Microsatellite and sequence-tagged site markers diagnostic for the rice bacterial leaf blight resistance gene xa-5

 Microsatellite and sequence-tagged site (STS) markers tightly linked to the bacterial leaf blight (BLB) resistance gene xa-5 were identified in this study. A survey was conducted to find molecular markers that detected polymorphisms between the resistant (IRBB5) and susceptible (‘IR24’) nearly isogenic lines for xa-5, and between Chinsurah Boro II (CBII), an alternative source of xa-5, and a widely planted variety (‘IR64’) that lacks xa-5. Two F₂ populations, from the crosses ‘IR24’×IRBB5 and CBIIבIR64’, were used to estimate linkage based on marker genotype and reaction to disease inoculation with Xanthomonas oryzae pv. oryzae. Two RFLP clones, RZ390 and RG556, were found to co-segregate with xa-5 and were converted into STS markers. A microsatellite marker, RM390, was developed based on a simple sequence repeat in the 5′ untranslated region of the cDNA probe, RZ390, and found to co-segregate with resistance. Two other microsatellites, RM122 and RM13, were located 0.4 cM and 14.1 cM away from xa-5. A germplasm survey of diverse lines containing BLB resistance genes using automated fluorescent detection indicated the range of allelic diversity for each of the microsatellite loci linked to xa-5 and confirmed their usefulness in following genes through the narrow crosses typical of a breeding program. The limited number of alleles observed at the microsatellite loci linked to the resistance gene in 35 xa-5-containing accessions suggested either a single ancestral origin or a few independent origins of the xa-5 gene. PCR-based markers, like the ones developed in this study, are economical and easy to use, and have applicability in efforts to pyramid the recessive xa-5 gene with other BLB resistance genes. Received: 27 September 1996/Accepted: 7 February 1997     

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.     

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.     

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     

Effective strategy for pyramiding three bacterial blight resistance genes into fine grain rice cultivar, Samba Mahsuri, using sequence tagged site markers

Bacterial leaf blight (BB) of rice is a major disease limiting rice production in several rice growing regions of the world. The pathogen, Xanthomonas oryzae pv oryzae, causing the disease is highly virulent to rice crops and is capable of evolving new races. Breeding efforts to incorporate single BB resistant gene often leads to resistance breakdown within a short period. To overcome such breakdown of resistance and develop germplasm with durable disease resistance, we have introgressed three bacterial blight resistance genes, xa5, xa13, and Xa21 into a fine grain rice variety, Samba Mahsuri, using sequence tagged site (STS) markers linked to these genes. Since the efficiency of the STS markers linked to recessive genes to detect homozygotes is less than 100%, we adopted four different pyramiding schemes to minimize loss of recessive resistance genes in advanced backcross generations. Pyramiding scheme A in which a two-gene Samba Mahsuri pyramid line containing Xa21 and xa5 genes was crossed with the Samba Mahsuri line having xa13 gene alone was found to be most effective in preventing the loss of an important recessive gene xa13. We further demonstrated that there was no yield penalty due to pyramiding of multiple genes into the elite indica rice variety.     

Induction of systemic resistance in rice to bacterial blight by 1,2,3-benzothiadiazole 7-carbothioic acid-S-methyl ester (BTH) treatments

Use of BTH to evaluate the disease severity and induction of systemic resistance in rice to bacterial blight caused by Xanthomonas oryzae pv. oryzae is investigated. A new batch of 25 isolates of Xanthomonas oryzae pv. oryzae was obtained from infected rice lead tissues collected from Pattambi, Kerala, south India. Their identification was confirmed by the plant inoculation test on to IR24 rice plants which produced characteristic bacterial blight lesions. Among the 25 of X.o. pv. oryzae, four of the isolates were also virulent to IRBB21 rice plants (a near isogenic line of IR24) which carry the Xa-21 gene for BB resistance. The results confirm that there are pathogen strains in India which can overcome Xa-21. Development of BB lesions developed in IR24 (BB susceptible) plants after they were treated with BTH applications either as seed treatment or as foliar spray at 0.1, 0.5, 0.1 and 2.0 mM concentrations showed that even at 2.0 mM concentrations, IR24 plants were still susceptible to the pathogen. There was very little or marginal effect of BTH on the induction of resistance to BB in IR24 rice plants. When the same concentrations of BTH were applied to IRBB21 (Xa-21) rice plants, they showed pronounced triggering of systemic resistance to BB pathogen even at 0.1 mM concentration of BTH applied either as seed treatment or as foliar spry. Disease severity index was reduced to 5 (against a score of 9 in untreated) and there was 85–86% reduction in BB incidence in plants that received 0.1 mM BTH. These results provide evidence that BTH-induced systemic resistance complements the R-gene resistance in IRBB21 plants but not in IR24 rice plants.     

Evaluation of resistance genes in rice against local isolates of Xanthomonas oryzae pv. oryzae in Punjab Province of Pakistan

Absence of resistance/tolerance against bacterial leaf blight (BLB), incited by Xanthomonas oryzae pv. oryzae, in famous basmati varieties is one of the main reason for BLB epidemic in Punjab in 2007–2008. For developing resistance against BLB, the response of 26 IRBB lines of IRRI including 10 near isogenic lines (NILs) and 16 gene pyramids carrying two to five resistance genes (Xa series) was evaluated against 61 indigenous Xoo isolates under artificial inoculation field conditions. None of the NILs or gene pyramid provides complete protection against all the isolates. However, Xa21 and xa13 were found resistant against the majority of Xoo isolates, followed by Xa14 and Xa7. Of the 16 gene pyramids used in this study, IRBB-54 (Xa5 + Xa21), IRBB-55 (Xa13 + Xa21) followed by IRBB-58 (Xa4 + Xa13 + Xa21) were found effective against the majority of the Xoo isolates. These resistance genes (individually and in combinations) can be incorporated for the improvement of basmati rice cultivars cultivated in Punjab province of Pakistan. Effectiveness of gene combination supports the strategy of pyramiding appropriate resistance genes. Newly identified resistant genes may also be evaluated for achieving broad spectrum resistance against more Xoo isolates of the area.     

RFLP- and physical mapping of resistance gene homologues in rice (O. sativa) and Barley (H. vulgare)

 The deduced peptide sequences of 25 gene fragments of NBS-LRR resistance (R) gene homologues from rice and barley and of characterized R genes were compared, revealing a string of six conserved motifs. Mapping of the R-gene candidates in rice showed linkage to genes conferring race-specific resistance to rice blast (Pi-k, Pi-f and Pi-1) and bacterial blight disease (Xa-1, Xa-3 and Xa-4), in barley to powdery mildew (Mla) and the rust fungus (Rpg1). In rice four mixed clusters were detected, each harboring at least two highly dissimilar NBS-LRR genes. A YAC-contig was established for one of these mixed clusters. YAC fragmentation experiments revealed the presence of at least five NBS-LRR genes within 200 kb in head-to-tail orientation. Received: 24 July 1998 / Accepted: 14 August 1998     

Targeting xa13, a recessive gene for bacterial blight resistance in rice

Bacterial blight, caused by Xanthomonas oryzae pv. oryzae (Xoo), is one of the most serious diseases of rice worldwide. Thirty bacterial blight resistance (R) genes (21 dominant genes and 9 recessive genes) in rice have been identified. They are the main sources for the genetic improvement of rice for resistance to Xoo. However, little is known about the recessive R genes. To clone and characterize the recessive R genes, we fine-mapped xa13, a fully recessive gene for Xoo resistance, to a DNA fragment of 14.8 kb using the map-based cloning strategy and a series of sequence-based molecular markers. Sequence analysis of this fragment indicated that this region contains only two apparently intact candidate genes (an extensin-like gene and a homologue of nodulin MtN3) and the 5′ end of a predicted hypothetical gene. These results will greatly facilitate the isolation and characterization of xa13. Four PCR-based markers, E6a, SR6, ST9 and SR11 that were tightly linked to the xa13 locus, were also developed. These markers will be useful tools for the marker-assisted selection of xa13 in breeding programs.     

Identification of a family of avirulence genes from Xanthomonas oryzae pv. oryzae.

Races of Xanthomonas oryzae pv. oryzae, the causal agent of bacterial blight of rice, interact with cultivars of rice in a gene-for-gene specific manner. Multiple DNA fragments of various sizes from all strains of X. o. pv. oryzae hybridized with avrBs3, an avirulence gene from Xanthomonas campestris pv. vesicatoria, in Southern blots; this suggests the presence of several homologs and possibly a gene family. A genomic library of a race 2 strain of X. o. pv. oryzae, which is avirulent on rice cultivars carrying resistance genes xa-5, Xa-7, and Xa-10, was constructed. Six library clones, which hybridized to avrBs3, altered the interaction phenotype with rice cultivars carrying either xa-5, Xa-7, or Xa-10 when present in a virulent race 6 strain. Two avirulence genes, avrXa7 and avrXa10, which correspond to resistance genes Xa-7 and Xa-10, respectively, were identified and partially characterized from the hybridizing clones. On the basis of transposon insertion mutagenesis, sequence homology, restriction mapping, and the presence of a repeated sequence, both genes are homologs of avirulence genes from dicot xanthomonad pathogens. Two BamHI fragments that are homologous to avrBs3 and correspond to avrXa7 and avrXa10 contain a different number of copies of a 102-bp direct repeat. The DNA sequence of avrXa10 is nearly identical to avrBs3. We suggest that avrXa7 and avrXa10 are members of an avirulence gene family from xanthomonads that control the elicitation of resistance in mono- and dicotyledonous plants.     

Combining bacterial blight resistance and Basmati quality characteristics by phenotypic and molecular marker-assisted selection in rice

Bacterial Blight (BB) caused by Xanthomonas oryzae pv. oryzae is a major disease of rice in tropical Asia. Since all the Basmati varieties are highly susceptible and the disease is prevalent in the entire Basmati growing region of India, BB is a severe constraint in Basmati rice production. The present study was undertaken with the objective of combining the important Basmati quality traits with resistance to BB by a combination of phenotypic and molecular marker-assisted selection (MAS). Screening of 13 near-isogenic lines of rice against four isolates of the pathogen from Basmati growing regions identified the Xa4, xa8, xa13 and Xa21 effective against all the isolates tested. Two or more of these genes in combination imparted enhanced resistance as expressed by reduced average lesion length in comparison to individual genes. The two-gene pyramid line IRBB55 carrying xa13 and Xa21 was found equally effective as three/four gene pyramid lines. The two BB resistance genes present in IRBB55 were combined with the Basmati quality traits of Pusa Basmati-1 (PB-1), the most popular high yielding Basmati rice variety used as recurrent parent. Phenotypic selection for disease resistance, agronomic and Basmati quality characteristics and marker-assisted selection for the two resistance genes were carried out in BC₁F₁, BC₁F₂ and BC₁F₃ generations. Background analysis using 252 polymorphic amplified fragment length polymorphism (AFLP) markers detected 80.4 to 86.7% recurrent parent alleles in BC₁F₃ selections. Recombinants having enhanced resistance to BB, Basmati quality and desirable agronomic traits were identified, which can either be directly developed into commercial varieties or used as immediate donors of BB resistance in Basmati breeding programs.     

Genetic enhancement of host plant-resistance of the Lalat cultivar of rice against bacterial blight employing marker-assisted selection

To incorporate durable resistance against bacterial blight, a major disease rice, three resistance genes, xa 5, xa13 and Xa21, from IRBB 60 were transferred through marker-assisted backcrossing using RG 556, RG 136 and pTA248 markers linked to the three genes to supplement the Xa4 gene present in Lalat, a popular rice cultivar. Effective selection enabled the transfer in three back-crosses and a generation of selfing and background selection employing morphological and grain quality traits and molecular markers, led to >90 % recovery of the recurrent parental genome. The gene pyramids exhibited high levels of resistance against the pathogen in multi-location evaluation trials conducted over several locations of bacterial blight in India. IL-2 (CRMAS2621-7-1), a gene pyramid, was identified as being promising for several endemic regions of bacterial blight and was released as Improved Lalat in one of the identified regions. The success of the study demonstrates the vast potential of marker-assisted selection for gene stacking and recovery of the parental genome with high precision.     

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.     

Identification of a YAC clone carrying the Xa-1 allele, a bacterial blight resistance gene in rice

Map-based cloning methods have been applied for isolation of Xa-1, one of the bacterial blight resistance genes in rice.Xa-1 was previously mapped on chromosome 4 using molecular markers. For positional cloning of Xa-1, a high-resolution genetic map was made for theXa-1 region using an F₂ population of 402 plants and additional molecular markers. Three restriction fragment length polymorphism (RFLP) markers, XNpb235, XNpb264 and C600 were found to be linked tightly to Xa-1, with no recombinants, and U08 ₇₅₀ was mapped 1.5 cM from Xa-1. The screening of a yeast artificial chromosome (YAC) library using theseXa-1-linked RFLP markers resulted in the identification of ten contiguous YAC clones. Among these, one YAC clone, designated Y5212, with an insert of 340 kb, hybridized with all three tightly linked markers. This YAC was confirmed to possess the Xa-1 allele by mapping the Xa-1 gene between both end clones of this YAC (Y5212R and Y5212L).     

水稻抗白叶枯病基因Xa-25的分子定位

Xa-25是从体细胞突变体HX-3中鉴定出的水稻抗白叶枯病基因。通过花药培养构建了02428(粳稻)和HX-3(籼稻)的双单倍体(DH)群体,该群体包含了129个稳定株系,以我国长江流域水稻白叶枯病的代表菌株浙173对DH群体进行抗病性鉴定,抗病株系数和感病株系数分别为62和67。共选用覆盖水稻12条染色体的300对SSR引物对02428和HX-3进行多态性分析,有74对引物在双亲之间表现差异。利用这些差异引物对DH群体进行连锁分析,从而将抗白叶枯病基因Xa-25定位到第4染色体长臂末端的两个SSR标记RM6748和RM1153之间,连锁距离分别为9．3cM和3．0cM。     

Fine genetic mapping of <Emphasis Type="Italic">xa24</Emphasis>, a recessive gene for resistance against <Emphasis Type="Italic">Xanthomonas oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzae</Emphasis> in rice

Bacterial blight, caused by Xanthomonas oryzae pv. oryzae (Xoo), is the most devastating plant bacterial disease worldwide. Different bacterial blight resistance (R) genes confer race-specific resistance to different strains of Xoo. We fine mapped a fully recessive gene, xa24, for bacterial blight resistance to a 71-kb DNA fragment in the long arm of rice chromosome 2 using polymerase chain reaction-based molecular markers. The xa24 gene confers disease resistance at the seedling and adult stages. It mediates resistance to at least the Philippine Xoo races 4, 6 and 10 and Chinese Xoo strains Zhe173, JL691 and KS-1-21. Sequence analysis of the DNA fragment harboring the dominant (susceptible) allele of xa24 suggests that this gene should encode a novel protein that is not homologous to any known R proteins. These results will greatly facilitate the isolation and characterization of xa24. The markers will be convenient tools for marker-assisted selection of xa24 in breeding programs. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Population structure analysis and association mapping of bacterial blight resistance in <Emphasis Type="Italic">indica</Emphasis> rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) accessions

Bacterial blight, caused by Xanthomonas oryzae pv. oryzae (Xoo), is a serious disease in rice production worldwide. To understand the genetic diversity of bacterial blight resistance a population consisting of 175 indica accessions from nine countries was collected and detected their association between SSR (Simple Sequence Repeat) markers and resistance to six bacterial races. The resistance phenotypes of various rice accessions were evaluated through artificial inoculation under controlled conditions in 2013 and 2014. Association analysis showed that 17 SSR markers were significantly associated with resistance to four bacterial races and the phenotypic variations explained (PVE) ranged from 7.43 to 15.05%. Among the 17 associated SSR markers, two SSR markers located in previously reported genes regions, and 15 SSR markers were newly identified in this study. These results validated a new approach to map resistance genes of rice to bacterial blight. These markers could be used for marker-assisted selection (MAS) in rice bacterial blight resistance breeding programs.     

Pyramiding Xa23 and Rxo1 for resistance to two bacterial diseases into an elite indica rice variety using molecular approaches

Rice bacterial leaf blight (BB) caused by Xanthomonas oryzae pv. oryzae and bacterial leaf streak (BLS) caused by X. oryzae pv. oryzicola (Xoc) are two important diseases of rice that often outbreak simultaneously and constrain rice production in much of Asia and parts of Africa. Developing resistant cultivars has been the most effective approach to control BB, however, most single resistance genes have limited value in breeding programs because of their narrow-spectrum of resistance to the races of the pathogen. By contrast, there is little progress in breeding varieties resistant to Xoc since BLS resistance in rice was a quantitative trait and so far only a few quantitative resistance loci have been identified. We reported here the development of a high yield elite line, Lu-You-Zhan highly resistant to both BB and BLS by pyramiding Xa23 with a wide-spectrum resistance to BB derived from wild rice and a non-host maize resistance gene, Rxo1, using both marker assisted selection (MAS) and genetic engineering. Our study has provided strong evidence that non-host R genes could be a valuable source of resistance in combating those plant diseases where no single R gene controlling high level of resistance exists and demonstrated that MAS combined with transgenic technologies are an effective strategy to achieve high level of resistance against multiple plant diseases. Y-L Zhou and J-L Xu contributed equally to this work.     

Marker-assisted breeding of Thai fragrance rice for semi-dwarf phenotype, submergence tolerance and disease resistance to rice blast and bacterial blight

Rice is a staple food crop for more than half of the world’s population. However, rice production is affected by many types of abiotic and biotic stress. Genetic breeding by utilizing natural resistance or tolerance genes is the most economic and efficient way to combat or adapt to these stresses. Khao Dawk Mali 105 (KDML 105) is an elite cultivar of aromatic rice mainly grown in Thailand. However, the production of KDML 105 is affected by lodging problems due to its tall plant type, regular flash floods or short-term submergence during the monsoon season, and diseases such as blast and bacterial blight. Here we report the pyramiding of semi-dwarf gene sd1, submergence tolerance gene Sub1A, blast resistance gene Pi9 and bacterial blight resistance genes Xa21 and Xa27 in KDML 105 by marker-assisted selection. The improved line, designated T5105, has a semi-dwarf phenotype with improved lodging resistance and a greater harvest index. T5105 survives after 2 weeks of complete submergence without significant loss of viability. T5105 confers high resistance to all five Magnaporthe oryzae isolates tested and provides resistance or moderate resistance to 25 of the 27 Xanthomonas oryzae pv. oryzae strains tested. In addition, T5105 produced higher yield than KDML 105 in two field trials and retains similar good grain quality to KDML 105. The development of T5105 provides a new line to boost the production of high-quality aromatic rice in tropical regions.     

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.