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.     

Tagging and combining bacterial blight resistance genes in rice using RAPD and RFLP markers

Four genes of rice,Oryza sativa L., conditioning resistance to the bacterial blight pathogenXanthomonas oryzae pv.oryzae (X. o. pv.oryzae), were tagged by restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD) markers. No recombinants were observed betweenxa-5 and RFLP marker lociRZ390, RG556 orRG207 on chromosome 5.Xa-3 andXa-4 were linked to RFLP locusXNpb181 at the top of chromosome 11, at distances of 2.3 cM and 1.7 cM, respectively. The nearest marker toXa-10, also located on chromosome 11, was the RAPD locusO07 ₂₀₀₀ at a distance of 5.3 cM. From this study, the conventional map [19, 28] and two RFLP linkage maps of chromosome 11 [14, 26] were partially integrated. Using the RFLP and RAPD markers linked to the resistance genes, we selected rice lines homozygous for pairs of resistance genes,Xa-4 +xa-5 andXa-4 +Xa-10. Lines carryingXa-4 +xa-5 andXa-4 +Xa-10 were evaluated for reaction to eight strains of the bacterial blight pathogen, representing eight pathotypes and three genetic lineages. As expected, the lines carrying pairs of genes were resistant to more of the isolates than their single-gene parental lines. Lines carryingXa-4 +xa-5 were more resistant to isolates of race 4 than were either of the parental lines (quantitative complementation). No such effects were seen forXa-4 +Xa-10. Thus, combinations of resistance genes provide broader spectra of resistance through both ordinary gene action expected and quantitative complementation.     

Pyramiding of bacterial blight resistance genes in rice: marker-assisted selection using RFLP and PCR

 DNA marker-assisted selection was used to pyramid four bacterial blight resistance genes, Xa-4, xa-5, xa-13 and Xa-21. Breeding lines with two, three and four resistance genes were developed and tested for resistance to the bacterial blight pathogen (Xanthomonas oryzae pv. oryzae). The pyramid lines showed a wider spectrum and a higher level of resistance than lines with only a single gene. To speed up the gene pyramiding process and to facilitate future marker-aided selection, we developed PCR markers for the two recessive genes, xa-5 and xa-13, and used these to survey a range of rice germplasm. The results of the germplasm survey will be useful for the selection of parents in breeding programs aimed at transferring these bacterial blight resistance genes from one varietal background to another. Received: 6 December 1996/Accepted: 20 December 1996     

云南30个水稻品种对水稻白叶枯病的抗性评价

【目的】水稻白叶枯病是一种严重危害水稻的细菌性病害,培育抗性品种是治理该病害的重要途径。因此,明确云南水稻材料对检疫性病害水稻白叶枯病的抗性,可以为该病害的防治与监测、水稻栽培的合理布局和良好抗性资源的获取提供依据。【方法】采用剪叶接种法测定云南稻区30个品种对7个不同致病型白叶枯病菌的抗性。【结果】在供试的30个云南水稻品种中,2个品种(玉粳16和JS42糯稻)对7个不同致病型菌株均表现为抗性;15个品种对7个致病型菌株均表现感病;对HEN11、SCYC-6、YN7、YN11、FUJ、YN241和PX099等7个致病型菌株表现抗性的水稻品种分别占26.67%、16.67%、23.33%、13.33%、6.67%、10.00%和20.00%。此外,区试材料的抗性比例高于主栽品种,地方稻未发现抗性品种。【结论】现在生产上的大部分水稻品种对优势致病型病原菌入侵的抵抗能力降低甚至丧失。针对云南地区的优势致病小种FUJ筛选得到2个抗性品种:玉粳16和JS42糯稻。     

Characterization of quantitative trait loci (QTLs) in cultivated rice contributing to field resistance to sheath blight (Rhizoctonia solani)

Sheath blight, caused by Rhizoctonia solani, is one of the most important diseases of rice. Despite extensive searches of the rice germ plasm, the major gene(s) which give complete resistance to the fungus have not been identified. However, there is much variation in quantitatively inherited resistance to R. solani, and this type of resistance can offer adequate protection against the pathogen under field conditions. Using 255 F₄ bulked populations from a cross between the susceptible variety Lemont and the resistant variety Teqing, 2 years of field disease evaluation and 113 well-distributed RFLP markers, we identified six quantitative trait loci (QTLs) contributing to resistance to R. solani. These QTLs are located on 6 of the 12 rice chromosomes and collectively explain approximately 60% of the genotypic variation or 47% of the phenotypic variation in the LemontxTeqing cross. One of these resistance QTLs (QSbr4a), which accounted for 6% of the genotypic variation in resistance to R. solani, appeared to be independent of associated morphological traits. The remaining five putative resistance loci (QSbr2a, QSbr3a, QSbr8a, QSbr9a and QSbr12a) all mapped to chromosomal regions also associated with increased plant height, three of which were also associated with QTLs causing later heading. This was consistent with the observation that heading date and plant height accounted for 47% of the genotypic variation in resistance to R. solani in this population. There were also weak associations between resistance to R. solani and leaf width, which were likely due to linkage with a QTL for this trait rather than to a physiological relationship.     

RFLP mapping of major and minor genes for important agronomic characters in rice

A molecular genetic map with 233 RFLP markers which covered about 2070 cM of rice genome was constructed based on a doubled haploid (DH) population derived from anther culture of a cross between an indica variety Gui630 and a japonica variety 02428. Quantitative trait loci (QTLs) for agronomic characters such as number of panides, heading date, plant height, number of spikelets, number of grains, fertility and 1 000-grain weight were analyzed using interval mapping approach. 8 major genes and 29 minor genes were identified associating with these traits. The results also indicated that great phenotypic difference between parents was profitable in detection of major genes.     

RAPD and RFLP mapping of the bacterial blight resistance gene xa-13 in rice

Bacterial blight (BB) caused by Xanthomonas oryzae pv oryzae (Xoo) is one of the most serious diseases of rice. The recessive gene xa-13 confers resistance to Philippine race 6 of Xoo. To tag xa-13 with molecular markers, RAPD analysis was conducted with the combined use of near-isogenic lines and bulked segregant analysis. From the survey of 260 arbitrary 10-nucleotide primers, one primer (OPAC05) was detected to amplify specifically a 0.9-kb band from the DNA of susceptible plants. The distance between the RAPD marker OPAC05-900 and xa-13 was estimated to be 5.3 cM. The RAPD marker was then mapped on chromosome 8 using a mapping population of doubled haploid lines derived from the cross of IR64/Azucena. The linkage between RFLP markers and the RAPD marker was analyzed using an F₂ population of 135 plants derived from a cross between a near-isogenic line for xa-13, IR66699-5-5-4-2, and IR24. No recombinants were found between RZ28 and CDO116 and their distance from xa-13 was estimated to be 4.8 cM. RG136 was located at 3.7 cM on the other side of xa-13. The mapping of xa-13 with closely linked DNA markers provides the basis for marker-aided selection for rice improvement.Department of Agronomy, South China Agricultural University, Guangzhou, China     

Induction of bacterial blight (Xanthomonas oryzae pv. oryzae) resistance in rice by treatment with acibenzolar-S-methyl

The role of the plant defence activator, acibenzolar‐S‐methyl (ASM), in inducing resistance in rice against bacterial leaf blight caused by Xanthomonas oryzae pv. oryzae (Xoo) was studied. Application of ASM induced resistance in rice to infection by Xoo. When the pathogen was clip‐inoculated to the rice plants, it caused bacterial leaf blight symptoms in the untreated control. However, in the rice plants pretreated with ASM, infection was significantly reduced. Induced systemic resistance was found to persist for up to 3 days in the pretreated rice plants. Increased phenolic content and accumulation of pathogenesis‐related (PR) proteins, viz. chitinase, β‐1,3‐glucanase and thaumatin‐like protein (TLP; PR 5) were observed in rice plants pretreated with ASM followed by inoculation with Xoo. Immunoblot analysis using rice TLP and tobacco chitinase antiserum revealed rapid induction and over‐expression of 25 and 35 kDa TLP and chitinase, respectively, in rice in response to pretreatment with ASM followed by Xoo inoculation. Based on these experiments, it is evident that induction of disease resistance in rice was accelerated following treatment with ASM.     

RFLP mapping of QTLs for yield and related characters in rice (Oryza sativa L.)

Quantitative triat loci (QTLs) for yield and related traits in rice were mapped based on RFLP maps from two indica/indica F₂ populations, Tesanai 2/CB and Waiyin 2/CB. In Tesanai 2/CB, 14 intervals carrying QTLs for eight traits were detected, including 3 for grain weight per plant (GWT), 2 for number of panicles per plant (NP), 2 for number of grains per panicle (NG), 1 for total number of spikelets per panicle (TNS), 1 for spikelet fertility (SF), 3 for 1000-grain weight (TGWT), 1 for spikelet density (SD), and 1 for number of first branches per main panicle. The 3 QTLs for GWT were located on chromosomes 1, 2, and 4, with 1 in each chromosome. The additive effect of the single locus ranged from 2.0 g to 9.1 g. A major gene (np4) for NP was detected on chromosome 4 within the interval of RG143–RG214, about 4cM for RG143, and this locus explained 26.1% of the observed phenotypic variance for NP. The paternal allele of this locus was responsible for reduced panicles per plant (3 panicles per plant). In another population, Waiyin 2/CB, 12 intervals containing QTLs for six of the above-mentioned traits were detected, including 3 for GWT, 2 for each of NP, TNS, TGWT and SD, 1 for SF. Three QTLs for GWT were located on chromosome 1, 4, and 5, respectively. The additive effect of the single locus for GWT ranged from 6.7 g to 8.8 g, while the dominance effect was 1.7–11.5 g. QTL mapping in two populations with a common male parent is compared and discussed.     

Pyramiding transgenic resistance in elite indica rice cultivars against the sheath blight and bacterial blight

Elite indica rice cultivars were cotransformed with genes expressing a rice chitinase (chi11) and a thaumatin-like protein (tlp) conferring resistance to fungal pathogens and a serine-threonine kinase (Xa21) conferring bacterial blight resistance, through particle bombardment, with a view to pyramiding sheath blight and bacterial blight resistance. Molecular analyses of putative transgenic lines by polymerase chain reaction, Southern Blot hybridization, and Western Blotting revealed stable integration and expression of the transgenes in a few independent transgenic lines. Progeny analyses showed the stable inheritance of transgenes to their progeny. Coexpression of chitinase and thaumatin-like protein in the progenies of a transgenic Pusa Basmati1 line revealed an enhanced resistance to the sheath blight pathogen, Rhizoctonia solani, as compared to that in the lines expressing the individual genes. A transgenic Pusa Basmati1 line pyramided with chi11, tlp, and Xa21 showed an enhanced resistance to both sheath blight and bacterial blight. S. Maruthasalam and K. Kalpana have contributed to this article equally.     

Genetic mapping of resistance to bacterial blight disease in cassava (Manihot esculenta Crantz)

Cassava bacterial blight (CBB), caused by Xanthomonas axonopodis pv. manihotis (Xam), is a major disease of cassava (Manihot esculenta Crantz) in Africa and South America. Planting resistant varieties is the preferred method of disease control. Recent genetic mapping of an F₁ cross (TMS 30572 × CM 2177–2) led to the construction of the first molecular genetic map of cassava. To better understand the genetics of resistance to CBB, we evaluated individuals of the F₁ cross for CBB resistance by controlled greenhouse inoculations and visually assessed symptoms on days 7, 15, and 30 days after inoculation, using a scale where 0 = no disease and 5 = maximum susceptibility. Five Xam strains were used: CIO-84, CIO-1, CIO-136, CIO-295, and ORST X-27. Area under the disease progress curve (AUDPC) was used as a quantitative measure of resistance in QTL analysis by single-marker regression. Based on the AUDPC values, eight QTLs (quantitative trait loci), located on linkage groups B, D, L, N, and X of the female-derived framework map, were found to explain 9–20% of the phenotypic variance of the crop’s response to the five Xam strains. With the male-derived framework map, four QTLs on linkage groups G and C explained 10.7–27.1% of the variance. A scheme to confirm the usefulness of these markers in evaluating segregating populations for resistance to CBB is proposed. Received: 20 September 1999 / Accepted: 30 December 1999     

Identification of QTLs involved in the resistance to South American leaf blight (Microcyclus ulei) in the rubber tree

South American leaf blight (SALB) is a disease of the rubber tree caused by the fungus Microcyclus ulei. Quantitative trait loci (QTLs) for resistance were mapped using 195 F₁ progeny individuals derived from the cross between a susceptible cultivated clone, PB260, and a resistant clone, RO38, derived from interspecific hybridization. The resistance level of the progeny individuals was evaluated in controlled conditions. The reaction type (RT) and the lesion diameter (LD) were measured on immature leaves after artificial inoculation of the fungus. Five different strains of the fungus were used, all highly sporulating on PB260. Among those, four did not sporulate and one sporulated partially on RO38. Both pseudo-testcross parental genetic maps and the consensus map were constructed. The search for QTLs was performed using the Kruskal-Wallis marker-by-marker test and the Interval-Mapping method for the three maps. Eight QTLs for resistance were identified on the RO38 map. Only one QTL was detected on the PB260 map. The analysis of the F₁ consensus map confirmed results obtained with the parental maps. A common QTL was detected for resistance to the five strains for both RT and LD. Two QTLs were common for complete resistance to four strains, for RT and LD respectively. Resistance determinism for complete and partial resistance, and perspectives for breeding for durable resistance to SALB are discussed. Received: 1 August 1999 / Accepted: 27 August 1999     

Races of Xanthomonas citri subsp. malvacearum,the causal organism of bacterial blight of cotton in northern Nigeria

Xanthomonas citri subsp. malvacearum (Xcm) causes severe qualitative and quantitative losses to farmers in cotton-growing areas of the world. Isolates of Xcm were extracted from cotton seeds obtained from five ginneries located in Funtua, Malumfashi, Gusau and Zaria and standardised to 10^?5?cfu/ml. One isolate per location was used to inoculate three sets of 10 cotton differential lines known to differentiate races of Xcm through possession of B-genes for resistance to bacterial blight. Each cotton differential line was inoculated with the isolates at the six-leaf stage and SDW was inoculated as control. One hundred and sixty pots used were arranged in completely randomised design on the screen house bench. Four different pathogenic races were identified namely race 1, race 12, race 13 and race 16. This confirms the existence of an evolution of the species across northern Nigeria.     

Bacterial leaf blight resistance genes (Xa21, xa13 and xa5) pyramiding through molecular marker assisted selection into rice cultivars

Marker assisted selection was employed to pyramid three bacterial blight resistance genes Xa21, xa13 and xa5 into high yielding susceptible rice cultivars ADT43 and ADT47. With the assistance of PCR markers, homozygous and heterozygous genotypes were identified in F₂ generation of two crosses (ADT43 × IRBB60 and ADT47 × IRBB60) and goodness of fit was tested. Eighty nine plants from F₃ generation of ADT43 × IRBB60 were also screened for resistance genes. The genotypes carrying resistance genes in different combinations were identified. The pyramided lines showed a wider spectrum and higher level of resistance against two Xoo isolates under field conditions.     

Integrating marker-assisted background analysis with foreground selection for pyramiding bacterial blight resistance genes into Basmati rice

Bacterial leaf blight (BB), caused by the bacterium Xanthomonas oryzae pv. Oryzae (Xoo), is the major constraint amongst rice diseases in India. CSR-30 is a very popular high-yielding, salt-tolerant Basmati variety widely grown in Haryana, India, but highly susceptible to BB. In the present study, we have successfully introgressed three BB resistance genes (Xa21, xa13 and xa5) from BB-resistant donor variety IRBB-60 into the BB-susceptible Basmati variety CSR-30 through marker-assisted selection (MAS) exercised with stringent phenotypic selection without compromising the Basmati traits. Background analysis using 131 polymorphic SSR markers revealed that recurrent parent genome (RPG) recovery ranged up to 97.1% among 15 BC₃F₁ three-gene-pyramided genotypes. Based on agronomic evaluation, BB reaction, aroma, percentage recovery of RPG, and grain quality evaluation, four genotypes, viz., IC-R28, IC-R68, IC-R32, and IC-R42, were found promising and advanced to BC₃F₂ generation.     

水稻对白叶枯病和细菌性条斑病的抗性遗传研究进展

水稻白叶枯病和水稻细菌性条斑病是由稻黄单胞细菌(Xanthomonas oryzae)不同致病变种引起的两种最重要的水稻细菌性病害。发掘和利用抗性基因,培育抗病品种是防治这两种病害的最有效手段之一。本文分别综述了这两种高度相关的病害的抗性遗传研究进展,包括已发掘和利用的主效抗性基因特点及目前国内外对这两种病害的抗性QTL定位研究进展,为水稻抗白叶枯病和细菌性条斑病育种研究提供有用信息。     

Mapping of QTLs conferring resistance to bacterial leaf streak in rice

A large F₂ and a RI population were separately derived from a cross between two indica rice varieties, one of which was highly resistant to bacterial leaf streak (BLS) and the other highly susceptible. Following artificial inoculation of the RI population and over 2 years of testing, 11 QTLs were mapped by composite interval mapping (CIM) on six chromosomes. Six of the QTLs were detected in both seasons. Eight of the QTLs were significant following stepwise regression analysis, and of these, 5 with the largest effects were significant in both seasons. The detected QTLs explained 84.6% of the genetic variation in 1997. Bulked segregant analysis (BSA) of the extremes of the F₂ population identified 3 QTLs of large effect. The 3 QTLs were dentical to 3 of the 5 largest QTLs detected by CIM. The independent detection of the same QTLs using two methods of analysis in separate mapping populations verifies the existence of the QTLs for BLS and provides markers to ease their introduction into elite varieties. Received: 13 October 1999 / Accepted: 29 October 1999     

Pyramiding three bacterial blight resistance genes (xa5, xa13 and Xa21) using marker-assisted selection into indica rice cultivar PR106

Bacterial blight (BB) of rice caused by Xanthomonas oryzae pv. oryzae (Xoo) is a major disease of rice in several countries. Three BB resistance genes, xa5, xa13 and Xa21, were pyramided into cv. PR106, which is widely grown in Punjab, India, using marker-assisted selection. Lines of PR106 with pyramided genes were evaluated after inoculation with 17 isolates of the pathogen from the Punjab and six races of Xoo from the Philippines. Genes in combinations were found to provide high levels of resistance to the predominant Xoo isolates from the Punjab and six races from the Philippines. Lines of PR106 with two and three BB resistance genes were also evaluated under natural conditions at 31 sites in commercial fields. The combination of genes provided a wider spectrum of resistance to the pathogen population prevalent in the region; Xa21 was the most effective, followed by xa5. Resistance gene xa13 was the least effective against Xoo. Only 1 of the BB isolates, PX04, was virulent on the line carrying Xa21 but avirulent on the lines having xa5 and xa13 genes in combination with Xa21. Received: 26 May 2000 / Accepted: 16 August 2000     

Identification and fine mapping of a new bacterial blight resistance gene,Xa43(t), in Zhangpu wild rice (Oryza rufipogon)

• Bacterial blight (BB) is currently considered one of the most serious rice diseases and is caused by Xanthomonas oryzae pv. oryzae (Xoo). Numerous studies have shown that breeding resistant rice varieties is one of the most effective methods to prevent BB, and it is important to identify and isolate more BB resistance (R) genes from different rice resources.
• Using a map-based approach, we identified a new QTL/gene, Xa43(t), from ZhangPu wild rice, which was highly resistant to the BB isolate PX099. We performed bulked segregant analysis combined with candidate gene prediction to identify the candidate gene.
• The Xa43(t) gene was narrowed down to a 29-kb region containing four putative genes. More importantly, the candidate gene Xa43(t) did not affect the main agronomic traits of rice. We also identified a widely applicable molecular marker, namely Inde1-18, which co-segregates with the Xa43(t) gene.
• The Xa43(t) gene is a new broad-spectrum BB resistance gene without identified alleles and has good application prospects for rice disease resistance breeding.