抗性等位点，少数（6个）携有Xa23（RM206）抗性等位点。在杂交水稻恢复系抗性改良中，应采用分子标记辅助选择（MAS）加快引入利用除工卅外的其它儿个显性抗性基因。     

水稻抗白叶枯病基因xa5的原核表达及蛋白纯化

水稻是重要的粮食作物,其产量高低与病虫害等因素密切相关,由黄单胞杆菌引起的水稻白叶枯病是导致水稻产量减少的主要病害之一。控制白叶枯病最安全、经济有效的方法是培育抗病品种,隐性抗病基因xa5是重要的水稻白叶枯病抗性基因。通过构建显性及隐性xa5基因的原核表达载体(PGEX-4T-1-Xa5和PGEX-4T-1-xa5),转化大肠杆菌BL21,经0.2 mmol/L IPTG 28℃ 诱导4 h,表达显性Xa5和隐性xa5蛋白;结合western-blotting分析,结果表明表达的蛋白是带有GST标签的融合蛋白,最后利用谷胱甘肽亲和层析柱将蛋白纯化,为后期研究xa5基因的功能提供基础。     

水稻抗白叶枯病微效QTL的定位分析

以不携有抗白叶枯病主效基因的中感籼粳双亲(窄叶青8号和京系17）及其花培DH群体为材料,接种白叶枯病菌浙9612后,考察了该DH群体的白叶枯病抗性,并进行了数量性状座位（QTL）分析。共检测到控制白叶枯病的4个QTL,分别位于第3、4、5和6染色体上,其中第3和第4染色体上的qBBR-3和qBBR-4,其加性效应为正值;而位于第5和第6染色体上的为负值,4个QTL的总效应是38.6%。方差分析和差异显著性比较表明,具有4个QTL抗性等位基因水稻株系的白叶枯病抗性与不含QTL抗性等位基因的株系相比,差异达极显著水平（P<0.01)。说明通过微效基因的聚合可以获得对白叶枯病的一定抗性。     

分子标记辅助选择改良水稻光温敏核不育系华201S的白叶枯病抗性

 以华201S为受体亲本,以含Xa21和Xa7基因的抗白叶枯病材料华恢20为供体亲本,进行1次杂交、3次回交和3次以上自交,每个世代用基因内标记PTA248对Xa21进行分子检测,用连锁标记STSP3（与Xa7的遗传距离为0.9 cM）对Xa7进行检测。新选育的4个株系YR7009(Xa21)、YR7014(Xa21)、YR7016(Xa7)和YR7023(Xa7/Xa21)在生育期、主要农艺性状、育性转换特性方面都与华201S基本相似。YR7009(Xa21)和YR7014(Xa21)对PXO61、PXO99、ZHE173和GD1358等4个菌株具有良好的抗性,而对FuJ和YN24不具有抗性。YR7016(Xa7)和YR7023(Xa7/Xa21)对6个菌株都表现良好的抗性,尤其是聚合双基因的YR7023(Xa7/Xa21)对6个菌株都表现高抗。表明已经将广谱白叶枯病抗性基因Xa21和Xa7渗入到华201S中,成功地改良了华201S的白叶枯病抗性。     

CecropinB与Xa21基因共表达提高转基因水稻白叶枯病抗性

为了探明CecropinB与Xa21这两种不同抗病途径的基因单独或共表达后的田间抗白叶枯病表现,利用农杆菌转化技术,获得了这两个基因的单独和共表达后的植株。利用PCR等分子鉴定技术,证明这两个基因已经整合到水稻基因组中。抗病性检测结果表明,Xa21和CecropinB均能提高水稻对白叶枯病的抗性,且Xa21的抗病性较CecropinB明显。Xa21和CecropinB基因共表达后的植株的抗病性比单基因的更强,说明将不同抗性机制的基因共表达,可以在育种上利用以提高水稻的白叶枯病抗性。     

疣粒野生稻抗白叶枯病新基因的初步鉴定

以10个菲律宾白叶枯病菌小种和1个中国白叶枯病菌小种为供试菌系,以高感白叶枯病水稻品种IR24及携有不同抗白叶枯病基因的近等基因系IRBB1等16个材料作为参照,对粳稻品种8411/疣粒野生稻体细胞杂交获得的两个抗白叶枯病新种质SH5和SH76进行了白叶枯病抗谱鉴定。结果表明SH5和SH76在苗期的抗谱较广,并且与已知抗病基因的抗谱不同,但与IRBB5(xa5)和IRBB7(Xa7)相似。分别用xa5和Xa7的分子标记2F_1R和M5进行检测,确定SH5和SH76中不含有xa5和Xa7基因。初步推测SH5、SH76可能含有一个新的抗病基因或者一个连锁的基因簇群。     

水稻品种五丰占2号的白叶枯病抗性遗传分析

研究了五丰占2号的白叶枯病抗性遗传及在回交世代中的抗性表现。结果表明,用白叶枯病菌株浙173（Ⅳ型）接种,五丰占2号表现中抗,IRBB5表现抗;五丰占2号的白叶枯病抗性受微效多基因控制,基因效应分析表明,该性状符合加性-显性模型,以加性效应为主;隐性主效基因xa5控制的IRBB5对白叶枯病菌株浙173的遗传符合隐性主基因的分离比。对白叶枯病菌株浙173的抗性反应与xa5基因的PCR检测结果一致。在五丰占2号2/IRBB5 B1F1群体中,基因型Xa5Xa5与Xa5xa5的分离比为1∶1;在五丰占2号2/蜀恢162 B1F1群体中,白叶枯病抗性达到五丰占2号水平的植株数占群体总数的68.3%。如果要将IRBB5中的xa5基因与五丰占2号的微效基因聚合,用五丰占2号回交1次是必要的。     

水稻抗白叶枯病的新基因

1978年,在田间研究了水稻(OryzaSativa L.)58个抗白叶枯病Xanthomonas campestris pv Oryzae(Ishi yama1972Dye)品种的遗传方式和基因的等位关系。由抗病品种和感病品种“TNI”杂交组合F_1和F_2群体的分析揭示了48个品种的抗性变由单一显性基因控制,其余的10个品种的抗性变单一隐性基因操纵。等位基因测验表明这48个品种具有Xa4抗性基因,8个品种的隐性抗性基因与Xa5等位。然而,“Khao Lay Nhay”和“Sateny”品种的隐性抗性基因彼此是等位的,与Xa3、Xa4、Xa5、Xa7和Xa8不等位且可独立分离。这个新的隐性基因同Xa6连锁,其同型染色体局部交换值为5.9％。因而,品种“Khao Lay Nhay”和“Sateng”有新的抗性基因,这种基因定名为Xag。水稻白叶枯病(Xanthomonasc ampestris pv.oryzae)是亚洲分布最广的一种水稻病害。早在1930年代,日本就开始系统研究寄主植物对白叶枯病的抗性并鉴定出许多抗日本菌系的品种(Mizukami1966,Ezuka和Horino1974)。研究了对日本菌系的抗性遗传并鉴定出三个抗性位点(Nishimura和Sakaguchi 1959,Sakaguchi 1967和Ezuka等1975)。把这三个位点叫做Xa1、Xa2、Xa3。在热带地区,早在1960年代,国际水稻研究所(IRRI)开始系统研究水稻白叶枯病并鉴定了3000多个抗病品种。近10年间,在进行遗传研究的基础上,鉴定出抗菲律宾菌系的另外5个抗性基因(Xa4、Xa5、Xa6、Xa7和Xa8)(Petpisit等1977;Olufowote等1977;Sidhu和Khushl978;Siolhu等1978)。其中,Xa4和Xa5分布最广。这两个基因彼此独立分离。Xa6与Xa4连锁(Sidhu和Khush 1978)。在IRRI和其它地方Xa4、Xa5和Xa6被广泛地用于育种计划,为了鉴定另外的抗性基因,进行了本研究。     

Breeding of R8012,a Rice Restorer Line Resistant to Blast and Bacterial Blight Through Marker-Assisted Selection

Genetic improvement is one of the most effective strategies to prevent rice from blast and bacterial blight(BB) diseases,the two most prevalent diseases jeopardizing rice production.Rice hybrids with dural resistance to blast and BB are needed for sustainable production of food.An incomplete diallele design resulted in 25 crosses between five blast and five BB resistant germplasm accessions.Only one pair of parents,DH146 × TM487,showed polymorphism for all the markers to identify one blast resistance gene Pi25 and three BB resistance genes,Xa21,xa13 and xa5,thus it was used in the marker-assisted selection(MAS).F2 individuals of DH146 × TM487 were genotyped using flanking markers of RM3330 and sequence tagged site(STS) marker SA7 for Pi25.The resistant F2 plants with Pi25 were used for pyramiding BB resistance genes Xa21,xa13 and xa5 identified by the markers pTA248,RM264 and RM153,respectively in subsequent generations.Finally,after selection for agronomic traits and restoration ability among 12 pyramided lines,we acquired an elite restorer line,R8012 including all four target genes(Pi25+Xa21+xa13+xa5).Hybrid Zhong 9A/R8012 derived from the selected line showed stronger resistance to blast and BB,and higher grain yield than the commercial checks uniformally in experimental plots,2007 state-wide yield trial and 2008 nation-wide yield trial.This study provides a paradigmatic example to show that MAS is a practically feasible tool in effectively pyramiding multiple resistance genes.The resultant restoring line and its hybrid would play an important role in securing rice production in China.     

A Single-Tube,Functional Marker-Based Multiplex PCR Assay for Simultaneous Detection of Major Bacterial Blight Resistance Genes Xa21,xa13 and xa5 in Rice

In marker-assisted breeding for bacterial blight(BB) resistance in rice, three major resistance genes, viz., Xa21, xa13 and xa5, are routinely deployed either singly or in combinations. As efficient and functional markers are yet to be developed for xa13 and xa5, we have developed simple PCR-based functional markers for both the genes. For xa13, we designed a functional PCR-based marker, xa13-prom targeting the In Del polymorphism in the promoter of candidate gene Os8N3 located on chromosome 8 of rice. With respect to xa5, a multiplex-PCR based functional marker system, named xa5 FM, consisting of two sets of primer pairs targeting the 2-bp functional nucleotide polymorphism in the exon II of the gene TFIIA5(candidate for xa5), has been developed. Both xa13-prom and xa5 FM can differentiate the resistant and susceptible alleles for xa13 and xa5, respectively, in a co-dominant fashion. Using these two functional markers along with the already reported functional PCR-based marker for Xa21(p TA248), we designed a single-tube multiplex PCR based assay for simultaneous detection of all the three major resistance genes and demonstrated the utility of the multiplex marker system in a segregating population.     

Application of Functional Markers to Identify Genes for Bacterial Blight Resistance in Oryza rufipogon

Field resistances of nine accessions of common wild rice (Oryza rufipogon Griff.) and one rice variety (IR24) were evaluated by using nine strains of bacterial blight pathogen (Xanthomonas oryzae pv. oryzae) from the Philippines. IR24 was highly susceptible to all the strains, and six common wild rice accessions resisted all the nine strains, with a resistance frequency of 67%. The accessions Yulin and Wanning were only susceptible to PXO280 and PXO71, respectively. The accession Gaozhou was susceptible to the three strains PXO79, PXO99 and PXO339, whereas resistant to the other six strains. It could be concluded that there is at least one resistance gene in each common wild rice accession. The functional markers of the genes xa5, xa13, Xa21 and Xa27 were used to detect the presence of these resistance genes in the nine tested wild rice accessions, and it was found that four wild rice accessions contained heterozygous xa13. Among the nine common wild rice accessions, five were homozygous for Xa27 and three homozygous for xa27, and the accession Laibin contained neither xa27 nor Xa27. In addition, there were no xa5 and Xa21 in all of these accessions.     

Improvement of Bacterial Blight Resistance in Rice Cultivars Jyothi and IR50 via Marker-Assisted Backcross Breeding

Abstract

In pathogen population analysis of 208 Xanthomonas oryzae pv. oryzae(Xoo) strains that were assembled from different parts of India, 21 pathotypes were identified on the basis of disease reactions on near-isogenic lines (NILs) and 13 pathotypes, on rice differentials. Rice cultivars, Jyothi and IR50, which are high yielding but highly prone to bacterial blight (BB) caused by pathogen populations of Xanthomonas oryzae pv. oryzae in India, were chosen. To improve the BB resistance of these two varieties, a pyramid line, NH56, containing four R-genes, Xa4, xa5, xa13, and Xa21, was selected as the R-donor based on resistance to existing pathogen population. The four R-genes were successfully transferred to cultivars through a traditional backcross method and their presence was documented with marker-aided selection (MAS). Thirty BC₄F₂ plants derived from JxNH56 (cv. Jyothi) and 45 BC₄F₂ plants derived from IR50xNH56 (cv. IR50) had all four resistance genes (Xa4, xa5, xa13, and Xa21), which should be useful resistance donors for breeding other BB-resistant elite indica varieties.     

分子标记辅助选择抗稻白叶枯病基因在选育水稻广亲和恢复系上的应用

 以IRBB21为Xa21基因供体,广亲和恢复系4183为受体，进行1次杂交并回交3次，逐代用分子标记检测手段，导入广谱抗白叶枯病基因Xa21,在保持广亲和恢复系4183优良经济性状的基础上，增强其抗白叶枯病的能力，育成抗白叶枯病的广亲和恢复系抗4183，其抗性达到了IRBB21的抗性水平，且保持了4183的广亲和性、恢复性及优良的经济性状。并就杂交水稻白叶枯病的抗性改良及育种对策进行了讨论。     

Genetic Improvement of Rice for Bacterial Blight Resistance: Present Status and Future Prospects

The production and productivity of rice has been challenged due to biotic and abiotic factors. Bacterial blight (BB) disease, caused by Xanthomonas oryzae pv. oryzae, is one of the important biotic stress factors, which reduces rice production by 20%–50%. The deployment of host plant resistance is the most preferred strategy for management of BB disease, and breeding disease resistant varieties remains a very economical and effective option. However, it is difficult to develop rice varieties with durable broad-spectrum resistance against BB using conventional approaches alone. Modern biotechnological tools, particularly the deployment of molecular markers, have facilitated the cloning, characterization and introgression of BB resistance genes into elite varieties. At least 46 BB resistance genes have been identified and mapped from diverse sources till date. Among these, 11 genes have been cloned and characterized. Marker-assisted breeding remains the most efficient approach to improve BB resistance by introducing two or more resistance genes into target varieties. Among the identified genes, xa5, xa13 and Xa21 are being widely used in marker-assisted breeding and more than 70 rice varieties or hybrid rice parental lines have been improved for their BB resistance alone or in combination with genes/QTLs conferring tolerance to other stress. We review the developments related to identification and utilization of various resistance genes to develop BB resistant rice varieties through marker-assisted breeding.     

Marker-Assisted Pyramiding of Genes Conferring Resistance Against Bacterial Blight and Blast Diseases into Indian Rice Variety MTU1010

Two major bacterial blight(BB) resistance genes(Xa21 and xa13) and a major gene for blast resistance(Pi54) were introgressed into an Indian rice variety MTU1010 through marker-assisted backcross breeding. Improved Samba Mahsuri(possessing Xa21 and xa13) and NLR145(possessing Pi54) were used as donor parents. Marker-assisted backcrossing was continued till BC2 generation wherein PCR based functional markers specific for the resistance genes were used for foreground selection and a set of parental polymorphic microsatellite markers were used for background selection at each stage of backcrossing. Selected BC2F1 plants from both crosses, having the highest recoveries of MTU1010 genome(90% and 92%, respectively), were intercrossed to obtain intercross F1(ICF1) plants, which were then selfed to generate 880 ICF2 plants possessing different combinations of the BB and blast resistance genes. Among the ICF2 plants, seven triple homozygous plants(xa13xa13Xa21Xa21Pi54Pi54) with recurrent parent genome recovery ranging from 82% to 92% were identified. All the seven ICF2 plants showed high resistance against the bacterial blight disease with a lesion lengths of only 0.53–2.28 cm, 1%–5% disease leaf areas and disease scoring values of ‘1' or ‘3'. The seven ICF2 plants were selfed to generate ICF3, which were then screened for blast resistance, and all were observed to be highly resistant to the diseases. Several ICF3 lines possessing high level of resistance against BB and blast, coupled with yield, grain quality and plant type on par with MTU1010 were identified and advanced for further selection and evaluation.     

Identification of QTLs underlying resistance to a virulent strain of Xanthomonas oryzae pv. oryzae in rice cultivar DV85

Bacterial blight (BB), caused by Xanthomonas oryzae pv. oryzae (Xoo), is one of the most devastating diseases of rice in China. A strongly virulent Xoo strain, designated Z-173, is widely distributed across China and Southeast Asia. Indica rice DV85 is known to carry the two resistance genes, xa5 and Xa7. However, their effectiveness against Z-173 is unknown. Using a recombinant inbred line (RIL) population derived from a cross between DV85 and the susceptible cultivar Kinmaze, we have identified the quantitative trait loci (QTLs) responsible for the resistance of DV85 to Z-173. Following 2 years of phenotyping, three QTLs associated with the resistance were detected. These were linked to RFLP markers X362, X292 and G1091 on chromosomes 3, 5, and 6, respectively. Qxa-5 and Qxa-6 probably correspond to xa5 and Xa7, respectively. Both the xa5 and Xa7 resistances are stable over different years, and act independently of one another in determining resistance. The effect of xa5 was larger than that of Xa7. Efficient ways to improve the resistance to Z-173 are discussed.