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.     

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.     

Are the dominant and recessive plant disease resistance genes similar? A case study of rice R genes and Xanthomonas oryzae pv. oryzae races.

The resistance of rice to its bacterial blight pathogen Xanthomonas oryzae pv. oryzae (Xoo) has both qualitative and quantitative components that were investigated using three near-isogenic line sets for four resistance (R) genes (Xa4, xa5, xa13, and Xa21) and 12 Xoo races. Our results indicate that these two resistance components of rice plants were associated with the properties of the R genes. The qualitative component of the R genes was reflected by their large effects against corresponding avirulent Xoo races. The quantitative component of the R genes was their residual effects against corresponding virulent races and their epistatic effects, which together could lead to high-level resistance in a race-specific manner. Our results revealed important differences between the different types of R genes. Two R genes, Xa4 and Xa21, showed complete dominance against the avirulent Xoo races and had large residual effects against virulent ones. They acted independently and cumulatively, suggesting they are involved in different pathways of the rice defensive system. The third R gene, xa5, showed partial dominance or additivity to the avirulent Xoo races and had relatively small but significant residual effects against the virulent races. In contrast, xa13 was completely recessive, had no residual effects against the virulent races, and showed more pronounced race specificity. There was a strong interaction leading to increased resistance between xa13 and xa5 and between either of them and Xa4 or Xa21, suggesting their regulatory roles in the rice defensive pathway(s). Our results indicated that high-level and durable resistance to Xoo should be more efficiently achieved by pyramiding different types of R genes.     

Pathotype and Genetic Diversity amongst Indian Isolates of Xanthomonas oryzae pv. oryzae

A number of rice resistance genes, called Xa genes, have been identified that confer resistance against various strains of Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of bacterial blight. An understanding of pathotype diversity within the target pathogen population is required for identifying the Xa genes that are to be deployed for development of resistant rice cultivars. Among 1024 isolates of Xoo collected from 20 different states of India, 11 major pathotypes were distinguished based on their reaction towards ten Xa genes (Xa1, Xa3, Xa4, xa5, Xa7, xa8, Xa10, Xa11, xa13, Xa21). Isolates belonging to pathotype III showing incompatible interaction towards xa8, xa13 and Xa21 and compatible interaction towards the rest of Xa genes formed the most frequent (41%) and widely distributed pathotype. The vast majority of the assayed Xoo isolates were incompatible with one or more Xa genes. Exceptionally, the isolates of pathotype XI were virulent on all Xa genes, but have restricted distribution. Considering the individual R-genes, Xa21 appeared as the most broadly effective, conferring resistance against 88 % of the isolates, followed in decreasing order by xa13 (84 %), xa8 (64 %), xa5 (30 %), Xa7 (17 %) and Xa4 (14 %). Fifty isolates representing all the eleven pathotypes were analyzed by southern hybridization to determine their genetic relatedness using the IS1112 repeat element of Xoo. Isolates belonging to pathotype XI were the most divergent. The results suggest that one RFLP haplotype that is widely distributed all over India and is represented in strains from five different pathotypes might be an ancestral haplotype. A rice line with xa5, xa13 and Xa21 resistance genes is resistant to all strains, including those belonging to pathotype XI. This three gene combination appears to be the most suitable Xa gene combination to be deployed in Indian rice cultivars.     

Gene silencing using the recessive rice bacterial blight resistance gene xa13 as a new paradigm in plant breeding

Resistant germplasm resources are valuable for developing resistant varieties in agricultural production. However, recessive resistance genes are usually overlooked in hybrid breeding. Compared with dominant traits, however, they may confer resistance to different pathogenic races or pest biotypes with different mechanisms of action. The recessive rice bacterial blight resistance gene xa13, also involved in pollen development, has been cloned and its resistance mechanism has been recently characterized. This report describes the conversion of bacterial blight resistance mediated by the recessive xa13 gene into a dominant trait to facilitate its use in a breeding program. This was achieved by knockdown of the corresponding dominant allele Xa13 in transgenic rice using recently developed artificial microRNA technology. Tissue-specific promoters were used to exclude most of the expression of artificial microRNA in the anther to ensure that Xa13 functioned normally during pollen development. A battery of highly bacterial blight resistant transgenic plants with normal seed setting rates were acquired, indicating that highly specific gene silencing had been achieved. Our success with xa13 provides a paradigm that can be adapted to other recessive resistance genes.     

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.     

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.     

高覆盖率水稻ＢＡＣ库的构建及抗病基因相关克隆的筛选

利用含Ｘa4、xa5和xa13 3个水稻白叶枯病抗性基因的累加系ＩＲＢＢ５６构建了一个水稻细菌人工染色体文库,该文库包含５５２９６个克隆,平均插入升段为１３２kb。按水稻基因组为４５０Ｍb计,该文库覆盖１４倍基因组,筛选出任一水稻基因或序列的概率为９９．９９％。用均匀分布的３个叶绿体基因和４个线粒体基因克隆作探针筛选文库,结果显示该文库中含细菌器基因组ＤＮＡ同源序列的克隆数小于１％、用分布于水稻３条不同染色体、分别与Ｘa4、xa5和xa13连锁的ＤＮＡ标记筛选文库,分别检测出１１－１０６个阳性克隆,为克隆这些基因打下了基础。该文库对水稻基因组的高度覆盖率和较大的插入片段,非常适合于物理作图和基因的分离和克隆。     

Genetic relationship,population structure analysis and pheno-molecular characterization of rice (Oryza sativa L.) cultivars for bacterial leaf blight resistance and submergence tolerance using trait specific STS markers

Rice is an important source of calorie for the growing world population. Its productivity, however is affected by climatic adversities, pest attacks, diseases of bacterial, viral and fungal origin and many other threats. Developing cultivars that are high yielding and stress resilient seems a better solution to tackle global food security issues. This study investigates the potential resistance of 24 rice cultivars against Xanthomonas oryzae pv. Oryzae (Xoo) infection that causes bacterial leaf blight disease and submergence stress. Bacterial leaf blight (BLB) resistance genes (Xa4, xa5, xa13, Xa21, Xa38) and submergence tolerance (Sub1) gene specific markers were used to determine the allelic status of genotypes. The results displayed presence of Xa4 resistance allele (78.95%), xa5 (15.79%) but xa13 and Sub1 tolerance allele were not found in any genotype. However, a new allele for Xa21 (84.21%) and Xa38 (10.52%) were identified in several genotypes. Phenotypic screening for both stress conditions was done to record the cultivars response. None of the genotypes showed resistance against Xoo, although varieties viz., Tapaswini and Konark showed moderate susceptibility. Likewise, survival percentage of genotypes under submergence stress varied from 0 to 100%. Tolerant checks FR13A (100%) and Swarna Sub1 (97.78%) exhibited high survival rate, whereas among genotypes, Gayatri (57.78%) recorded high survivability even though it lacked Sub1 tolerant its genetic background. A total of six trait specific STS and two SSR markers generated an average of 2.38 allele per locus. Polymorphism information content (PIC) value ranged from 0.08 to 0.42 with an average of 0.20. Structure analysis categorized 24 genotypes into two sub-populations, which was in correspondence with Nei’s genetic distance-based NJ tree and principal co-ordinate analysis (PCoA). Swarna Sub1 could be differentiated clearly from BLB resistant check, IRBB60 and other 22 genotypes without having Sub1 gene. Analysis of molecular variance (AMOVA) revealed more genetic variation within population than among population. Principal component analysis (PCA) showed that 9 morphological traits collectively explained 76.126% of total variation among all the genotypes studied. The information from this study would be useful in future breeding programs for pyramiding trait specific genes into high yielding cultivars that fall behind with respect to stress resilience. Supplementary InformationThe online version contains supplementary material available at 10.1007/s12298-021-00951-1.     

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     

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.     

Genotypic and Pathotypic Diversity of Xanthomonas oryzae pv. oryzae Strains in Taiwan

Rice bacterial leaf blight, caused by Xanthomonas oryzae pv. oryzae [(Ishiyama) Swings et al. 1990] (Xoo), is a major rice disease of the second crop season in Taiwan. A total of 88 Xoo strains collected from 10 major rice cultivating areas in Taiwan from 1986, 1997, 2000, 2004, and 2011 were characterized by repetitive‐element PCR (REP‐PCR) fingerprinting and virulence analyses. Among the five genetic clusters identified by the pJEL1/pJEL2 (IS1112‐based) and REP1R‐Dt/REP2‐D [repetitive extragenic palindromic (REP)‐based] primer sets, clusters A, C and D contained Xoo strains from geographically distant regions, which suggests a high frequency of Xoo dispersal in Taiwan. The 88 Xoo strains were evaluated by inoculations on IRBB near‐isogenic lines and five Taiwan rice cultivars. A subset of 45 moderately or highly virulent strains were classified into 15 pathotypes by their compatible or incompatible reactions on IR24 and 12 IRBB near‐isogenic lines, each containing a single resistance gene. Analysis of molecular haplotypes and pathotypes revealed a partial relationship. IRBB5, IRBB21 and IRBB4 were incompatible with 96%, 96% and 73% of the strains, so xa5, Xa21 and Xa4 can recognize most of the Xoo strains in Taiwan and elicit resistance. In contrast, IRBB3 (Xa3), IRBB8 (xa8), IRBB10 (Xa10), IRBB11 (Xa11), IRBB13 (xa13) and IRBB14 (Xa14) were susceptible to almost all of the 45 Xoo strains. Inoculation trials revealed significant differences in the susceptibility of five Taiwan cultivars to Xoo (from high to low susceptibility: Taichung Sen 10 >  IR24, Taichung Native 1 >  Taichung 192, Taikeng 9, Tainan 11). This study provides useful information for resistance breeding and the development of disease management strategies against bacterial blight disease of rice.     

Genetic analysis of bacterial blight resistance in seventy-four cultivars of rice,Oryza sativa L.

Summary The genetics of resistance to bacterial blight, Xanthomonas oryzae (Uyeda and Ishiyama) Dowson, for 74 cultivars of rice, Oryza sativa L., was studied. The PX061 isolate of bacterial blight from the Philippines was used for inoculation of parental and hybrid populations. Single dominant genes at the Xa 4 locus convey resistance in 38 cultivars. Of these, 18 are resistant at all stages of plant growth and thus have the Xa 4 ^aallele for resistance. However, 20 are susceptible up to maximum tillering stage but are resistant at booting and flowering stages. These cultivars have the Xa 4 ^ballele for resistance. Thirty-two cultivars have single recessive genes for resistance which are allelic to xa 5.The resistance in DV85, DV86 and DZ78 is conditioned by two genes. At maximum tillering stage xa 5 conveys resistance. However, at later growth stages an additional dominant gene, designated Xa 7 in DZ78, also gives resistance. The dominant genes of DV85 and DV86 are probably allelic to Xa 7. Xa 7 segregates independently of Xa 4, xa 5 and Xa 6, however like Xa 6, it conveys resistance at booting and post-booting stages only.The resistance in PI 231129 is conditioned by a single recessive gene, designated xa 8. It also segregates independently of Xa 4, xa 5 and Xa 6.     

A paralog of the MtN3/saliva family recessively confers race-specific resistance to Xanthomonas oryzae in rice

Approximately one third of the identified 34 rice major disease resistance (R) genes conferring race-specific resistance to different strains of Xanthomonas oryzae pv. oryzae (Xoo), which causes rice bacterial blight disease, are recessive genes. However, only two of the recessive resistance genes have been characterized thus far. Here we report the characterization of another recessive resistance gene, xa25, for Xoo resistance. The xa25, localized in the centromeric region of chromosome 12, mediates race-specific resistance to Xoo strain PXO339 at both seedling and adult stages by inhibiting Xoo growth. It encodes a protein of the MtN3/saliva family, which is prevalent in eukaryotes, including mammals. Transformation of the dominant Xa25 into a resistant rice line carrying the recessive xa25 abolished its resistance to PXO339. The encoding proteins of recessive xa25 and its dominant allele Xa25 have eight amino acid differences. The expression of dominant Xa25 but not recessive xa25 was rapidly induced by PXO339 but not other Xoo strain infections. The nature of xa25-encoding protein and its expression pattern in comparison with its susceptible allele in rice-Xoo interaction indicate that the mechanism of xa25-mediated resistance appears to be different from that conferred by most of the characterized R proteins.     

Analysis of Pathogenic Diversity of the Rice Bacterial Blight Pathogen (Xanthomonas oryzae pv. oryzae) in the Andaman Islands and Identification of Effective Resistance Genes

Bacterial blight (BB) caused by Xanthomonas oryzae pv. oryzae (Xoo) is a major disease of rice in the tropics for which genetic resistance in the host plants is the only effective solution. This study aimed at identification of resistance gene combinations effective against Xoo isolates and fingerprinting of the Xoo isolates of Andaman Islands (India). Here, we report the reaction of 21 rice BB differentials possessing Xa1 to Xa21 genes individually and in different combinations to various isolates of pathogen collected from Andaman Islands. Pathological screening results of 14 isolates revealed that among individual genes tested across 2 years, Xa4, Xa7 and Xa21 conferred resistance reaction across all isolates, whereas among combinations, IRBB 50 (Xa4 + xa5), IRBB 52 (Xa4 + Xa21) and IRBB 60 (Xa4 + xa5 + xa13 + Xa21) conveyed effective resistance against tested isolates. The nature of genetic diversity among four isolates selected on the basis of geographical isolation in the islands was studied through DNA finger printing. The RAPD primers S111, S119, S1117, S1109, S1103, S109 and S105 were found to be better indicators of molecular diversity among isolates than JEL primers. The diversity analysis grouped 14 isolates into three major clusters based on disease reaction wherein isolate no. 8 was found the most divergent as well as highly virulent. The remaining isolates were classified into two distinct groups. The importance of the study in the context of transfer of resistance gene(s) in the local cultivars specifically for tropical island conditions is presented and discussed.     

来源于疣粒野生稻的白叶枯病新抗源的鉴定

疣粒野生稻对白叶枯病高抗甚至免疫。粳稻品种8411与疣粒野生稻体细胞杂交获得了2个高抗所有栽培稻白叶枯病抗性基因均不能抵御的国际广致病菌系P6的新种质SH5和SH76,遗传分析鉴定出新种质含有1个抗P6小种的显性基因。本研究以高感白叶枯病水稻品种IR24及携带不同抗性基因的16个材料为参照,对分蘖期、孕穗期的SH5和SH76分别接种11个白叶枯病小种,抗性分析表明SH5和SH76的抗谱广,与IRBB21(Xa21)抗谱一致,与IRBB5(xa5)、IRBB7(Xa7)和Asominori(Xa17)的较相近。用Xa21的分子标记pTA248和XA21检测,确定SH5和SH76不携带Xa21基因,前期研究结果证实新种质中不含xa5和Xa7;与Asominori的杂交试验表明其抗性基因与Xa17基因不等位。这些结果表明SH5和SH76中存在1个抗P6小种的新基因。     

Transformation to telephonic genetic counselling during SARS-CoV-2 pandemic: challenges and suggested protocol in Indian scenario

To combat the dreaded diseases in rice like bacterial blight (BB) and blast, host plant resistance has been advocated as the most suitable and sustainable method. Through the present study, we have successfully incorporated three major BB resistance genes, namely Xa21, xa13 and xa5 into NLR3449, a high yielding, blast resistant, fine-grain type, popular rice variety through marker-assisted backcross breeding. Foreground selection was carried out using polymerase chain reaction based, gene-specific markers, namely pTA248 (Xa21), xa13prom (xa13) and xa5FM (xa5) at each generation of backcrossing, while 127 polymorphic SSR markers spanning on 12 chromosomes were used for background selection and backcrossing was limited to two rounds. At BC₂F₁ generation, a single plant (NLR-87-10) with 89.9% recovery, possessing all the three BB resistance genes was forwarded to BC₂F₂ generation. A solitary BC₂F₂ plant, namely NLR-87-10-106 possessing all the three resistance genes and 96% genome recovery was identified and advanced through selfing until BC₂F₄ generation by adopting pedigree-method of selection. Three best BC₂F₄ lines, possessing high level of resistance against BB and blast, and equivalent or superior to NLR 34449 in terms of yield, grain quality and agro-morphological traits were identified and advanced for multi-location trials.