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.     

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.     

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.     

水稻白叶枯病抗性基因鉴定进展及其利用

 1982~1987年，日本热带农业研究中心（TARC）和国际水稻研究所（IRRI）合作采取统一研究方案，创建了国际水稻白叶枯病抗性鉴别系统。统一了命名，删去了重复（包括与Xa3相同的Xa4b、Xa6和xa9）。截至2005年6月，经国际注册确认和期刊报道的水稻白叶枯病抗性基因共30个，其中 Xa22(t)、Xa26(t)、xa26(t)、Xa27(t)、xa28(t)、Xa29(t)和3个Xa25(t)为暂定名基因，有待订正。在30个基因中，21个为显性基因（Xa），9个为隐性基因（xa）；13个表现全生育期抗性，15个为成株期抗性，Xa21和Xa25(t) (O. minuta)两个基因在分蘖后期表达抗性。已被定位的抗性基因有17个，即第11染色体上有7个，Xa3、Xa4、Xa10、Xa21、Xa22(t)、Xa23和Xa26；第4染色体上有4个，Xa1、Xa2、Xa12和Xa14;第5染色体上有2个，xa5和xa13；第6染色体上有Xa7和Xa27；第12染色上有Xa25(t)；第1染色体上有Xa29（t）。包括Xa1、xa5、Xa21、Xa26五个基因已被克隆。并讨论了合理利用抗性基因等问题。     

Improvement of Upland Rice Variety by Pyramiding Drought Tolerance QTL with Two Major Blast Resistance Genes for Sustainable Rice Production

Varalu is an early maturing rice variety widely grown in the rainfed ecosystem preferred for its grain type and cooking quality. However, the yield of Varalu is substantially low since it is being affected by reproductive drought stress along with the blast disease. The genetic improvement of Varalu was done by introgressing a major yield QTL, qDTY_12.1, along with two major blast resistance genes i.e. Pi54 and Pi1 through marker-assisted backcross breeding. Both traits were transferred till BC₂ generation and intercrossing was followed to pyramid the two traits. Stringent foreground selection was carried out using linked markers as well as peak markers (RM28099, RM28130, RM511 and RM28163) for the targeted QTL (qDTY_12.1), RM206 for Pi54 and RM224 for Pi1. Extensive background selection was done using genome-wide SSR markers. Six best lines (MSM-36, MSM-49, MSM-53, MSM-57, MSM-60 and MSM-63) having qDTY_12.1 and two blast resistance genes in homozygous condition with recurrent parent genome of 95.0%-96.5% having minimal linkage drag of about 0.1 to 0.7 Mb were identified. These lines showed yield advantage under drought stress as well as irrigated conditions. MSM-36 showed better performance in the national coordinated trials conducted across India, which indicated that improved lines of Varalu expected to replace Varalu and may have an important role in sustaining rice production. The present study demonstrated the successful marker-assisted pyramiding strategy for introgression of genes/QTLs conferring biotic stress resistance and yield under abiotic stress in rice.     

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.     

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.     

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。     

利用分子标记技术聚合3个稻瘟病基因改良金23B的稻瘟病抗性

以C101LAC和C101A51为稻瘟病抗性基因的供体亲本,金23B为受体亲本,通过杂交、复交及一次回交,在分离世代,利用分子标记辅助选择技术结合特异稻瘟病菌株接种鉴定和农艺性状筛选,获得6个导入Pi 1、Pi 2和Pi 33基因的金23B导入系,其中导入系W1对稻瘟病的抗病频率为96.7%,明显高于携带单个基因的C104LAC（Pi 1）、C101A51（Pi 2）和北京糯（Pi 33）。基因聚合后抗病频率提高,说明基因聚合是培育稻瘟病持久抗性的有效方法之一。     

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。     

分子标记辅助选择改良水稻恢复系香5的病虫抗性

香5是由湖北省农科院选育的优质两系杂交稻恢复系,所配组合广两优5号(广占63-4S/香5)于2013年通过了湖北省审定.利用回交和分子标记辅助选择技术,将供体亲本MD12086-1351中的抗稻瘟病基因Pi9、抗褐飞虱基因Bph14、Bph15和抗白叶枯病基因Xa23渗入到香5背景中,育成了3个同时携带Pi9、Bph14、Bph15和Xa23基因的新株系.鉴定结果表明,新株系的叶瘟抗性明显提高,穗颈瘟抗性部分提高,苗期抗褐飞虱,分蘖盛期高抗白叶枯病;产量、主要农艺性状、香味和稻米品质主要指标与香5相似.新株系所配的组合在产量、主要农艺性状上与香5所配的组合相似.表明新株系可以作为香5的替代系用于培育抗稻瘟病、抗褐飞虱和抗白叶枯病的两系杂交稻新组合.     

具抗稻瘟病基因Pi25杂交稻恢复系的分子标记辅助选育

 从组合中156/谷梅2号所衍生的重组自交系群体中选择携带抗稻瘟病基因Pi25的3个株系,分别与高产恢复系9308和3 11配组,通过单粒传法获得的6个F6重组自交系群体用于研究。在Pi25先前定位的遗传图谱上,新增加与该基因紧密连锁的分子标记RM3330和A7,并结合RM3330和A7分子标记辅助选择的结果,共筛选到了109个Pi25基因纯合的株系。用现有的与恢复基因连锁的标记对这109个株系进行二次筛选,最终获得20个Pi25基因和恢复基因均纯合的株系。对育性恢复力鉴定试验表明,选育出的抗病恢复系具有较好的恢复能力,并已应用于育种实践;人工稻瘟病接种试验证实,所用标记不同,分子标记辅助选择的效率差异显著,单个标记辅助选择符合率均不高,而采用目标基因两侧连锁的标记同时进行辅助选择,则可以明显提高分子标记辅助选择符合率。     

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。     

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。     

Breeding Rice Restorer Lines with High Resistance to Bacterial Blight by Using Molecular Marker-Assisted Selection

Rice bacterial blight, one of the major diseases of rice caused by Xanthomonas oryzae pv. oryzae, Xoo, jeopardizes rice diversely. It causes leaf wilting, affects photosynthesis and reduces 1000-grain weight and generally results in yield loss by 20-30 pe…     

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.     

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。     

籼型水稻中稻瘟病抗性基因分布及抗性研究

稻瘟病严重威胁着水稻的安全生产。目前,培育抗性品种是控制稻瘟病危害最经济有效的途径之一。本研究利用11个主效稻瘟病抗性(R)基因(Pi2、Piz-t、Pi9、Pi54、Pik-m、Pid3、Pib、Pit、Pi5、Ptr和Pita)的分子标记对48个常规稻、15个不育系和129个杂交稻进行了检测。结果表明,在常规稻中,Ptr和Pi5的分布频率均为35.42%,Pib、Pi2、Piz-t、Pit的分布频率介于20.0%~30.0%之间,其余均在15%以下;在不育系中,Pita的分布频率为40.0%,其余均在20%以下;杂交稻中,Pita、Pib、Pi54、Ptr和Pi5的分布频率在40.31%~55.04%之间,其余均在20%以下;Pita在各个品系中均广泛存在,频率介于35.42%~51.16%。田间抗性评价表明,R基因较多的品种具有较高的抗性。综上所述,在参试的水稻材料中,不育系中存在抗性基因较少,所有类型材料中广谱抗性基因分布较少,抗性基因聚合可以有效提高稻瘟病田间抗性。     

Avoidance of Linkage Drag Between Blast Resistance Gene and the QTL Conditioning Spikelet Fertility Based on Genotype Selection Against Heading Date in Rice

Previous study showed that a linkage drag between a blast resistance gene Pi25(t) and QTLs conditioning spikelet fertility (qSF-6) and number of filled grains per panicle (qNFGP-6) was detected on the short arm of chromosome 6. A larger population was used for further verification, and the results confirmed the linkage drag between the blast resistance gene and QTL conditioning spikelet fertility, other than QTL conditioning number of filled grains per panicle. Breakdown or avoidance of the linkage drag could be achieved by selection against the genotype background of a heading-date gene (qHD-7) that resided in the region between RM2 and RM214 on chromosome 7. For further validation, two lines with almost identical genotypes on all chromosomal regions except the Pi25(t) region on chromosome 6 were chosen to develop a new population. The results showed that qSF-6 could be further subdivided into qSF-6-1 and qSF-6-2. When the genotype of the region between RM2 and RM214 was from rice variety Zhong 156, the linkage drag between Pi25(t) and qSF-6-2 was detected and the allele of qSF-6-2 from rice variety Gumei 2 reduced the spikelet fertility. When the genotype of the region between RM2 and RM214 was from Gumei 2, no linkage drag was detected. This indicates that the linkage drag between the blast resistance gene and the QTL conditioning spikelet fertility could be broken down or avoided under a certain background genotype selection against heading-date and provides a marker aided solution for high level of blast resistance and yield breeding in rice and other crops as well.     

Race Specificity of Major Rice Blast Resistance Genes to Magnaporthe grisea Isolates Collected from indica Rice in Guangdong, China

Race-specific resistance and field resistance of 30 rice blast resistance monogenic lines dedved from different resources were evaluated. The spectra of resistance to 163 Magnaporthe grisea isolates collected from indica rice in Guangdong Province, China ranged from 0.6% to 89.6%. Most of the monogenic lines showed a narrow resistance spectrum and high susceptibility in rice blast area, whereas the lines with Pikh and Pi1(t) had the broad resistance spectra of 89.6% and 82.2% respectively, showing a high and stable blast resistance in fields. According to the cluster analysis of specific resistance to 163 blast isolates tested, the 30 monogenic lines were divided into 15 groups, and based on the principal factor analysis, nine kinds of race-specific resistance were identified. Pik, Piz5, Pi9 and Pish can be used as candidate resistance genes for rice breeding since their specific resistance differed from those of the backbone parents in Guangdong, China. Gene pyramiding of Pikh [or Pi1(t)], Pi9 (or Piz5) and Pish (or Pita2) will be effective to obtain broad-spectrum blast resistance in rice breeding program in Guangdong, China. The strategies for studying and application of rice blast resistance genes were discussed.