Genetic,physiological and molecular interactions of rice and its major dipteran pest,gall midge

The gall midge, Orseolia oryzae, is a major dipteran pest of rice affecting most rice growing regions in Asia, Southeast Asia and Africa. Chemical and other cultural methods for control of this pest are neither very effective nor environmentally safe. The gall midge problem is further compounded by the fact that there are many biotypes of this insect and new biotypes are continuously evolving. However, resistance to this pest is found in the rice germ plasm. Resistance is generally governed by single dominant genes and a number of non-allelic resistance genes that confer resistance to different biotypes have been identified. Genetic studies have revealed that there is a gene-for-gene interaction between the different biotypes of gall midge and the various resistance genes found in rice. This review discusses different aspects of the process of infestation by the rice gall midge and its interaction with its host. Identification of the gall midge biotypes by conventional methods is a long and tedious process. The review discusses the PCR-based molecular markers that have been developed recently to speed up the identification process. Similarly, molecular markers have been developed for two gall midge resistance genes in rice – Gm2 and Gm4t – and these markers are now being used for marker-assisted selection. The mapping, tagging and map-based gene cloning of one of these genes – Gm2 – has also been discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Identification of flanking SSR markers for a major rice gall midge resistance gene <Emphasis Type="Italic">Gm1</Emphasis> 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.     

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.     

Identification and mapping of an AFLP marker linked to Gm7, a gall midge resistance gene and its conversion to a SCAR marker for its utility in marker aided selection in rice

We have identified an AFLP marker SA598 that is linked to Gm7, a gene conferring resistance to biotypes 1, 2 and 4 of the gall midge ( Orseolia oryzae), a major dipteran pest of rice. A set of PCR primers specific to an RFLP marker, previously identified to be linked to another gall midge resistance gene Gm2, also amplified a 1.5-kb (F8LB) fragment that is linked to Gm7. Gm7 is a dominant gene and non-allelic to Gm2. Hybridization experiments with clones from a YAC library of Nipponbare, a japonica variety, a BAC library of IR-BB21, an indica variety, and cosmid clones encompassing Gm2 from Phalguna, an indica variety, with F8LB and SA598 as probes, revealed that Gm7 is tightly linked to Gm2 and is located on chromosome 4 of rice. SA598 was sequenced and the sequence information was used to design sequence-characterized amplified region (SCAR) primers. The potential use of these SCAR primers in marker-aided selection of Gm7 in a rice breeding program has been demonstrated.     

Tagging and mapping of a rice gall midge resistance gene, <Emphasis Type="Italic">Gm8</Emphasis>, and development of SCARs for use in marker-aided selection and gene pyramiding

Using amplified fragment length polymorphisms (AFLPs) and random amplified polymorphic DNAs (RAPDs), we have tagged and mapped Gm8, a gene conferring resistance to the rice gall midge (Orseolia oryzae), a major insect pest of rice, onto rice chromosome 8. Using AFLPs, two fragments, AR257 and AS168, were identified that were linked to the resistant and susceptible phenotypes, respectively. Another resistant phenotype-specific marker, AP19₅₈₇, was also identified using RAPDs. SCAR primers based on the sequence of the fragments AR257 and AS168 failed to reveal polymorphism between the resistant and the susceptible parents. However, PCR using primers based on the regions flanking AR257 revealed polymorphism that was phenotype-specific. In contrast, PCR carried out using primers flanking the susceptible phenotype-associated fragment AS168 produced a monomorphic fragment. Restriction digestion of these monomorphic fragments revealed polymorphism between the susceptible and resistant parents. Nucleotide BLAST searches revealed that the three fragments show strong homology to rice PAC and BAC clones that formed a contig representing the short arm of chromosome 8. PCR amplification using the above-mentioned primers on a larger population, derived from a cross between two indica rice varieties, Jhitpiti (resistant parent) and TN1 (susceptible parent), showed that there is a tight linkage between the markers and the Gm8 locus. These markers, therefore, have potential for use in marker-aided selection and pyramiding of Gm8 along with other previously tagged gall midge resistance genes [Gm2, Gm4(t), and Gm7].The nucleotide sequence data reported here will appear in the EMBL, GenBank and DDBJ nucleotide sequence databases under the accession numbers AY545920–AY545923     

Natural and human‐mediated selection in a landrace of Thai rice (Oryza sativa)

Landrace rice in Thailand consists of managed populations grown under traditional and long‐standing agricultural practices. These populations evolve both in response to environmental conditions within the local agro‐ecosystem and in response to human activities. Single landraces are grown across varying environments and recently have experienced temporal changes in local environments due to climate change. Here we assess the interplay between natural selection in a changing climate and human‐mediated selection on the population genetic structure of Muey Nawng, a local landrace of Thai rice. Genetic diversity and population structure of landrace rice were assessed by a STRUCTURE analysis of 20 microsatellite loci. The first exon–intron junction of the waxy gene was sequenced to determine genotypes for glutinous or non‐glutinous grain starch. Muey Nawng rice is genetically variable and is structured based on starch grain types and the level of resistance to gall midge pest. A strong positive correlation was found between genetic diversity and the percentage of gall midge infestation. Variation in the waxy locus is correlated with starch quality; selection for non‐glutinous rice appears to involve additional genes. The dynamics of genetic diversity within Muey Nawng rice depends on three factors: (a) a genetic bottleneck caused by strong selection associated with gall midge infestation, (b) selection by local farmers for starch quality and (c) variation introduced by farmer practices for cultivation and seed exchange. These results, when taken in total, document the ability of landrace rice to quickly evolve in response to both natural and human‐mediated selection.     

Molecular mapping of a resistance-specific PCR-based marker linked to a gall midge resistance gene (Gm4t) in rice

 A PCR-based marker (E20₅₇₀) linked to the gene Gm4t, which confers resistance to a dipteran pest gall midge (Orseolia oryzae), has been mapped using the restriction fragment length polymorphism (RFLP) technique in rice. Gm4t is a dominant resistance gene. We initially failed to detect useful polymorphism for this marker in a F₃ mapping population derived from a cross between two indica parents, ‘Abhaya’בShyamala’, with as many as 35 restriction enzymes. ‘Abhaya’ carries the resistance gene Gm4t and ‘Shyamala’ is susceptible to gall midge. Subsequently, E20₅₇₀ was mapped using another mapping population represented by a F₂ progeny from a cross between ‘Nipponbare’, a japonica variety, and ‘Kasalath’, an indica variety, in which the gene Gm4t was not known to be present. Gm4t mapped onto chromosome 8 between markers R1813 and S1633B. Our method, thus, presents an alternative way of mapping genes which otherwise would be difficult to map because of a lack of polymorphism between closely related parents differing in desired agronomic traits. Received: 1 April 1997 / Accepted: 13 May 1997     

Isolation and FISH mapping of Yeast Artificial Chromosomes (YACs) encompassing an allele of the Gm2 gene for gall midge resistance in rice

 Ten yeast artificial chromosomes (YACs) spanning the Gm2 locus have been isolated by screening high-density filters containing a total of approximately 7000 YAC (representing six genome equivalents) clones derived from a japonica rice, Nipponbare. The screening was done with five RFLP markers flanking a gall midge resistance gene, Gm2, which was previously mapped onto chromosome 4 of rice. This gene confers resistance to biotype 1 and 2 of gall midge (Orseolia oryzae), a major insect pest of rice in South and Southeast Asia. The RFLP markers RG214, RG329 and F8 hybridized with YAC Y2165. Two overlapping YAC clones (Y5212 and Y2165) were identified by Southern hybridization, with Gm2-flanking RFLP markers, and their inserts isolated. The purified YACs and RFLP markers flanking Gm2 were labeled and physically mapped by the fluorescence in situ hybridization (FISH) technique. All of them mapped to the long arm of chromosome 4 of the resistant variety of rice, ‘Phalguna’, confirming the previous RFLP mapping data. Received: 15 December 1997 / Accepted: 5 March 1998     

Flanking Microsatellite Markers for Breeding Varieties Against Asian Rice Gall Midge

The Asian rice gall midge, Orseolia oryzae Wood-Mason (Cecidomyiidae: Diptera) is a serious pest of wet season rice in South and Southeast Asia. Due to internal feeding habit and presence of biotypes of the pest, the most feasible way to control is breeding varieties resistant against multiple biotypes through marker-assisted breeding (MAB). But very few versatile co-dominant markers linked to the gall midge resistance genes are available. We used a set of F₉ recombinant inbred lines (RILs) of the cross TN1/PTB10 and identified microsatellite markers for the gall midge resistance gene in cv. PTB10 on short arm of rice chromosome 8. Markers RM22550 and RM547 flank the gene at a distance of 0.9 and 1.9 cM, respectively. Amplification of the markers in gall midge resistant and susceptible cultivars showed that these markers can be successfully used in MAB for development of gall midge resistant varieties.     

An AFLP marker that differentiates biotypes of the Asian rice gall midge (Orseolia oryzae, Wood-Mason) is sex-linked and also linked to avirulence

In an attempt to identify a specific marker for biotype 2 of the Asian rice gall midge (Orseolia oryzae, Wood-Mason), we used AFLP (amplified fragment length polymorphism) fingerprinting. We identified an AFLP marker that is specifically amplified in biotypes 1, 2 and 5 of the rice gall midge, but not in biotype 4. Biotypes 1, 2 and 5 are avirulent to hosts bearing the Gm2 resistance gene (found in rice variety Phalguna), whereas biotype 4 is virulent to Gm2. Based on the sequence of this AFLP marker, SCAR (sequence characterized amplified region) primers were designed and used in combination with previously developed SCAR primers to distinguish effectively all five biotypes in a multiplex PCR-based assay. The inheritance pattern of this marker in the progenies of inter-biotype crosses between biotypes 1, 2 and 4 shows that the marker can be amplified by PCR from all F₁ females, irrespective of the biotype status of their parents. However, the marker is present only in those male progenies whose mother was of a Gm2 avirulent biotype. The specific amplification of this marker in the avirulent biotypes and its pattern of inheritance show that avirulence with respect to carriers of the Gm2 gene in rice gall midge is sex-linked. Received: 16 August 1999 / Accepted: 27 December 1999     

Rice–gall midge interactions: Battle for survival

Gall midges are insects specialized in maneuvering plant growth, metabolic and defense pathways for their benefit. The Asian rice gall midge and rice share such an intimate relationship that there is a constant battle for survival by either partner. Diverse responses by the rice host against the midge include necrotic hypersensitive resistance reaction, non-hypersensitive resistance reaction and gall-forming compatible interaction. Genetic studies have revealed that major R (resistance) genes confer resistance to gall midge in rice. Eleven gall midge R genes have been characterized so far in different rice varieties in India. In addition, no single R gene confers resistance against all the seven biotypes of the Asian rice gall midge, and none of the biotypes is virulent against all the resistance genes. Further, the interaction of the plant resistance gene with the insect avirulence gene is on a gene-for-gene basis. Our recent investigations involving suppressive subtraction hybridization cDNA libraries, microarray analyses, gene expression assays and metabolic profiling have revealed several molecular mechanisms, metabolite markers and pathways that are induced, down-regulated or altered in the rice host during incompatible or compatible interactions with the pest. This is also true for some of the pathways studied in the gall midge. Next generation sequencing technology, gene expression studies and conventional screening of gall midge cDNA libraries highlighted molecular approaches adopted by the insect to feed, survive and reproduce. This constant struggle by the midge to overcome the host defenses and the host to resist the pest has provided us with an opportunity to observe this battle for survival at the molecular level.     

Marker-trait association analysis for gall midge (Orseolia oryzae) resistance in a diverse rice population

Damage caused by insect herbivores, notably Asian rice gall midge, Orseolia oryzae is more prevalent in the rice-growing belts of India's southern and north-eastern states. As a prelude to resistant cultivar development, the identification of genomic regions for resistance in the source population is crucial. In the present investigation, 202 rice genotypes were phenotyped and assayed with genomic markers reported for gall midge resistance. Positive skewness and platykurtic distribution of response scores suggested the inheritance of gall midge resistance in the study population. The marker gm3del3 contributed the most genetic variation, followed by RM28574 and marker RM22709 explained minimal variation. A marker-trait association analysis with a single marker-trait linear regression approach was performed to discover gall midge resistant genomic region/genes. The marker RM17480 on chromosome 4 reported to be linked with gm3 gene was found significantly associated with the gall midge resistance genomic region with allelic effects in a negative direction favouring resistance reaction. The allelic effects of significantly associated markers were correlated significantly with the phenotypic variation of gall midge damage scores. Genes identified in the vicinity of this marker contribute to stress response reactions in rice plants. The 200 bp allele of the marker was associated with susceptibility, while the 250 bp allele was associated with resistance expression. This allelic association with trait variation suggests the importance of associated marker for utilisation in marker-assisted selection programmes to incorporate resistance alleles into elite rice genotypes.     

PCR-based DNA markers linked to a gall midge resistance gene, Gm4t, has potential for marker-aided selection in rice

Rice DNAs from a gall midge resistant variety, Abhaya, a susceptible variety, Tulsi and their F₃ progeny were screened using 500 random primers in conjunction with bulked-segregant analysis in a polymerase chain reaction (PCR) with a view to detecting random amplified polymorphic DNAs (RAPDs) linked to the gene, Gm4t, which confers resistance to gall midge, a dipteran insect pest of rice. A total of 454 primers were able to produce a distinct amplification pattern, and 3695 bands/loci were amplified between the phenotypically different parents. Of these, 304 bands were polymorphic between the parents, with 19 being phenotypespecific. One of these primers, E20, amplified 2 bands, E20₅₇₀ and E20₅₈₃, which are tightly linked to resistance and susceptibility, respectively. These specific bands were cloned and sequenced, and a 94% sequence homology was found between the two fragments. Two specific 20-mer oligonucleotides were synthesized, based on the sequence information of E20₅₈₃, for use in PCR amplification directly from genomic DNAs. These PCR primers were able to amplify phenotype-specific bands, a 583-bp fragment in susceptible F₃ lines and a 570-bp fragment in resistant F₃ lines that had been derived from a cross between the parents, indicating their potential and utility for marker-aided selection of the Gm4t gene in rice. Its use would facilitate the early and efficient selection of resistant genes in plant breeding programmes and even in those areas where the insect is not known to occur. These phenotype-specific bands are single-copy sequences and are being mapped to ascertain their chromosomal location in rice.     

Molecular mapping of gene Gm-6(t) which confers resistance against four biotypes of Asian rice gall midge in China

The Chinese rice cultivar Duokang #1 carries a single dominant gene Gm-6(t) that confers resistance to the four biotypes of Asian rice gall midge (Orseolia oryzae Wood-Mason) known in China. Bulked segregant analysis was performed on progeny of a cross between Duokang #1 and the gall midge-susceptible cultivar Feng Yin Zhan using the RAPD method. The RAPD marker OPM06₍₁₄₀₀₎ amplified a locus linked to Gm-6(t). The locus was subsequently mapped to rice chromosome 4 in a region flanked by cloned RFLP markers RG214 and RG163. Fine mapping of Gm-6(t) revealed that markers RG214 and RG476 flanked the gene at distances of 1.0 and 2.3 cM, respectively. Another gall midge resistance gene, Gm-2, mapped previously to chromosome 4, is located about 16 cM from Gm-6(t), to judge by data from a segregating population derived from a cross between Duokang #1 and the Indian cultivar Phalguna that carries Gm-2. We developed a PCR-based marker-assisted selection kit for transfer of the Gm-6(t) gene into Ming Hui 63 and IR50404, two parental lines commonly used in hybrid rice production in China. The kit contains PCR primer pairs based on the terminal sequences of the RG214 and RG476 clones. Polymorphism between Duokang #1 and the hybrid parental lines was found at these markers after digestion of the PCR products with specific restriction endonucleases. The kit will accelerate introduction of gall midge resistance into hybrid rice in China. Received: 18 May 2000 / Accepted: 9 March 2001     

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.     

Microbiome responses during virulence adaptation by a phloem‐feeding insect to resistant near‐isogenic rice lines

The microbiomes of phloem‐feeding insects include functional bacteria and yeasts essential for herbivore survival and development. Changes in microbiome composition are implicated in virulence adaptation by herbivores to host plant species or host populations (including crop varieties). We examined patterns in adaptation by the green leafhopper, Nephotettix virescens, to near‐isogenic rice lines (NILs) with one or two resistance genes and the recurrent parent T65, without resistance genes. Only the line with two resistance genes was effective in reducing leafhopper fitness. After 20 generations on the resistant line, selected leafhoppers attained similar survival, weight gain, and egg laying to leafhoppers that were continually reared on the susceptible recurrent parent, indicating that they had adapted to the resistant host. By sequencing the 16s rRNA gene, we described the microbiome of leafhoppers from colonies associated with five collection sites, and continually reared or switched between NILs. The microbiomes included 69–119 OTUs of which 44 occurred in ≥90% of samples. Of these, 14 OTUs were assigned to the obligate symbiont Candidatus sulcia clade. After 20 generations of selection, collection site had a greater effect than host plant on microbiome composition. Six bacteria genera, including C. sulcia, were associated with leafhopper virulence. However, there was significant within‐treatment, site‐related variability in the prevalence of these taxa such that the mechanisms underlying their association with virulence remain to be determined. Our results imply that these taxa are associated with leafhopper nutrition. Ours is the first study to describe microbiome diversity and composition in rice leafhoppers. We discuss our results in light of the multiple functions of herbivore microbiomes during virulence adaptation in insect herbivores.     

Inducible expression of a fusion gene encoding two proteinase inhibitors leads to insect and pathogen resistance in transgenic rice

Plant proteinase inhibitors (PIs) are considered as candidates for increased insect resistance in transgenic plants. Insect adaptation to PI ingestion might, however, compromise the benefits received by transgenic expression of PIs. In this study, the maize proteinase inhibitor (MPI), an inhibitor of insect serine proteinases, and the potato carboxypeptidase inhibitor (PCI) were fused into a single open reading frame and introduced into rice plants. The two PIs were linked using either the processing site of the Bacillus thuringiensis Cry1B precursor protein or the 2A sequence from the foot‐and‐mouth disease virus (FMDV). Expression of each fusion gene was driven by the wound‐ and pathogen‐inducible mpi promoter. The mpi‐pci fusion gene was stably inherited for at least three generations with no penalty on plant phenotype. An important reduction in larval weight of Chilo suppressalis fed on mpi‐pci rice, compared with larvae fed on wild‐type plants, was observed. Expression of the mpi‐pci fusion gene confers resistance to C. suppressalis (striped stem borer), one of the most important insect pest of rice. The mpi‐pci expression systems described may represent a suitable strategy for insect pest control, better than strategies based on the use of single PI genes, by preventing insect adaptive responses. The rice plants expressing the mpi‐pci fusion gene also showed enhanced resistance to infection by the fungus Magnaporthe oryzae, the causal agent of the rice blast disease. Our results illustrate the usefulness of the inducible expression of the mpi‐pci fusion gene for dual resistance against insects and pathogens in rice plants.     

Genetic mapping of the rice resistance-breaking gene of the brown planthopper Nilaparvata lugens

Host plant resistance has been widely used for controlling the major rice pest brown planthopper (BPH, Nilaparvata lugens). However, adaptation of the wild BPH population to resistance limits the effective use of resistant rice varieties. Quantitative trait locus (QTL) analysis was conducted to identify resistance-breaking genes against the anti-feeding mechanism mediated by the rice resistance gene Bph1. QTL analysis in iso-female BPH lines with single-nucleotide polymorphism (SNP) markers detected a single region on the 10th linkage group responsible for the virulence. The QTL explained from 57 to 84% of the total phenotypic variation. Bulked segregant analysis with next-generation sequencing in F₂ progenies identified five SNPs genetically linked to the virulence. These analyses showed that virulence to Bph1 was controlled by a single recessive gene. In contrast to previous studies, the gene-for-gene relationship between the major resistance gene Bph1 and virulence gene of BPH was confirmed. Identified markers are available for map-based cloning of the major gene controlling BPH virulence to rice resistance.