A new gene for resistance in rice to Asian rice gall midge (Orseolia oryzae Wood Mason) biotype 1 population at Raipur, India

The Asian rice gall midge, Orseolia oryzae Wood Mason (Diptera: Cecidomyiidae), is a major pest of rice in several South and South East Asian countries. The maggots feed internally on the growing tips of the tillers and transform them into tubular galls, onion leaf-like structures called ‘silver shoots’ resulting into severe yield loss to the rice crop. We studied the mode of inheritance and allelic relationships of the resistance genes involved in resistant donor Line 9, a sib of a susceptible cultivar ‘Madhuri’. The segregation behaviour of F₁, F₂ and F₃ populations of the cross between Line 9 and susceptible cultivar MW10 confirmed the presence of a single dominant gene for resistance. Tests of allelism with all the known genes giving resistance to this population indicated that Line 9 possessed a new gene which was designated Gm 9 This revised version was published online in July 2006 with corrections to the Cover Date.     

Genetic analysis and pyramiding of two gall midge resistance genes (Gm-2 and Gm-6t) in rice ( Oryza sativa L.)

Asian rice gall midge (Orseolia oryzae) is a major pest across much of south and southeast Asia. This pest is genetically diverse and many gall midge biotypes are known to exist in each country. During the last three decades, host plant resistance has proved to be the most effective mechanism of controlling the Asian rice gall midge. Seven genes conditioning resistance to gall midge larvae have been identified in rice (Oryza sativa) and are being used in cultivar improvement programs. However, some of these genes are rendered ineffective by new gall midge biotypes. Increased understanding of genetics, inheritance, allelic relationships and linkage is necessary to maximise the durability of major gene resistance by the pyramiding of these genes. The two genes, Gm-2 and Gm-6(t), are known to confer resistance against a number of biotypes in India and China, respectively. An F₃ population derived from a cross between Duokang #1 (donor of Gm-6(t)) and Phalguna (donor of Gm-2) was screened against Chinese gall midge biotype 4 at Guangdong, China, and Indian gall midge biotype 1 at Raipur, India. At each location, separately,a single gene governed resistance. The parallel segregation of 417 F₃progenies for both biotypes at two locations revealed that recombination had occurred between the two genes, establishing that the two genes are not allelic. However, the two genes Gm-2 and Gm-6(t), were found to be linked with a distance of ∼16.3 cM. A number of lines homozygous at one locus and segregating for the other locus were identified and selected. These lines were selfed to obtain lines homozygous for the favourable alleles at both loci (two locus pyramids). This is the first report on use of conventional host-pest interaction method for pyramiding two closely located Gm-resistance loci of dissimilar effects. The implications of deployment of these pyramids within and across country borders, with reference to the prevailing gall midge populations are discussed. This revised version was published online in August 2006 with corrections to the Cover Date.     

A new rice gall midge resistance gene in the breeding line CR57-MR1523, mapping with flanking markers and development of NILs

Gall midge is the third most destructive insect pests of rice after stem borers and planthoppers. Host plant resistance has been recognized as the most effective and economic, means for gall midge management. With the characterization of a new gall midge biotype (GMB) 4M, unique feature of gall midge resistance in the breeding line CR57-MR1523 was highlighted. Multi-location evaluation of F₃ families derived from the cross TN1 × CR57-MR1523 against different gall midge biotypes helped to identify a new dominant gene conferring resistance against GMB4. This gene has been designated as Gm11t. Though CR57-MR1523 has been extensively used in breeding gall midge resistant rice varieties like Suraksha, neither the genetics of resistance nor chromosomal location of the resistance gene(s) is known. In the present study we have tagged and mapped the new gall midge resistance gene, Gm11t, on chromosome 12, using SSR markers. To map the gene locus, 466 F₁₀ generation Recurrent Inbred Lines (RILs), from the cross of TN1 × CR57-MR1523 were used. Of the 471 SSR markers spread across the rice genome, 56 markers showed polymorphism and were used to screen a subset of the mapping population consisting of 10 resistant (R) and 10 susceptible (S) F₁₀ RILs. Six SSR markers, RM28706, RM235, RM17, RM28784, RM28574 and RM28564 on chromosome 12 were initially found to be associated with resistance and susceptibility. Based on the linkage analysis in selected 158 RILs, we were able to map the locus between two flanking SSR markers, RM28574 and RM28706, on chromosome 12 within 4.4 and 3.8 cM, respectively. Further, two NILs with 99% genetic similarity, were identified from the RILs which differed in gall midge resistance. The tightly linked flanking SSR markers will facilitate marker-assisted gene pyramiding and map-based cloning of the resistant gene. NILs would be valuable materials for functional analysis of the identified candidate gene.     

Molecular markers for yellow stem borer Scirpophaga incertulas (Walker) resistance in rice

Breeding for yellow stem borer resistance in rice is difficult owing to the complex genetics of the trait and inherent difficulties in screening. Identification of molecular markers linked to the trait would enhance phenotypic evaluation for the trait. An F₂ population was developed using parents contrasting in their reaction to yellow stem borer resistance. Random Amplified Polymorphic DNA (RAPD) analysis,in conjunction with bulked segregant analysis, enabled us to identify four phenotype-specific RAPD markers. The markers C1₃₂₀ and K6₆₉₅ were linked with resistance and AH5₆₆₀ and C4₁₃₀₀ with susceptibility. The markers K6₆₉₅ and AH5₆₆₀ were linked to the gene(s) at distances of 12.8 cM and 14.9 cM, respectively. Scoring of these markers in a set of germplasm confirmed their reproducibility and their association with the trait. This revised version was published online in August 2006 with corrections to the Cover Date.     

Mapping and marker-assisted breeding of a gene allelic to the major Asian rice gall midge resistance gene <Emphasis Type="Italic">Gm8</Emphasis>

Host plant resistance is the preferred management strategy for Asian rice gall midge (Orseolia oryzae), a serious pest in many rice-growing countries. Identification of simple sequence repeat (SSR) markers that are tightly linked to pest resistance genes can accelerate development of gene pyramids for durable/multiple resistance. Based on conventional and molecular allelism tests, we report herein that rice genotype Aganni possesses Gm8 gene, conferring hypersensitive independent (HR– type) resistance to gall midge biotypes GMB1, GMB2, GMB3, GMB4, and GMB4M. The gene Gm8 was mapped to chromosome 8 within a 400-kbp region, and the SSR markers RM22685 and RM22709 flank the gene closely. Using these closely linked flanking markers, nine other gall midge-resistant genotypes were identified as carrying the same gene Gm8. Through marker-assisted selection, Gm8 has been introgressed into an elite bacterial blight-resistant cultivar, Improved Samba-Mahsuri (IS).     

The rice cultivar Baixiangzhan harbours a recessive gene xa42(t) determining resistance against Xanthomonas oryzae pv. oryzae

Bacterial leaf blight (BB) is among the “top 3” diseases in rice production. Breeding resistant cultivars has been the most effective strategy for BB management. The inbred cultivar Baixiangzhan (BXZ) showed a broad spectrum of resistance to five Xoo pathotypes in China, including the prevalent and highly virulent Xoo pathotypes Chinese Race V (CV), which can overcome the resistance of Xa4 and Xa21. The resistance heredity of BXZ has been explored in this study. A single recessive major resistance gene, which designated as xa42(t), confers resistance against the tested Xoo pathotypes CV. Linkage analysis lands xa42(t) on chromosome 6, and genetic mapping confines it to 3.9 cM region flanked by RM20558/RM20547 and RM20580. A further seven markers were developed from this interval for high‐resolution mapping, and the xa42(t) locus was narrowed to 34.8 Kb segment bounded by RM20572 and DT46. The only functionally predicted gene included in the target region is LOC_Os06g45960 if based on the ‘Nipponbare’ reference sequence. This candidate gene is predicted to encode a cytochrome P450 protein.     

Development and validation of class I SSR markers targeting (GATA) n repeat motifs in rice

SSR markers targeting (GATA) _n motifs are known to be highly polymorphic and useful in many organisms. (GATA) _n motif specific SSR markers covering the whole rice genome are not available. The present study was carried out with an objective to identify class I rice microsatellites in the rice genome with (GATA)_n motifs, in-silico, and validate their potential as molecular markers. A total of 243 such motifs were identified; 65 of these were present in the genic region, 59 were in the upstream region and the remaining motifs were found in the intergenic regions. Many of the (GATA) _n motifs were found within and/or upstream of genes associated with biotic or abiotic stress tolerance. A total of 230 PCR-based markers targeting all the class I (GATA) _n microsatellites were developed and 35 of these markers spread across the rice genome were validated in a set of 24 representative rice varieties belonging to five distinct cultivar groups. All the markers were polymorphic, with average polymorphism information content (PIC) value of 0.61, and the rice cultivars could be uniquely distinguished into different cultivar groups based on marker analysis. These informative markers targeting (GATA) _n motifs representing a new set of markers in rice will be highly useful for genetic studies and marker-assisted selection. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Passoupathy Rajendrakumar, Akshaya Kumar Biswal and Kannabiran Sakthivel contributed equally.     

Confirming a major QTL and finding additional loci responsible for field resistance to brown spot (Bipolaris oryzae) in rice

Brown spot is a devastating rice disease. Quantitative resistance has been observed in local varieties (e.g., ‘Tadukan’), but no economically useful resistant variety has been bred. Using quantitative trait locus (QTL) analysis of recombinant inbred lines (RILs) from ‘Tadukan’ (resistant) × ‘Hinohikari’ (susceptible), we previously found three QTLs (qBS2, qBS9, and qBS11) that conferred resistance in seedlings in a greenhouse. To confirm their effect, the parents and later generations of RILs were transplanted into paddy fields where brown spot severely occurred. Three new resistance QTLs (qBSfR1, qBSfR4, and qBSfR11) were detected on chromosomes 1, 4, and 11, respectively. The ‘Tadukan’ alleles at qBSfR1 and qBSfR11 and the ‘Hinohikari’ allele at qBSfR4 increased resistance. The major QTL qBSfR11 coincided with qBS11 from the previous study, whereas qBSfR1 and qBSfR4 were new but neither qBS2 nor qBS9 were detected. To verify the qBSfR1 and qBSfR11 ‘Tadukan’ resistance alleles, near-isogenic lines (NILs) with one or both QTLs in a susceptible background (‘Koshihikari’) were evaluated under field conditions. NILs with qBSfR11 acquired significant field resistance; those with qBSfR1 did not. This confirms the effectiveness of qBSfR11. Genetic markers flanking qBSfR11 will be powerful tools for marker-assisted selection to improve brown spot resistance.     

QTLs and candidate genes for chlorate resistance in rice (Oryzasativa L.)

Chlorate resistance is one of the reliable characters in Indica/Japonica classification. To understand the genetic basis of chlorate resistance is very important for revealing the evolutionary mechanism of Indica/Japonica differentiation. In this study, a doubled haploid (DH) population derived from anther culture of ZYQ8/JX17, a typical Indica and Japonica hybrid, was used as the genetic material to investigate chlorate sensitivity of the parents and DH lines. The quantitative trait loci (QTLs) of chlorate resistance were analyzed based on the molecular linkage map of this population. Total of 3 QTLs (qCHR-2, qCHR-8 and qCHR-10) for chlorate resistance were detected on chromosomes 2, 8 and 10, respectively. A QTL × QTL epistatic interaction was detected between qCHR-2 and qCHR-10. Genes involved in nitrogen assimilation, such as nitrate reduction, molybdenum cofactor biosynthesis and nitrate transport were strong candidates of QTLs for chlorate resistance. A putative nitrate reductase gene (8611.t00011), and two putative nitrate reductase genes (9319.t00010 and 9319.t00012) were in the genomic region of qCHR-2, and qCHR-8, respectively, and a putative nitrate transporter gene (756.t00011) was in the region of qCHR-10. The expression of 8611.t00011, 9319.t00010 and 756.t00011 were confirmed by the corresponding cDNAs, and 2 in/del and 12 SNPs in the coding regions of these three genes were found between Indica (cv. 9311) and Japonica (cv. Nipponbare) in silico. These results indicated that these three genes were candidates of the chlorate resistance QTLs. An in/del in the coding region of 8611.t00011 was used to develop a new PCR marker. A polymorphism was detected between JX17/Nipponbare and ZYQ8/9311. This polymorphism corresponds to the chlorate sensitivity of Nipponbare and 9311. This marker was located between Y8007R and RM250 on chromosome 2 in the DH population, where qCHR-2 was also located.     

Molecular markers for the identification of resistance genes and marker-assisted selection in breeding wheat for leaf rust resistance

The resistance genes Lr9, Lr24, Lr25, Lr29, Lr35 and Lr37, which were not previously utilised in Hungary, have been incorporated into four Martonvásár winter wheat cultivars using marker-assisted selection with PCR-based markers. In the course of a backcross programme, the genes were transferred into Martonvásár wheat varieties and various BC generations were produced. Work aimed at pyramiding resistance genes is currently underway in Martonvásár, and plants containing the gene combinations Lr9 + Lr24, Lr9 + Lr25 and Lr9 + Lr29 are now available. From the BC₂F₄ generation of the ‘Mv Emma’*3/’R.L.6010’ combination (‘R.L.6010’ is the donor of the Lr9 gene) 287 lines were tested for leaf rust resistance in an artificially inoculated nursery. A co-dominant primer combination was designed to identify both resistant and susceptible offsprings. The results of resistance tests and molecular marker detection agreed in most cases. Designated leaf rust resistance genes were identified with molecular markers in wheat varieties and breeding lines. The Lr26 and Lr34 resistance genes occur frequently in the Martonvásár gene pool, and the presence of the Lr37 gene has also been detected in a number of Hungarian genotypes.     

The host background of rice influences the resistance expression of a three genes pyramid (xa5 + xa13 + Xa21) to bacterial blight (Xanthomonas oryzae pv. oryzae) pathotypes of Indian mainland and Bay islands

Bacterial blight (BB) is a major disease of rice for which host resistance is the only effective solution. The three genes pyramid xa5 + xa13 + Xa21 is recently the most utilized combination for developing resistant varieties through marker‐assisted breeding. Our study was carried out to elicit the detailed response of twenty lines possessing these three genes in five genetic backgrounds to twelve diverse BB pathotypes in India. The lines developed from ADT 47 variety showed incomplete resistance to most of the pathotypes, whereas susceptibility varied from 8.3% to 16.6% in ADT 43 and IR24, respectively. However, in IMP ASD16/60 and Improved Samba Mahsuri, complete resistance against all pathotypes was observed. The overall results confirmed that genetic background plays crucial role for the effective expression of xa5 + xa13 + Xa21 combination. Molecular studies did not reveal correlation between origin of pathotypes and their virulence potential. It is suggested to deploy Improved Samba Mahsuri, IMP ASD 16/60 and AD1306 varieties in the bacterial blight prone areas or use them as donors for realizing wider and durable resistance.     

Using molecular markers to pyramid genes for resistance to ascochyta blight and anthracnose in lentil (Lens culinaris Medik)

Ascochyta blight caused by the fungus Ascochyta lentis Vassilievsky and anthracnose caused by Colletotrichum truncatum [(Schwein.) Andrus & W.D. Moore] are the most destructive diseases of lentil in Canada. The diseases reduce both seed yield and seed quality. Previous studies demonstrated that two genes, ral1 and AbR1, confer resistance toA. lentis and a major gene controls the resistance to 95B36 isolate of C. truncatum. Molecular markers linked to each gene have been identified. The current study was conducted to pyramid the two genes for resistance to ascochyta blight and the gene for resistance to anthracnose into lentil breeding lines. A population (F_6:7) consisting of 156 recombinant inbred lines (RILs) was developed from across between ‘CDC Robin’ and a breeding line ‘964a-46’. The RILs were screened for reaction to two isolates (A1 and 3D2) ofA. lentis and one isolate (95B36) ofC. truncatum. χ² analysis of disease reactions demonstrated that the observed segregation ratios of resistant versus susceptible fit the two gene model for resistance to ascochyta blight and a single gene model for resistance to anthracnose. Using markers linked to ral1 (UBC 227₁₂₉₀), to AbR1(RB18₆₈₀) and to the major gene for resistance to anthracnose (OPO6₁₂₅₀),respectively, we confirmed that 11 RILs retained all the three resistance genes. More than 82% of the lines that had either or both RB18₆₈₀ and UBC227₁₂₉₀markers were resistant to 3D2 isolate and had a mean disease score lower than 2.5. By contrast, 80% of the lines that had none of the RAPD markers were susceptible and had a mean disease score of 5.8. For the case of A1 isolate of A. lentis, more than 74% of the lines that carriedUBC227₁₂₉₀ were resistant, whereas more than 79% of the lines that do not have the marker were susceptible. The analysis of the RILs usingOPO6₁₂₅₀ marker demonstrated that 11out of 72 resistant lines carried the marker, whereas 66 out of 84 susceptible lines had the marker present. Therefore, selecting materials with both markers for resistance to ascochyta blight and a marker for resistance to anthracnose can clearly make progress toward resistance in the population. This revised version was published online in July 2006 with corrections to the Cover Date.     

Developing gene-tagged molecular markers for functional analysis of starch-synthesizing genes in rice (Oryza sativa L.)

The granule-bound starch synthase(GBSS), starch branching enzymes 1 (SBE1)and 3 (SBE3) are major enzymes involved in starch biosynthesis in rice endosperm. Available sequences of Sbe1 and Sbe3 genes encoding corresponding SBE1 and SBE3 have been used to identify homologous regions from genome databases of both the indica rice 93-11 and the japonica rice Nipponbare. Sequence diversities were exploited to develop gene-tagged markers to distinguish an indica allele from a japonica allele for both Sbe1 and Sbe3 loci. With these newly developed gene-tagged markers and available Wx gene markers, the roles of these starch-synthesizing genes (Sbe1, Sbe3, and Wx) in determining amylose content (AC) in the rice endosperm were evaluated using a double-haploid (DH)population derived from a cross between the indica rice cv. Nanjing11 and the japonica rice cv. Balilla. Only the Wx and Sbe3 loci had significant effects on the AC variation. On average, indica Wx ^a genotypes showed ∼9.1% higher AC than japonica Wx ^b genotypes, while indica Sbe3 ^a genotypes showed ∼1.0% lower AC than japonica Sbe3 ^b genotypes. A significant interaction was also observed between Wx and Sbe3 loci whereby the amylose content was 0.3% higher in Sbe3 ^a than Sbe3 ^b genotypes in the presence of the Wx ^a allele, but it was lower by 2.3% in the presence of the Wx ^b allele. Overall, polymorphisms at the Wx and Sbe3 loci together could account for 79.1% of the observed AC variation in the DH population. The use of gene-tagged markers in marker-assisted selection and gene functional analysis was also discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

SSR markers associated for late leaf spot disease resistance by bulked segregant analysis in groundnut (Arachis hypogaea L.)

Late leaf spot (LLS) caused by Phaeoisariopsis personata is the major foliar disease that reduces the pod yield and severely affects the fodder and seed quality in groundnut. Molecular markers linked with LLS can improve the process of identification of resistant genotypes. In the present study, a LLS susceptible genotype (TMV 2) and the LLS resistant genotype (COG 0437) were crossed and their F₂ population was used for marker analysis. The phenotypic mean data on F_2:3 progenies were used as phenotype. Parents were surveyed with 77 SSR (Simple Sequence Repeat) primers to identify polymorphic markers. Among SSR markers, nine primers were found polymorphic between the parents TMV 2 and COG 0437. These markers were utilized for bulked segregant analysis (BSA). Among the polymorphic SSR markers, three primers viz., PM 375₁₆₂, pPGPseq5D5₂₂₀ and PM 384₁₀₀ were able to distinguish the resistant and susceptible bulks and individuals for LLS. In single marker analysis, the markers PM 375, PM 384, pPGPseq5D5, PM 137, PM 3, PMc 588 and Ah 4-26 were linked with LLS severity score. The phenotypic variation explained by these markers ranged from 32 to 59?%. The markers identified through BSA were also confirmed with single marker analysis. While validating the three primers over a set of resistant and susceptible genotypes, the primer PM 384₁₀₀ allele had association with resistance. Hence PM 384 could be utilized in the marker assisted breeding programme over a wide range of genetic background.     

Development of cleaved amplified polymorphic sequence (CAPS) markers linked to a green rice leafhopper resistance gene, Grh3(t)

The chromosomal location of the resistance gene for green rice leafhopper (GRLH), an injurious insect for rice, has been determined and RFLP markers closely linked to this gene have been identified. The susceptible japonica rice variety ‘Nipponbare’ was crossed with a resistant japonica rice line ‘Aichi42’, in which green rice leaf hopper resistance had been introduced from an indica variety ‘Rantaj‐emas2’, and the 100 F₂ plants obtained were used for linkage analysis. The green rice leafhopper resistance gene, Grh3(t), was mapped between RFLP markers C288B and C133A on chromosome 6 and co‐segregated with C81. Of the RFLP markers tightly linked to Grh3(t), C81 was converted to a SCAR marker and C133A to a cleaved amplified polymorphic sequence marker that could distinguish the heterozygous genotype to establish an effective marker‐aided selection system for the GRLH resistance gene.     

Exploring genetic diversity of rice cultivars for the presence of brown planthopper (BPH) resistance genes and development of SNP marker for Bph18

Brown planthopper (BPH) is the most devastating insect pest in rice‐growing areas. Information on availability of BPH resistance alleles and their sources enhances BPH‐resistant breeding programmes. In this study, 260 highly diversified rice cultivars or breeding lines were screened for the presence of five major BPH resistance genes (Bph10, Bph13, Bph18, Bph20 and Bph21) using gene‐specific markers. The analysis revealed that 137 of the 260 cultivars possess at least one BPH resistance gene. Bph10 was predominant while Bph20 was the least distributed. Moreover, two and three different resistance gene combinations were found in the cultivars. Molecular markers play an important role in molecular breeding programmes. A tightly linked PCR‐based co‐dominant Bph18 marker was developed, which is cost effective and time effective and simpler than available Bph18 CAPS marker (7312.T4A). We strongly believe that the identified BPH‐resistant cultivars can be used as alternative resistance gene sources and also as resource for novel BPH resistance genes. The developed Bph18 marker will be highly useful in molecular breeding applications of BPH‐resistant breeding programmes.