Field and agroinoculation screening of national collection of urd bean (Vigna mungo) germplasm accessions for new sources of mung bean yellow mosaic virus (MYMV) resistance.

Yellow mosaic disease (YMD) is a major problem in Urd bean (Vigna mungo L.) in India, which causes huge yield losses. Breeding for wide spectrum and durable Mungbean yellow mosaic virus (MYMV) resistance and cultivating resistant cultivars is the most appropriate and effective approach. However, the task has become challenging with the report of at least two species of the virus, viz., Mungbean yellow mosaic virus (MYMV) and Mungbean yellow mosaic India virus (MYMIV) and their recombinants; the existence of various isolates of these species with varied virulence and rapid mutations noted in the virus as well as in the whitefly vector population. Thus the present study was carried out to identify and characterize novel and diverse sources of YMV resistance and develop linked molecular markers for breeding durable and broadspectrum resistant urdbean cultivars against YMV. Towards this goal, we have screened 998 accessions of urdbean national collection of germplasm against YMD Hyderabad isolate both in a field under the natural level of disease incidence and through agro inoculation in the laboratory using viruliferous clones of the same isolate. Ten highly resistant accessions identified through repeated testing have been characterized in terms of reported linked markers. We attempted to see diversity among the ten resistant accessions reported here using earlier reported resistance-linked SCAR marker YMV1 and SSR CEDG180 marker. SCAR marker YMV1 did not amplify with any of the 10 accessions. But with CEDG180, results suggested that 10 accessions shortlisted through field and laboratory tests do not carry PU31 allele and this shows that it may be likely to carry novel gene(s). Further studies are needed to genetically characterize these new sources.

Polymorphisms in the hypervariable control region of the mitochondrial DNA differentiate BPH populations.

The brown planthopper (BPH; Nilaparvata lugens) is one of India's most destructive pests of rice. BPH, a monophagous migratory insect, reported from all major rice-growing ecosystems of the country, is capable of traversing large distances and causing massive crop loss. A crucial step for developing viable management strategies is understanding its population dynamics. Very few reliable markers are currently available to screen BPH populations for their diversity. In the current investigation, we developed a combinatorial approach using the polymorphism present within the mitochondrial Control Region of BPH and in the nuclear genome (genomic simple sequence repeats; gSSRs) to unravel the diversity present in BPH populations collected from various rice-growing regions of India. Using two specific primer pairs, the complete Control Region (1112 to 2612 bp) was PCR amplified as two overlapping fragments, cloned and sequenced from BPH individuals representing nine different populations. Results revealed extensive polymorphism within this region due to a variable number of tandem repeats. The three selected gSSR markers also exhibited population-specific amplification patterns. Overall genetic diversity between the nine populations was high (>5%). Further, in silico double-digestion of the consensus sequences of the Control Region, with HpyCH4IV and Tsp45I restriction enzymes, revealed unique restriction fragment length polymorphisms (digital-RFLPs; dRFLPs) that differentiated all the nine BPH populations. To the best of our knowledge, this is the first report of markers developed from the Control Region of the BPH mitogenome that can differentiate populations. Eventually, such reliable and rapid marker-based identification of BPH populations will pave the way for an efficient pest management strategy.

RNA-Sequencing Reveals Differentially Expressed Rice Genes Functionally Associated with Defense against BPH and WBPH in RILs Derived from a Cross between RP2068 and TN1.

BACKGROUND: Rice is staple food for over two billion people. Planthoppers like BPH and WBPH occur together in most of rice growing regions across Asia and cause extensive yield loss by feeding and transmission of disease-causing viruses. Chemical control of the pest is expensive and ecologically disastrous; breeding resistant varieties is an acceptable option. But most of such efforts are focused on BPH with an assumption that these varieties will also be effective against WBPH. No critical studies are available to understand rice resistance, common or otherwise, against these two planthoppers. RESULTS: Our studies aimed to understand the defense mechanisms in rice line RP2068 against BPH and WBPH through RNA sequencing analysis of a RIL line TR3RR derived from the cross TN1 (susceptible) and RP2068 (resistant) after infestation with BPH or WBPH. Results revealed higher number of differentially expressed genes (DEGs) in BPH infested plants than in WBPH infested plants when compared with the uninfested plants. These DEGs could be grouped into UPUP, DNDN, UPDN and DNUP groups based on whether the DEGs were up (UP) or down (DN) regulated against BPH and WBPH, respectively. Gene ontology analysis, specially of members of the last two groups, revealed differences in plant response to the two planthoppers. Abundance of miRNAs and detection of their target genes also indicated that separate sets of genes were suppressed or induced against BPH and WBPH. These results were validated through the analysis of expression of 27 genes through semi-quantitative and quantitative real-time RT-PCR using a set of five RILs that were genetically identical but with different reaction against the two planthoppers. Coupled with data obtained through pathway analysis involving these 27 genes, expression studies revealed common and differential response of rice RP2068 against BPH and WBPH. Trehalose biosynthesis, proline transport, methylation were key pathways commonly upregulated; glucosinolate biosynthesis, response to oxidative stress, proteolysis, cytokinesis pathways were commonly down regulated; photosynthesis, regulation of transcription, expression and transport of peptides and defense related pathways were exclusively upregulated against WBPH; MYB transcription factor mediated defense induction was exclusive to BPH. CONCLUSION: Rice defense against the two sympatric planthoppers: BPH and WBPH has distinct features in RP2068. Hence, a conscious combination of resistance to these two pests is essential for effective field management.

Map-based cloning and validation of a gall midge resistance gene, Gm8, encoding a proline-rich protein in the rice variety Aganni.

The Asian rice gall midge (ARGM), Orseolia oryzae is an important insect pest causing an annual yield loss of about US$ 80 million in India. Till now 11 R genes and seven biotypes of the pest have been characterized and reported. The indica rice variety Aganni, a landrace from the state of Kerala, is known to carry the gall midge resistance gene Gm8 with HR-type of resistance. This gene has been fine mapped within 0.43 Mb region with the flanking markers RM22685 and RM22709. We identified 63 possible candidate genes through in silico analysis in the reference Nipponbare rice genome between 7.5 and 9.5 Mb region. One of the markers targeting the proline rich protein (PRP) gene (LOC_Os08g15080) showed polymorphism between the parents and also exhibited complete co-segregation with the trait in 426 F10 RIL populations. Functional validation of this gene through RT-PCR in contrasting parents and Pre-NILs (near isogenic lines) revealed that this is an early responsive gene with rapid induction at 24 h after gall midge infestation (hai) followed by subsequent reduction in the expression levels at late hours. Validation of this gene in five gall midge resistant rice varieties carrying different resistance genes revealed that the induction was unique to Aganni rice carrying Gm8 gene. Further, cloning and sequencing of the alleles of this gene including promoter region from TN1 (susceptible parent) and Aganni (resistant parent) revealed 153 nucleotide substitution, four amino acid substitutions and three mutations at putative cis-acting elements in TN1 when compared to Aganni. In addition, we also developed a functional marker (PRP-del) for detection of the gene for use in marker-assisted introgression of Gm8.

Bacterial Community Structure in the Asian Rice Gall Midge Reveals a Varied Microbiome Rich in Proteobacteria.

The Asian rice gall midge (ARGM) has emerged as a model gall forming pest of rice. The ARGM infestation of rice results in failure of panicle formation and economic loss. Understanding the molecular basis of ARGM-rice interactions is very crucial in order to control this devastating pest of rice. The current investigation was devised to identify bacterial communities present in the ARGM and in addition the bacterial diversity in the maggots during their interaction with susceptible or resistant rice varieties. Sequencing of 16S rRNA bacterial gene (V3-V4 region) revealed differences in the microflora of the ARGM maggots feeding on susceptible or resistant rice hosts. Results revealed that Wolbachia was the predominant bacterium in pupae and adults while Pseudomonas was predominant in maggots. Further, we observed that members of proteobacteria were predominant across all the samples. There was high species diversity in maggots isolated from susceptible rice and a high representation of unclassified bacteria in maggots isolated from resistant rice. This is the first study that reports variation of microbiome of the ARGM, based on host phenotype from which it was isolated, and results suggest that these variations could have an important role in host's susceptibility.

Analysis of SSH library of rice variety Aganni reveals candidate gall midge resistance genes.

The Asian rice gall midge, Orseolia oryzae, is a serious insect pest causing extensive yield loss. Interaction between the gall midge and rice genotypes is known to be on a gene-for-gene basis. Here, we report molecular basis of HR- (hypersensitive reaction-negative) type of resistance in Aganni (an indica rice variety possessing gall midge resistance gene Gm8) through the construction and analysis of a suppressive subtraction hybridization (SSH) cDNA library. In all, 2,800 positive clones were sequenced and analyzed. The high-quality ESTs were assembled into 448 non-redundant gene sequences. Homology search with the NCBI databases, using BlastX and BlastN, revealed that 73% of the clones showed homology to genes with known function and majority of ESTs belonged to the gene ontology category 'biological process'. Validation of 27 putative candidate gall midge resistance genes through real-time PCR, following gall midge infestation, in contrasting parents and their derived pre-NILs (near isogenic lines) revealed induction of specific genes related to defense and metabolism. Interestingly, four genes, belonging to families of leucine-rich repeat (LRR), heat shock protein (HSP), pathogenesis related protein (PR), and NAC domain-containing protein, implicated in conferring HR+ type of resistance, were found to be up-regulated in Aganni. Two of the reactive oxygen intermediates (ROI)-scavenging-enzyme-coding genes Cytosolic Ascorbate Peroxidase1, 2 (OsAPx1 and OsAPx2) were found up-regulated in Aganni in incompatible interaction possibly suppressing HR. We suggest that Aganni has a deviant form of inducible, salicylic acid (SA)-mediated resistance but without HR.

Feeding on resistant rice leads to enhanced expression of defender against apoptotic cell death (OoDAD1) in the Asian rice gall midge.

BACKGROUND: The Asian rice gall midge (Orseolia oryzae) is a destructive insect pest of rice. Gall midge infestation in rice triggers either compatible or incompatible interactions leading to survival or mortality of the feeding maggots, respectively. In incompatible interactions, generation of plant allelochemicals/defense molecules and/or inability of the maggots to continue feeding on the host initiate(s) apoptosis within the maggots. Unraveling these molecular events, triggered within the maggots as a response to feeding on resistant hosts, will enable us to obtain a better understanding of host resistance. The present study points towards the likely involvement of a defender against apoptotic cell death gene (DAD1) in the insect in response to the host defense. RESULTS: The cDNA coding for the DAD1 orthologue in the rice gall midge (OoDAD1) consisted of 339 nucleotides with one intron of 85 bp and two exons of 208 and 131 nucleotides. The deduced amino acid sequence of OoDAD1 showed a high degree of homology (94.6%) with DAD1 orthologue from the Hessian fly (Mayetiola destructor)--a major dipteran pest of wheat. Southern hybridization analysis indicated that OoDAD1 was present as a single copy in the genomes of the Asian rice gall midge biotypes (GMB) 1, 4 and 4 M. In the interactions involving GMB4 with Jaya (susceptible rice host) the expression level of OoDAD1 in feeding maggots gradually increased to 3-fold at 96 hai (hours after infestation) and peaked to 3.5-fold at 96 hai when compared to that at 24 hai. In contrast, expression in maggots feeding on RP2068 (resistant host) showed a steep increase of more than 8-fold at 24 hai and this level was sustained at 48, 72 and 96 hai when compared with the level in maggots feeding on Jaya at 24 hai. Recombinant OoDAD1, expressed in E. coli cells, when injected into rice seedlings induced a hypersensitive response (HR) in the resistant rice host, RP2068, but not in the susceptible rice variety, Jaya. CONCLUSIONS: The results indicate that the expression of OoDAD1 is triggered in the feeding maggots probably due to the host resistance response and therefore, is likely an important molecule in the initial stages of the interaction between the midge and its rice host.

Expression of Orseolia oryzae nucleoside diphosphate kinase (OoNDPK) is enhanced in rice gall midge feeding on susceptible rice hosts and its over-expression leads to salt tolerance in Escherichia coli.

The Asian rice gall midge, Orseolia oryzae, is a major dipteran pest of rice, with many known biotypes. The present investigation was initiated to understand the molecular mechanisms of infestation for developing novel integrated pest management strategies. We isolated and characterized a gene, nucleoside diphosphate kinase (OoNDPK), from the rice gall midge, encoding a protein with 169 amino acid residues and with a secretory signal sequence - an observation that assumes significance as salivary gland secretions have been implicated to play a major role in insect-plant interactions. Furthermore, up-regulation (> 18 folds) of OoNDPK was observed in the salivary glands of maggots feeding on susceptible host in contrast to those feeding on resistant host. Phylogenetic analysis revealed similarity of OoNDPK with its dipteran orthologues. 3DLigandSite analysis, of the predicted OoNDPK and its orthologues, revealed phenylalanine and tyrosine residues to be specifically present in NDPK proteins from the plant feeders. Results suggest secretion of OoNDPK into the host plant and its probable involvement in gall midge-rice interaction. Using the coleoptile cell elongation assay, we demonstrated that the recombinant OoNDPK is capable of causing elongation of rice coleoptile cells. Additionally, heterologous expression of OoNDPK in Escherichia coli increased the tolerance of these cells to salt (NaCl; up to 1 mM), hinting at the involvement of this gene in abiotic stress response as well.

Compatible interaction with its rice host leads to enhanced expression of the gamma subunit of oligosaccharyl transferase in the Asian rice gall midge, Orseolia oryzae.

The Asian rice gall midge, Orseolia oryzae, is a fast evolving, damaging pest of rice. Understanding the underlying molecular mechanism of interaction between the gall midge and rice will help in devising strategies to control and manage the pest. The present study aims to identify rice-responsive genes in the gall midge that aid pest survival. The abundance of transcripts coding for enzymes related to glycosylation, in a cDNA library prepared from maggots of the rice gall midge feeding on susceptible hosts, indicated their probable involvement in the gall midge-rice interaction. Hence, a full-length transcript for a gamma subunit of the oligosaccharyl transferase gene (OoOST) from the gall midge was cloned and characterized. It has 72% similarity to its orthologue cloned from Aedes aegypti. Tissue-specific analysis of the expression of OoOST revealed an increase (> sevenfold) in the transcripts of the gene in the salivary glands of maggots in susceptible plants when compared with the transcript level in the salivary glands of maggots feeding on resistant hosts. Using quantitative PCR, performed on different developmental stages of the maggots in two susceptible and two resistant hosts, we observed similar expression patterns (i.e. overexpression in the compatible interaction). These results indicate the involvement of OoOST in maggot survival and establishment in the susceptible host. In order to identify polymorphism in the gene, OoOST was cloned from three gall midge biotypes GMB1, GMB4 and GMB4M.

Bt rice evaluation and deployment strategies.

Bacillus thuringiensis (Bt), a gram positive soil bacteria was first identified and named by Japanese microbiologist Shigetane Ishiwata in 1901. During sporulation Bt produces proteinaceous parasporal crystal proteins called δ-endotoxins, or Cry proteins, which are insecticidal. Numerous Cry proteins have been isolated and characterized from different Bt strains with activity against insects, mites and nematodes. Sprayable formulations containing these Cry proteins as active ingredients have contributed significantly in the field of insect pest management. Since the first cloning of cry genes from Bt,1 scientists have successively demonstrated that plants could be genetically engineered to express these cry genes for the control of dreadful insect pests. Eventually, the first transgenic crop expressing Btcry1Ac gene in cotton was approved in 1996 for commercial cultivation in the USA to manage bollworms.

Impact of small fitness costs on pest adaptation to crop varieties with multiple toxins: a heuristic model.

A deterministic two-locus model was used to examine how small fitness costs to individuals carrying resistance alleles could impact the risk of panmictic insect pest populations adapting to crop varieties that produced two distinct toxins. Parameters examined were (1) level of toxicity of each toxin, (2) initial frequencies of alleles for adaptation to the toxins, (3) percentage of population feeding on nontoxic plants, and (4) level of fitness cost associated with adaptation to each of the two toxins. Resistance to each toxin was assumed to be biochemically independent, controlled by a resistance coding allele at a single locus, and inherited as a partially recessive trait in the field. When plants are extremely toxic to the pest, effective refuge size is 10%, and there is a fitness cost to resistance alleles only when in homozygous form (5%), the pest population is never predicted to adapt to either toxin as long as the initial frequencies of the resistance alleles are below 0.05. Even if the initial frequency of the allele for adapting to one toxin is 0.95 when a two-toxin cultivar completely replaces a one-toxin cultivar, the model predicts that a low equilibrium allelic frequency will develop for both resistance alleles, as long as the frequency of the allele for adapting to the second toxin is initially 0.001 or less. If cultivars with one and two toxins are planted, the model predicts that resistance will develop. Nonrandom mating and stochastic variation within subpopulations also could lead to evolution of resistance.

Identification of flanking SSR markers for a major rice gall midge resistance gene Gm1 and their validation.

Host-plant resistance is the preferred strategy for management of Asian rice gall midge (Orseolia oryzae), a serious pest in many rice-growing countries. The deployment of molecular markers linked to gall midge resistance genes in breeding programmes can accelerate the development of resistant cultivars. In the present study, we have tagged and mapped a dominant gall midge resistance gene, Gm1, from the Oryza sativa cv. W1263 on chromosome 9, using SSR markers. A progeny-tested F2 mapping population derived from the cross W1263/TN1 was used for analysis. To map the gene locus, initially a subset of the F2 mapping population consisting of 20 homozygous resistant and susceptible lines each was screened with 63 parental polymorphic SSR markers. The SSR markers RM316, RM444 and RM219, located on chromosome 9, are linked to Gm1 at genetic distances of 8.0, 4.9 and 5.9 cM, respectively, and flank the gene locus. Further, gene/marker order was also determined. The utility of the co-segregating SSR markers was tested in a backcross population derived from the cross Swarna/W1263//Swarna, and allelic profiles of these markers were analysed in a set of donor rice genotypes possessing Gm1 and in a few gall midge-susceptible, elite rice varieties.

Frequency of alleles conferring resistance to a Bacillus thuringiensis toxin in a Philippine population of Scirpophaga incertulas (Lepidoptera: Pyralidae).

Using the F2 screen methodology, we estimated the frequency of alleles conferring resistance to the Cry1Ab toxin of Bacillus thuringiensis Berliner in a Philippine population of the stem borer Scirpophaga incertulas (Walker). Evaluation of >450 isofemale lines for survival of F2 larvae on cry1Ab plants did not detect the presence of an allele conferring a high level of resistance. The frequency of such an allele in the sampled population was conservatively estimated to be <3.6 x 10(-3) with 95% confidence and a detection probability of 94%. However, there was evidence of the presence of alleles conferring partial resistance to Cry1Ab. The frequency of alleles for partial resistance was estimated as 4.8 x 10(-3) with a 95% CI between 1.3 x 10(-3) and 1.04 x 10(-2) and a detection probability of 94%. Our results suggest that the frequency of alleles conferring resistance to Cry1Ab in the population of S. incertulas sampled is not too high to preclude successful implementation of the high dose/refuge resistance management strategy.

Biodiversity of Asian rice gall midge (Orseolia oryzae Wood Mason) from five countries examined by AFLP analysis.

Amplified fragment length polymorphism (AFLP) analysis was used to assess the biodiversity of one of the most important dipteran pests of cereals, the Asian rice gall midge (Orseolia oryzae Wood Mason). Larvae and pupae were collected at 15 locations in five Asian countries and preserved in 95% ethanol for storage, shipment, and DNA extraction using cetyltrimethylammonium bromide (CTAB). Although only approximately 1 microg of DNA was extracted from a single pupa or larva, the use of several AFLP primers in various combinations meant that this amount of DNA was sufficient to allow many DNA fingerprints to be made per individual. Fingerprints were sufficiently reproducible, especially during selective amplification, to allow the genetic diversity within a field population to be characterized. Extraction of DNA from a pool of 20 insects yielded AFLP fingerprints in which variation among individuals was sacrificed in favor of detecting differences among populations. For each location, pooled DNA was amplified with three primer pairs. A total of 261 distinct AFLP bands were identified for the 45 fingerprints. Cluster analysis, performed by the unweighted pair-group method (UPGMA), separated the populations into two distinct groups. Group I included two populations from Guangdong province of southern China and one each from Laos and Imphal in northeastern India, while group II was comprised of eleven populations from elsewhere in India (Assam, Orissa, Madhya Pradesh, Andhra Pradesh, and Kerala) and from Nepal and Sri Lanka. AFLP analysis provided insight into the origins of gall midge biotypes. In 1992, the prevailing biotype in Imphal changed from Indian biotype 3 to a new biotype 3M. Our data show that biotype 3M belongs to group I and did not arise by a recent mutation from biotype 3, which belongs to group II. By contrast, Indian biotypes 2 and 4 are likely to have diverged through recent mutation and selection, as are Chinese biotypes 1 and 4. The almost simultaneous emergence of new biotypes in Kerala and Sri Lanka during 1985-1988 was most probably coincidental, because these biotypes are not closely related. AFLP fingerprints were also able to detect sexual dimorphism in the DNA of adult gall midges and to distinguish gall midge from its major parasite Platygaster oryzae.

Variation in performance on cry1Ab-transformed and nontransgenic rice varieties among populations of Scirpophaga incertulas (Lepidoptera: Pyralidae) from Luzon Island, Philippines.

We quantified variation in performance under greenhouse conditions among seven populations of Scirpophaga incertulas (Walker) from Luzon Island, Philippines, on three rice varieties: 'IR58' transformed with the cry1Ab gene from Bacillus thuringiensis Berliner, and nontransgenic IR58 and IR62. On IR62, S. incertutas performance did not differ among provinces for any of the 10 parameters measured, but there was a significant effect of town within province for one parameter, 20-d-old larval weight. Larval survival after 48 h on cy1Ab-transformed IR58 did not differ significantly among provinces, but did differ significantly among towns within a province. There was no geographic variation in larval survival after 48 h on control plants of IR58. Surviving insects from the cry1Ab-transformed IR58 were transferred to IR62 to complete development. There was no geographic variation in the percentage of insects completing development to adult emergence and the time required by the transferred female insects to complete development. However, there was variation among provinces in male developmental time. The absence of geographic variation on nontransgenic IR58 and the very limited variation on IR62 indicated that there was little variation in general vigor among the S. incertulas populations and thus that the variation in performance oil cry1Ab-transformed IR58 was probably attributable to differences in susceptibility to Cry1Ab.

Highly repetitive DNA sequence elements from Orseolia oryzae (Wood-Mason) discriminate between the Indian isolates and the Asian rice gall midge and the paspalum midge.

We described multicopy DNA clones isolated from a partial genomic library of Orseolia oryzae, based on reverse genomic hybridization, suitable for studying genetic variation in the Asian rice gall midge and other isomorphic species. Three clones produced monomorphic DNA band patterns between biotypes of O. oryzae but polymorphic patterns were produced between O. oryzae and O. fluvialis, the paspalum midge. These probes detect changes in the repetitive sequence structure between species and constitute the first genetic markers for distinguishing between field isolates of rice gall midge and related species of Orseolia. These will be useful in identifying and perhaps eradicating alternative hosts for this pest, and detecting early-season outbreaks of O. oryzae from light trap collections.

Characterization of a DNA sequence that detects repetitive DNA elements in the Asian rice gall midge (Orseolia oryzae) genome: potential use in DNA fingerprinting of biotypes.

We have isolated based on reverse genome hybridization, and sequenced a DNA clone, pNZE25, from a partial genomic library of the Asian rice gall midge Orseolia oryzae (Wood-Mason) (O.o.). Clone pNZE25 is highly A+T rich (67%), lacks any open reading frame and does not display homology to sequences in GenBank. Clone pNZE25 detects a 120-bp repeat in the O.o. genome, as seen from the generation of a 120-bp ladder after Southern analysis of HinfI-digested genomic DNA. When used to probe O.o. genomic DNA digested with DraI, HaeIII or AluI, pNZE25 generates individual specific DNA fingerprints on target DNA isolated from gall midge biotypes collected from different parts of India.

DNA markers tightly linked to a gall midge resistance gene (Gm2) are potentially useful for marker-aided selection in rice breeding.

We have developed a polymerase chain reaction (PCR)-based assay that could effectively reduce the time period required to screen and select for Gall Midgeresistant rice lines under field conditions. The primers for the assay were designed on the basis of sequence information of two phenotype specific random amplified polymorphic DNA fragments which were found to be tightly linked to Gall Midge biotype-1 resistance gene (Gm2). The two RAPD fragments, F81700 in the susceptible parent 'ARC6650' and F10600 in the resistant parent 'Phalguna', were identified after screening 5450 loci using 520 random primers on genomic DNAs of 'ARC6650' and 'Phalguna'. These primers, when used in a multiplexed PCR, amplified specifically a 1.7-kb and 0.6-kb fragment in the susceptible and resistant parents, respectively. When this assay was performed on genomic DNAs of 44 recombinant inbred lines derived from 'ARC6650' x 'Phalguna' and 5 lines derived from other crosses where one of the parents was 'Phalguna', 'ARC6650' or their derivatives, the primers amplified a 1.7-kb fragment in all of the susceptible lines or a 0.6-kb fragment in all of the resistant ones. These markers can be of potential use in the marker-aided selection of Gall Midge biotype-1 resistant phenotypes. As screening for resistance can now be conducted independent of the availability of insects, the breeding of resistant varieties can be hastened.

RFLP and RAPD mapping of the rice gm2 gene that confers resistance to biotype 1 of gall midge (Orseolia oryzae).

Gm2 is dominant gene conferring resistance to biotype 1 of gall midge (Orseolia oryzae Wood-Mason), the major dipteran pest of rice. The gene was mapped by restriction fragment length polymorphism (RFLP) analysis of a set of 40 recombinant inbred lines derived from a cross between the resistant variety 'Phalguna' and the susceptible landrace 'ARC 6650'. The gene is located on chromosome 4 at a position 1.3 cM from marker RG329 and 3.4 cM from RG476. Since the low (28%) polymorphism of this indica x indica cross hindered full coverage of the genome with RFLP markers, the mapping was checked by random amplified polymorphic DNA (RAPD)/bulked segregant analysis. Through the use of 160 RAPD primers, the number of polymorphic markers was increased from 43 to 231. Two RAPD primers amplified loci that co-segregated with resistance/susceptibility. RFLP mapping of these loci showed that they are located 0.7 cM and 2.0 cM from RG476, confirming the location of Gm2 in this region of chromosome 4. Use of these DNA markers will accelerate breeding for gall midge resistance by permitting selection of the Gm2 gene independently of the availability of the insect.