Yogesh Vikal

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

Abstractu2002Bacterial 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.

A novel bacterial blight resistance gene from Oryza nivara mapped to 38 kb region on chromosome 4L and transferred to Oryza sativa L.

Bacterial blight (BB) of rice caused by Xanthomonas oryzae pv oryzae (Xoo) is one of the major constraints to productivity in South-East Asia. The strategy of using major genes, singly or in combination, continues to be the most effective approach for BB management. Currently, more than two dozen genes have been designated but not all the known genes are effective against all the prevalent pathotypes. The challenge, therefore, is to continue to expand the gene pool of effective and potentially durable resistance genes. Wild species constitute an important reservoir of the resistance genes including BB. An accession of Oryza nivara (IRGC 81825) was found to be resistant to all the seven Xoo pathotypes prevalent in northern states of India. Inheritance and mapping of resistance in O. nivara was studied by using F2, BC2F2, BC3F1 and BC3F2 progenies of the cross involving Oryza sativa cv PR114 and the O. nivara acc. 81825 using the most virulent Xoo pathotype. Genetic analysis of the segregating progenies revealed that the BB resistance in O. nivara was conditioned by a single dominant gene. Bulked segregant analysis (BSA) of F2 population using 191 polymorphic SSR markers identified a approximately 35 centiMorgans (cM) chromosomal region on 4L, bracketed by RM317 and RM562, to be associated with BB resistance. Screening of BC3F1 and BC2F2 progenies and their genotyping with more than 30 polymorphic SSR markers in the region, covering Bacterial artificial chromosome (BAC) clone OSJNBb0085C12, led to mapping of the resistance gene between the STS markers based on annotated genes LOC_Os04g53060 and LOC_Os04g53120, which is approximately 38.4 kb. Since none of the known Xa genes, which are mapped on chromosome 4L, are effective against the Xoo pathotypes tested, the BB resistance gene identified and transferred from O. nivara is novel and is tentatively designated as Xa30(t). Homozygous resistant BC3F3 progenies with smallest introgression region have been identified.

New PCR-based sequence-tagged site marker for bacterial blight resistance gene Xa38 of rice

Bacterial blight (BB), caused by Xanthomonas oryzae pv. oryzae, is a major disease of rice managed largely through the deployment of resistance genes. Xa38, a BB resistance gene identified from Oryza nivara acc. IRGC 81825, was mapped on chromosome 4L in a 38.4-kb region. The closely linked markers for this gene, identified earlier, were simple sequence repeat marker RM17499 and sequence-tagged site markers developed from loci Os04g53060 and Os04g53120. Marker Os04g53060 is dominant while the other two markers show smaller size differences difficult to resolve accurately on agarose gel. Based on gene annotation, three nucleotide binding site–leucine-rich repeat genes present in the target region were cloned from O. nivara and sequenced. One of the loci, LOC_Os04g53050, had a 48-base-pair deletion in O. nivara acc. IRGC 81825 compared to the cultivated rice. Primers were designed around the deletion and the resulting marker is codominant and easy to score in agarose gel. The newly designed marker co-segregated with Xa38, amplifying products of 269xa0bp in O. nivara and 317xa0bp in cultivated rice. This marker could be more useful for marker-assisted selection than ones reported earlier.

Development of high yielding IR64 × Oryza rufipogon (Griff.) introgression lines and identification of introgressed alien chromosome segments using SSR markers

Modern rice varieties that ushered in the green revolution brought about dramatic increase in rice production worldwide but at the cost of genetic diversity at the farmers’ fields. The wild species germplasm can be used for broadening the genetic base and improving productivity. Mining of alleles at productivity QTL from related wild species under simultaneous backcrossing and evaluation, accompanied by molecular marker analysis has emerged as an effective plant breeding strategy for utilization of wild species germplasm. In the present study, a limited backcross strategy was used to introgress QTL associated with yield and yield components from Oryza rufipogon (acc. IRGC 105491) to cultivated rice, O. sativa cv IR64. A set of 12 BC2F6 progenies, selected from among more than 100 BC2F5 progenies were evaluated for yield and yield components. For plant height, days to 50% flowering and tillers/plant, the introgression lines did not show any significant change compared to the recurrent parent IR64. For yield, 9 of the 12 introgression lines showed significantly higher yield (19–38%) than the recurrent parent IR64. Four of these lines originating from a common lineage showed higher yield due to increase in grain weight and another three also from a common lineage showed yield increase due to increase in grain number per panicle. For analyzing the introgression at molecular level all the 12 lines were analyzed for 259 polymorphic SSR markers. Of the total 259 SSR markers analyzed, only 18 (7.0%) showed introgression from O. rufipogon for chromosomes 1, 2, 3, 5, 6 and 11. Graphical genotypes have been prepared for each line and association between the introgression regions and the traits that increased yield is reported. Based on marker trait association it appears that some of the QTL are stable across the environments and genetic backgrounds and can be exploited universally.

DNA fingerprinting and virulence analysis of Xanthomonas oryzae pv. oryzae isolates from Punjab, northern India

AbstractDNAs of 693 isolates of bacterial blight pathogen of rice, Xanthomonas oryzae pv. oryzae (Xoo), were characterized using PCR-based primers pJEL1 and pJEL2. The pathogen populations were grouped into 97 haplotypes based on DNA-banding patterns. An un-weighted pair-group method using arithmetic averages (UPGMA) indicated a high level of diversity in the pathogen isolates (51 lineages of Xoo at a 70% similarity level). Among these, lineages 5, 7, 27, and 29 are widely distributed and others are localized in the northern region of India. The isolates represent lineage-27, were prevalent in the entire disease-prone area in the region except at Ferozepur. Pathotyping data of the representative isolates of each lineage also indicate 17 different reaction patterns on a set of isogenic lines. Resistance genesnxa8 and Xa21 were the most effective followed by xa5, and Xa7 against Xoo isolates prevalent in northern India. Different genes in combinations (xa5+xa13, xa5+Xa21, xa13+Xa21, and xa5+xa13+Xa21) in IR24 genetic background provided better protection against all the pathogen isolates tested in this study than did the component genes.

Identification of new sources of bacterial blight ( Xanthomonas oryzae pv. oryzae ) resistance in wild Oryza species and O. glaberrima

Bacterial blight (BB) of rice is a widespread disease in tropical Asia, contained largely through the deployment of race-specific resistance genes. Although more than 25 BB resistance genes have been identified, none are effective individually against all the pathotypes prevalent in north-western India. The response of a set of 327 accessions of 13 wild Oryza species and cultivated African rice, O. glaberrima, was evaluated to infection with seven pathotypes of Xanthomonas oryzae pv. oryzae over a period of 3‐4 years. Of these, 67 were resistant or moderately resistant to all pathotypes. These comprised 13 accessions of O. glaberrima ,5o f O. barthii ,1 0 ofO. rufipogon ,4o fO. longistaminata ,2 2 ofO. nivara ,6o fO. officinalis ,2 of O. rhizomatis and 5 of O. minuta. Inheritance studies, molecular mapping and transfer of some of these genes into O. sativa ssp. indica are in progress.

Tagging of an Aegilops speltoides Derived Leaf Rust Resistance Gene Lr 28 with a Microsatellite Marker in Wheat

Leaf rust resistance gene Lr28 has been transferred form Aegilops speltoides into bread wheat on chromosome 4AL. To identify the molecular markers linked to Lr28 the available microsatellite markers for wheat chromosome arm 4AL were surveyed on near isogenic lines (NILs) of Triticum aestivum cultivars having Lr28 gene, other Lrgenes and susceptible cultivars. A null allele of Xgwm 160 marker was found to be associated with Lr28. Linkage between the marker and the Lr28 resistance gene was confirmed using F2 mapping population of cross PBW343 and HD2329 + Lr28.

Molecular Markers Detect Redundancy and Miss-identity in Genetic Stocks with Alien Leaf Rust Resistance Genes Lr32 and Lr28 in Bread Wheat

Ten elite near-isogenic line (NIL) pairs of bread wheat (Triticum aestivum L em Thell) each carrying one of the two alien leaf rust resistance (Lr) genes Lr32 and Lr28, derived from Triticum tauschii and Triticum speltoides, respectively were tested for disease phenotype in controlled conditions. The disease phenotype of the NIL pair detected distinction between the Lr32 donor parent and its derivatives in ten cultivar backgrounds documented as carrying the gene Lr32. The RAPD and SCAR molecular markers identified earlier as linked to Lr32 amplified the critical marker bands identically in eight of the ten NIL pairs as well as the Lr28 donor parent. The critical bands were not amplified in the Lr32 donor parent. A Triticum speltoides specific microsatellite null allele marker located on chromosome 4AL, the genomic region associated with Lr28, expressed in an identical polymorphism as the RAPD and SCAR markers. The PCR product sequenced from a NIL pair revealed 100% homology. It is confirmed that eight of the ten elite Lr32 lines carry the gene Lr28. Molecular marker tools need to be employed to eliminate such miss-identities and reduce redundancy in Indian elite germplasm stocks of wheat possessing the alien Lr genes.

SSR marker-based DNA fingerprinting and cultivar identification of rice ( Oryza sativa L.) in Punjab state of India

In India, Protection of Plant Varieties and Farmers Rights Act (PPV&FRA, 2001) requires the registration and protection of new and notified/extant plant varieties based on the criteria of distinctness, uniformity and stability (DUS) of morphological characteristics. However, these morphological traits have not been able to resolve closely related genotypes. The molecular markers can very well support the DUS testing in such cases. In the present study, therefore, 14 varieties of rice cultivated in Punjab state of India were fingerprinted using 75 simple sequence repeat (SSR) primers. Out of these, 58 primers produced polymorphic profiles, while 13 were monomorphic, 2 revealed null allele and the remaining 2 amplified only from super basmati. In a screen of 7 cultivars, 16 SSR loci produced 17 rare/unique alleles, which provided an opportunity for their unambiguous identification. Cluster analysis based on SSR data clearly distinguished the cultivars into two dist inct groups: comprising non-basmati (group I) and basmati (group II). The cluster pattern was consistent with the pedigree and breeding history of the cultivars.

Mapping of bacterial blight resistance gene xa8 in rice (Oryza sativa L.)

Bacterial blight (BB) of rice, caused by Xanthomonas oryzae pv. oryzae (Xoo), is one of the major diseases causing yield losses in rice worldwide. Development and deployment of resistant rice cultivars and pyramiding two or more resistance genes into elite varieties is the only strategy available to combat the disease. The BB resistance gene, xa8 conferred complete resistance to only one pathotype and partial resistance against two pathotypes at seedling stage while complete resistance against five of the seven pathotypes prevalent in Punjab at adult plant stage. The gene was earlier mapped on rice chromosome 7 at 19.9 cM genetic distance from SSR marker, RM214 using a recombinant inbred line (RIL) population generated from the cross between PR106 and IRBB8. However, due to the paucity of markers in that region, the gap could not be narrowed. With the availability of new SSR markers in that region, the gene was placed between two consecutive SSR markers, RM21044 and RM21045 at 7.0 and 9.9 cM respectively. Analysis of the 9.5 kb DNA sequence harboring the xa8 gene revealed the presence of three intact genes, which codes for putative expressed proteins viz., Zinc finger A20 and AN1 domain-containing protein (LOC_Os07g07400), Oxidoreductase, 2OG-Fe oxygenase family protein (LOC_Os07g07410) and Gibberellin 20 oxidase 1-B protein (LOC_Os07g07420). All the three genes are involved in plant response to various stresses and could be the possible candidate genes for xa8.