WO2005056833A1 - Molecular markers for mapping and tagging gm8 gene - Google Patents

Abstract

A method for tagging and mapping Gm8, a gene conferring resistance to the rice gall midge (Orseolia oryzae), a major insect pest of rice, using AFLPs and RAPDs, onto chromosome 8 of rice is disclosed. Using AFLPs, two fragments, AR257 and AS168, were identified that were linked to the resistant and susceptible phenotypes, respectively. Another resistant phenotype-specific marker, AP 19587, was also identified using RAPDs. SCAR primers based on the sequence of the fragments AR257 and AS 168 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 AS 168, 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 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.

Description

MOLECULAR MARKERS FOR MAPPING AND TAGGING GM8 GENE

Filed of the invention The present invention relates to molecular markers for mapping and tagging 5 Gm8 gene, and their application in marker-assisted selection (MAS) of gall midge resistant/susceptible phenotypes. More particularly, the present invention relates to DNA markers linked to a gall midge resistance gene Gm8 for marker-aided selection in rice. The present invention also relates to novel primers for use in preparing the DNA markers linked to a gall midge resistance gene Gm8 for marker-aided selection in rice.

.0 The present invention also relates to a method for screening rice varieties for susceptibility and/or resitance to gall midge. Background of the invention Rice gall infestation is a serious rice disease caused by a dipteran insect pest known as gall midge (Orseolia oiyzae). The disease is prevalent in India, China, South

15 east Asia and Africa. In Asia alone, the damage caused by gall midge is more than US $550 million per year (Bentur et al. 2003). In India, the gall midge infestation is most prevalent in the states of Madhya Pradesh, Chhattisgarh, Bihar, Jharkhand, Orissa, Andhra Pradesh and Maharashtra. Recent reports show that it is becoming a serious threat in Kerala and some northeastern states (Bentur et al. 2003).

Z0 The gall midge problem in rice is further compounded by the fact that there are many biotypes of this insect and new biotypes keep continuously evolving. In India, till recently, five biotypes of gall midge were known to exist but now a new biotype has been reported from the northeast (Bentur et al. 2003). Different biotypes of gall midge are distributed in different regions of the country. Resistance in a rice variety for a

25 particular biotype is usually governed by single dominant gene and a total of nine non- allelic resistance genes have been identified from different varieties of rice that confer resistance against different biotypes of the pest (Kumar et al. 1998; Sardesai et al. 2001). Genetic studies have revealed that there is a gene-for-gene interaction between different resistance genes and their respective biotypes of gall midge (Harris et al.

30 2003). The deployment of these resistant genes will not only be environment-friendly but also likely to provide durable resistance. Molecular markers are used for tagging of desirable genes. Mapping and tagging of agriculturally important genes with molecular markers forms the foundation for marker-assisted selection (MAS) in crop plants. Traditional procedures of screening populations for desired traits are not only labour-intensive but also time consuming. Molecular markers have several advantages over the traditional phenotypic markers that were previously available to plant breeders. They offer great scope for improving the efficiency of conventional plant breeding by carrying out selection not directly on the trait of interest but on molecular markers linked to that trait. Availability of tightly linked genetic markers for resistance genes will help in identifying plants carrying these genes simultaneously without subjecting them to pathogen or insect attack in early generations. Biotyping of gall midge is done traditionally by observing the infectivity pattern on a set of rice differentials/varieties. It is not possible to morphologically differentiate the different biotypes of gall midge and thus biotyping has been solely based on differential infestation patterns on specific rice hosts. This has slowed down the process of biotype identification, and consequently the selection for rice plants resistant to more than one biotype of gall midge particularly since the natural occurrence of gall midge is restricted to a 2 to 4 month period every year. This also slows down the process of breeding new gall midge resistant rice varieties. An alternative to this labour-intensive and time-consuming screening procedure is the screening of the population of interest using genetic markers. The identification of suitable molecular markers closely linked to the gall midge resistance gene will allow one to easily follow the gene in a cross intended to breed new resistant varieties any time of the year without depending on the annual occurrence of the insects. The availability of molecular markers would enable breeders to ascertain which of the individuals from a cross are resistant or susceptible to gall midge. Thus the utility of MAS lies in the breeder being able to conduct many rounds of selection in a year without depending on the natural occurrence of the pest. The identification and development of DNA-based molecular markers that are tightly linked to a resistance gene enables one to follow the gene in a cross intended to breed new resistant varieties any time of the year without depending on the annual occurrence of insects (Mohan et al. 1997a). Of the total nine gall midge resistance genes that is known (Gml to Gm9) so far, four (Gm2, Gm4(t), Gm6(t) and GmT) have been tagged and mapped (Mohan et al. 1994, 1997b; Nair et al. 1995, 1996; Katiyar et al. 2001; Sardesai et al. 2002). The development of these markers has been made possible using various molecular marker techniques (Mohan et al 1994, 1997b; Nair et al 1995, 1996). The AFLP (Amplified Fragment Length Polymorphism) has been widely used as a DNA fingerprinting technique (Nos et al. 1995) in plant genetic studies (Hill et al. 1996; Mackill et al. 1996; Maughen et al. 1996; Hongtrakul et al. 1997; Zhu et al. 1998; Bonnema et al. 2002). Owing to its higher marker-index and the potential to scan a wider genome area for polymorphisms, AFLP technique was employed which is also known to produce highly specific and reproducible results (Ellis et al. 1997; Singh et al. 1999). In the present study, using AFLPs and RAPDs (Random Amplified Polymorphic DNA), we have developed two SCAR (Sequence Characterized Amplified Region) markers that show very tight linkage to a gall midge resistance gene locus, Gm8, in rice. Objects of the invention Accordingly, it is an object of the present invention to provide an alternative to the labour-intensive and time consuming screening procedures employed for tagging and mapping the rice gall midge resistance gene Gm8. It is another object of the present invention to provide a non-destructive method for screening rice populations of interest to select varieties, which are resistant to gall midge attack. It is yet another object of the present invention to provide a method for identification of suitable molecular markers closely linked to gall midge resistance gene

Gm8 to enable easy following of the gene in a cross intended to breed new resistance varieties any time of the year without having to depend upon the annual occurrence of insect infestation. It is a further object of the present invention to provide an AFLP marker linked to a gall midge resistance gene Gm8 for marker-aided selection in rice. It is yet another object of the present invention to provide a method for fine mapping and potential application of AFLP markers linked to a gall midge resistance gene Gm8, for marker-aided selection in rice. It is still further object of the present invention to novel primers for developing AFLP markers linked to a gall midge resistance gene Gm8. Summary of the invention The above and other objects of the present invention are achieved by two SCAR (Sequence Characterized Amplified Region) markers that show very tight linkage to a gall midge resistance gene locus, Gm8, in rice which were developed in accordance with the present invention using AFLPs and RAPDs (Random Amplified

Polymorphic DNA). The first step in marker-assisted selection (MAS) is the tagging of the gene by identifying molecular markers that are closely linked with it, which cosegregate with the desired phenotype. A cross between the gall midge resistant parent, Jhitpiti (carrying the Gm8 gene) and susceptible parent, TNI, has been developed and a F₅ progeny has been raised. A polymorphic band has been identified from the F₅ progeny, using AFLP that cosegregates with the susceptible phenotype. This band has been eluted from the gel and cloned. The cloned AFLP fragment has been sequenced and primers have been developed that can selectively amplify DNA of the susceptible phenotype, thus differentiating it from the resistant phenotype. This Gm8 gene linked marker has been mapped on chromosome 8 of rice and has also been shown to be linked to the Gm4 gene. This marker is present as a single copy in the resistant parent, Jhitpiti. Primers developed from this marker have also been shown to differentiate between the resistant and susceptible phenotypes in different crosses carrying different gall midge resistance genes. These primers are thus, of great use in marker-assisted selection as they show polymorphism in resistant and susceptible plants carrying different gall midge resistance genes. Detailed description of the invention The F₄ population used in the present study consisted of rice lines derived from a cross between the two indica rice varieties; 'Jhitpiti' (carrying Gm8; resistant to gall-midge biotype 1) and 'TNI' (susceptible to gall midge). Of the 608 F₂ plants, derived from the above cross, 265 random plants were sown as single-plant progeny in F₃. The reaction of each of the individual F₃ lines was recorded as homozygous resistant, segregating, or homozygous susceptible. From each scored F₃ progeny, one resistant or susceptible plant [i.e. (i) resistant plants from progenies showing homozygous resistance reaction; or, (ii) susceptible plants from progenies showing homozygous susceptible reaction; or (iii) resistant/susceptible plants from progenies showing segregation for resistance/susceptibility)] was selected for advancing to F₄. Thus, we had 265 F₃ progenies from which we selected 265 F₄ individual plants. These F₄ plants were grown again as individuals (lines). Individual plants were scored in each line (resistant plants were tagged from progenies showing homozygous resistance reaction, susceptible plants were tagged from progenies showing homozygous susceptible reaction) and DNA was isolated from leaves of scored individual resistant /susceptible plants. Plant reaction for resistance and susceptibility towards the gall midge was observed under field-conditions, based on the natural occurrence of the insect, at the Indira Gandhi Agricultural University, Raipur, Chhattisgarh, India. The plants were screened for the presence or absence of galls. The plants without any gall formation were scored as resistant and those with even one gall per plant were recorded as susceptible. The present invention will now be described with reference to the following Example and accompanying drawings, which illustrate a preferred embodiment of the invention. It will be clear to a person skilled in the art that various modifications of the invention are possible without departing from the spirit and scope of the present invention. In the accompanying drawings: Fig. la: AFLP fragment (AR257) segregating with the resistant phenotype (arrows), using primer combination Pst I-AT (5 ' GACTGCGT AC ATGC AAT 3 ' .... Seq ID # 1) and Mse I-CGT (5 ' GATGAGTCCTGAGTAACGT 3 ' .... Seq ID # 2). The first two lanes are the resistant (R) and susceptible (S) parents, Jhitpiti and TNI, respectively, followed by the resistant (Rp) and susceptible (Sp) bulks. Lanes / and ii represent lines 215S and 201 S, respectively. Labels at the bottom of the figure indicate individual F₄ line numbers. Fig. lb AFLP fragment (AS 168) segregating with the susceptible phenotype

(arrows), using primer combination Pst I-AA (5' GACTGCGTACATGCAAA 3 '....Seq ID # 3) and Mse I-CAC (5' GATGAGTCCTGAGTAACAC 3 '....Seq ID # 4). The first two lanes are the resistant parents, Jhitpiti and TNI, respectively, followed by the resistant (Rp) and susceptible (Sp) bulks. Labels at the bottom of the figure indicate individual F₄ line numbers. Fig. 2 Mapping of the gall midge resistance gene, Gm8, on the molecular linkage map of rice Chromosome 8. Numbers on the left show genetic distances (cM). AR257 and AS 168 are the AFLP, and AP19₅₈₇ is the RAPD, markers identified in this study. Map position of a previously mapped (see Mohan et al. 1997b) gall midge resistance gene Gm4(t) is also shown. The genetic distances are based on the Rice Genome Program map

(http://rgp.dna.affrc.go.jp/publicdata/geneticmap2000/chr08.html). Map not to scale. Fig. 3a PCR-based screening for gall midge resistant and susceptible progeny in the F₄ population, derived from a cross between Jhitpiti and TNI, using SCAR primers flanking the resistance phenotype specific marker, AR257. Lanes /^', /^' and in represent lines 198R, 20 IS and 215S, respectively. Lane M represent 1-kb DNA marker ladder. Figures on the left represent the molecular weights in bp. R, resistant parent; S, Susceptible parent; Rp and Sp, Resistant and Susceptible bulks, respectively. Labels at the bottom of the figure indicate individual F₄ line numbers. Fig. 3b Southern hybridization of the gel shown in (3a) using the susceptible- specific fragment, amplified by the SCAR primers flanking AR257, as probe. Lanes i, //^' and Hi represent 198R, 20 IS and 215S, respectively. Figures on the left represent the molecular weight in bp. R, resistant parent; S, Susceptible parent; Rp and Sp, Resistant and Susceptible bulks, respectively. Labels at the bottom of the figure indicate individual F₄ line numbers. Fig. 4. Restriction profile of the monomorphic PCR products generated using the SCAR primers flanking the susceptible specific AFLP fragment AS 168. These fragments, on double digestion with Mse I and Pst I, resulted in a polymorphism that distinguished the phenotypes. Lanes i, ii, Hi, iv and v represents 198R, 99S, 186S, 20 IS and 215S, respectively. Lane M represents 50-bp DNA marker ladder. Figures on the left represent the molecular weight in bp. R, resistant parent; S, Susceptible parent. Labels at the bottom of the figure indicate individual F₄ line numbers. Example DNA extraction and preparation of resistant- and susceptible-bulks Total genomic DNA was isolated from the leaves of 40 field-grown F₄ plants (10-week-old) along with parents using the modified CTAB method of Murray and Thompson (1980). For bulked segregant analysis, an equal quantity of DNA from 12 resistant and 12 susceptible F₄ individuals was pooled to form the resistant- and susceptible- bulks, respectively (Michelmore et al. 1991; Mohan et al. 1994). The concentration of DNA of the two bulks and the two parental DNAs was adjusted to 10 ng/μl. Random Amplified Polymorphic DNA analysis The amplification conditions were kept as described previously (Williams et al. 1990) with certain modifications (Mohan et al. 1994). A total of 1200 RAPD primers (Operon technologies, Alameda, California, USA) belonging to A to Z, AA to AZ and BA to BH series were used in this study. The RAPD products (7.5 μl out of 25 μl reaction volume) were separated on 1.1% agarose gels in IX TBE buffer and stained with ethidium bromide at a concentration of 0.5 μg/ml. The gels were visualized and photographed on a UN trans-illuminator using Polaroid film (Type 667). Amplified Fragment Length Polymorphism analysis AFLP reactions were performed as described by Nos et al. (1995) with some minor modifications (Sardesai et al. 2002). A total of 105 selective enzyme-primer combinations were tried in this study. After PCR, 20 μl of formamide dye (98% forma ide, 10 mM EDTA, 0.1% bromophenol blue and 0.1% xylene cyanol) was added to the reaction. The samples were heat-denatured for 5 min, snap-cooled on ice and loaded onto a 6% sequencing gel containing 8M urea. The gel was dried and exposed overnight to Bio Max MR film (Kodak) at -80 °C.

Isolation, cloning and sequencing of the phenotype-specific AFLP and RAPD fragments The phenotype-specific AFLP fragments were cut out from gels by first aligning the respective autoradiograms on the dried gels. DΝA from the cut gel-piece was isolated and reamplified as described by Behura et al. (2000). The PCR products were gel-purified using a Qiagen gel extraction kit (Qiagen, Hilder, Germany) and cloned into PCR-4-TOPO vector (Invitrogen, California, USA). Two clones each containing the resistant phenotype-specific fragment and susceptible phenotype- specific AFLP fragments were sequenced and named AR257 (254 bp) and AS 168 (168 bp), respectively. The resistant phenotype-specific RAPD fragment was directly gel purified and cloned as mentioned above. The clone was named AP19₅87 (587 bp) and was partially sequenced from the two termini. Sequencing of these clones was done by di-deoxy chain-termination method (Sanger et al. 1977) using Sequenase Ner. 2.0 sequencing Kit (USB, Cleveland, Ohio, USA). The sequences of the AFLP fragments were used to develop SCAR primers. Southern hybridization of the AFLP fragments Genomic DΝA (5 μg) of rice varieties, Jhitpiti and TΝ1 were digested with 10U of each oϊBamB I, Bgl II, Cla I, EcoR I, EcoR N, Hind III, Pst I, Sal I and Xba I at 37 °C overnight. The digested DΝA was run on a 0.8% agarose gel and blotted onto a nylon membrane (GeneScreen Plus, ΝΕΝ Life Sciences, Boston, USA) as described by Williams et al. (1991). The membrane was probed with AR257 and AS168 separately. The probes were labeled with [α^"32P]-dCTP using the Nick Translation Kit (Bethesda Research Laboratories, Life Technologies, USA). After hybridization for 20 h at 65 °C, the membrane was washed under stringent conditions (Mohan et al. 1994) and kept for autoradiography. Mapping of the phenotype-specific AFLP and RAPD fragments The sequences of both phenotype-specific AFLP fragments, AR257 and AS 168, and the RAPD fragment, AP19₅₈₇, were subjected to homology searches using the rice database at National Center for Biotechnology Information (NCBI http://www.ncbi.nlm.nih.gov), The Institute for Genomic Research (TIGR http://www.tigr.org/tdb/e2kl/osal) and Rice Genome Research Program (RGP http://rgp.dna.affrc.go.jp) to map them to the chromosomal location in the rice genome. Design of the SCAR primers and PCR For conversion of AFLP and RAPD fragments into PCR-based SCAR markers, forward and reverse SCAR primers were designed based on the sequence of the AFLP fragments, AR257 and AS 168 and the RAPD fragment AP19₅₈₇, using Oligo 4.0 software (National Biosciences) and were synthesized by Microsynth GmbH (Balagad, Switzerland). In addition, another set of SCAR primers (forward and reverse) were designed from the region flanking each AFLP fragment. These regions were identified using the rice database at RGP. The details of AFLP -based SCAR primers used in this study are given in Table 1 below:

Table 1 Sequences of the SCAR primers designed for the different phenotype specific markers used in this study

Marker Primer Sequence

AR257 5 '-ATC GAA GGA GGA GCC TTT GC-3 ' .... Seq ID # 5 (F) 5 '-AAC GTA TCA TAG CTT ACC CAT AAA CCA-3 ' .... Seq ID # 6 (R)

AS 168 5 '-ATA TTT ACT TGA ATT TAC AGA TG-3 ' .... Seq ID # 7 (F) 5 '-AAT AGG GCT TAG CTT GAT GAT G-3 ' .... Seq ID # 8 (R) Flanking

AR257 5 '-ACA AAA TCA AAT GTG AAA CTA GG-3 ' .... Seq ID # 9 (F) 5'-AGT CCG CTT CGT CCGTCGTT-3' Seq ID # 10 (R)

Flanking

AS168 5'-TGA TGT TTC CCT TGC TTT TCT T-3' .... Seq ID # 11 (F) 5'-TAC GGA CGGAGA TGAACT GT-3' Seq ID # 12 (R)

(F) = forward; (R) = reverse PCR was carried out using genomic DNA of the resistant and susceptible parents as well as resistant and susceptible individuals of F₄ progeny in a 50 μl reaction volume containing lOmM of Tris-Cl (pH 8.0), 50 mM of KCl, 1.5 mM of MgCl₂, 0.01% gelatin, 200 μM of each dNTP, 450 nM of each primer, 200 ng of template DNA and 2.5U of Taq DNA polymerase. The amplification conditions were 94 °C for 1 min, 56 °C for 1 min and 72 °C for 1 min for 30 cycles except that the annealing temperature was kept at 59 °C to amplify AP19₅87. The PCR products were electrophoresed on 1.2 -1.3% agarose gel in IX TBE. Restriction of AFLP-derived SCAR-amplified products Single and double-digestions of a SCAR product were carried out using Pst I and/or Mse I enzyme(s). Ten microlitres of the amplified products were digested using 10U of a restriction enzyme in a 20 μl restriction volume. In case of double digestions, 10U each of both the enzymes were used in a restriction reaction. Results RAPDs 1200 RAPD primers were screened to identify markers tightly linked to the Gall midge resistance gene, Gm8. We observed 1112 polymorphic bands between the parents. Of these, 115 were resistant/susceptible bulk-specific. Only one RAPD fragment, AP19₅₈₇, showed tight linkage with the resistance phenotype. It amplified a 587 bp fragment in the resistant parent and in the resistant bulk. When tested on F₄ individuals, 18 out of 19 resistant lines and 2 out of 20 susceptible lines amplified the fragment linked to the resistant phenotype (data not included). AFLPs The 105 enzyme-primer combinations used in this study revealed a total of 24 fragments that amplified in phenotype-specific manner. Of these, three fragments were found to be tightly linked to resistant/susceptible phenotype. While one enzyme- primer combination (Pst I- AT + Mse I-CGT) generated a 254 bp, resistant phenotype- specific fragment (AR257) (Fig. la); the other enzyme-primer combination (Pst I-AA + Mse I-CAC) yielded two fragments, 168 bp (AS 168) and 135 bp, associated with the susceptible phenotype (Fig. lb). The resistant phenotype-specific fragment, AR257, amplified only from the resistant parent and resistant bulk. Individual F₄ lines (total 40; including the 24 lines that constituted the bulks) when amplified using the same enzyme-primer combination revealed presence of AR257 in all resistant individuals and its absence in all susceptible individuals (except 20 IS and 215S) (Fig. la). In susceptible phenotype- specific enzyme-primer combination, the fragment AS 168, amplified in all susceptible individuals (i.e. TNI parent and all susceptible individuals that constituted the susceptible-bulk) whereas it was absent from all the resistant individuals (Fig. lb). The AFLP screening results showed that two lines (i.e. 20 IS and 215S) did not show phenotype-specific amplification (Fig. la). Cloning and Southern hybridization Two AFLP fragments i.e. resistance-associated (AR257) and susceptible- associated (AS 168) were eluted and cloned into PCR-4-TOPO vector and sequenced. Southern hybridization of digested genomic DNA of parents, Jhitpiti and TNI, using nine different restriction enzymes, with AR257 and AS 168 as probes, revealed polymorphisms between the parents. The hybridization signals using the AR257 as well as AS 168 revealed that these regions were present as single or low-copy sequences in both the parents (data not shown).

Chromosomal location and relative map-position of AFLP and RAPD fragments in the rice genetic map BLAST searches using sequences of the two cloned AFLP fragments showed tight homology to individual PAC clones belonging to the short arm of rice chromosome 8 at NCBI, TIGR and RGP databases. While AR257 showed 96% homology (Score of 432 and E value of e^"120) to a PAC clone (AP004705) at 48.8 cM position of chromosome 8 (short arm), AS 168 showed 95% homology (Score of 224 and E value of 2e^"57) to a PAC clone (AP004690) at 50.8 cM position of chromosome 8 (short arm) in the rice database. The RAPD fragment, AP19₅₈7, showed 99% homology to adjacent, overlapping, rice clones AP005440 and AP004646 (Score of 456 and E value of e^'127) in the rice databank. The map-positions of these markers along with other associated markers on chromosome 8 are shown in Fig. 2. In order to ascertain if these fragments were part of a gene, we also carried out a BLAST search using the phenotype-specific sequences against the sequences in the EST data bank. Of the three fragments identified in this study, only the resistant phenotype-specific AFLP marker, AR257, showed significant homology to a sequence in the EST library. This fragment had strong homology (96%; E value e^"120) to an EST clone # CB674118 from Or za sativa var japonica leaf cDNA library. This EST was identified as being involved in the interaction between rice and its fungal pathogen, Magnaporthe grisea. SCAR amplification and generation of phenotype-specific polymorphisms No polymorphism could be observed between parents when SCAR primer pairs, derived from the end-sequences of AR257 or AS 168, were used. Even restricting the monomorphic amplified products with five different restriction enzymes located within the sequences (data not shown) did not yield any phenotype-specific restriction length polymorphism. However, a distinct phenotype-specific SCAR amplification profile was obtained when primers based on the sequences flanking AR257 were used to amplify DNA from the parents and their F₄ progeny. All resistant individuals (Jhitpiti parent & resistant F₄ lines) showed the amplification of two bands, i.e. of 550 bp and 640 bp, in their profiles whereas all susceptible individuals (TNI parent & susceptible F lines) except 20 IS, showed the presence of only one band i.e. of 500 bp (Fig. 3a). Two lines i.e. one resistant (198R) and one susceptible (215S) showed the amplification of all of the three bands in a co-dominant manner. In contrast, when PCR was carried out using primers based on the sequences flanking (these sequences were obtained from the rice genome sequence data bank) AS 168, it did not show a phenotype-specific polymorphism, initially. However, the monomorphic amplification product (276 bp) from resistant and susceptible individuals (parents and their F₄ progeny) when restricted with Pst I and Mse I, resulted in a phenotype-specific restriction length polymorphism with all resistant individuals showing the presence of a distinct 225 bp fragment and all susceptible individuals showing the occurrence of a distinct 168 bp fragment (Fig. 4). Four F₄ lines, i.e. one resistant (198R) and three susceptible (99S, 186S, 215S), showed the amplification of both resistance- and susceptible-associated fragments (Fig. 4); and one susceptible line (20 IS) showed the presence of only resistance-associated fragment. It is interesting to note that these five F₄ lines included the three lines (198R, 20 IS and 215S) that had earlier shown similar results (co-dominant amplification in case of 198R and 215S; and resistance-associated amplification in case of 20 IS) with resistance-associated SCAR primers also. However, the SCAR primers designed for the RAPD marker AP19₅₈₇ failed to amplify in a phenotype-specific manner. Southern hybridization of resistance-derived SCAR (flanking AR2S7) amplified products The resistance-derived SCAR primers (flanking AR257) amplified the products in a co-dominant manner and it was necessary to ascertain whether the different fragments specifying the individual phenotypes amplified in an allele-specific manner. Since heterozygous lines were not available, sequence homology of resistant phenotype-specific and susceptible phenotype-specific products was tested as an alternate resort, by hybridizing gel-eluted susceptible phenotype-specific product (500 bp) as the probe to a blot carrying the SCAR primers-amplified PCR products. Results revealed that it hybridised well with both the fragments linked to resistant phenotype (640 bp and 550 bp) as well as to the fragment linked to susceptible phenotype (Fig. 3b). Discussion Recent genetic studies revealed that the gall-midge resistance gene, Gm8, present in the rice variety, Jhitpiti, is a dominant gene and is non-allelic to other known gall midge resistance genes (Gml, Gm2, grtι3, Gm4(t), Gm5, Gm6(t) and GmT) in rice (Kumar et al. 2000). The occurrence of resistant phenotype-specific fragment, AR257, in two susceptible lines, 20 IS and 215S, could be due to a recombination event(s) between the AFLP marker locus and the Gm8 locus. Thus, a high level of phenotype-specificity could be obtained using resistance- and susceptible-associated AFLP markers. BLAST searches using sequence information of resistant phenotype-specific fragment (AR257) and susceptible phenotype-specific fragment (AS168), showed near 100% homology to PAC clones AP004705 and AP004690, respectively, in the RGP database. Both PAC clones are located at a 2.0 cM relative genetic distance on the short arm of rice chromosome 8 (48.8 and 50.8 cM from the short arm end covering a region of approximately 400 kb) (Fig. 2). As both AFLP markers show tight linkage to the resistant and susceptible phenotypes, and show high homology to the above mentioned PAC clones, it is therefore inferred that the gall-midge resistance gene, Gm8, is located on the short arm of rice chromosome 8. Similarly, the RAPD fragment AP19₅₈₇ showed near 100% homology to adjacent overlapping BAC clones AP005440 and AP004646 and both these clones map to the short arm of chromosome 8 (36.8 cM). Initially, SCAR primers derived from the sequences of AFLP fragments (either AR257 or AS 168) failed to generate phenotype-specific amplification and instead produced a single monomorphic band in both parents. However, when SCAR primers flanking AR257 were used, a distinct phenotype-specific PCR amplification was revealed in parents and in their F₄ progeny in a co-dominant manner (Fig. 3 a). The occurrence of heterozygous-specific profile in a susceptible line, 215 S, concurs with the results of AFLP screening where this line showed the presence of resistant phenotype-specific AR257 fragment. This suggests that the susceptible line 215S contains a recombination event that seems to have occurred between the marker and the Gm8 loci or is a heterozygous individual mislabeled as susceptible in the field. Also, the presence of all of the three bands (both resistant phenotype-specific and one susceptible phenotype-specific) in the resistant line i.e. 198R indicates that the individual could actually be heterozygous. The conversion of the dominant AFLP marker (AR257 specific) to a co-dominant SCAR marker is, thus, advantageous since the latter can identify a heterozygous individual in the population and is, therefore, more informative than a dominant marker. It is interesting to observe that in the resistant phenotypes, SCAR primers (flanking AR257) amplified two fragments instead of one for which they were actually designed. Nonetheless, both fragments amplified in all the resistant individuals. The amplification of the two bands in the resistant phenotypes could be due to a duplication event of a micro-chromosomal segment associated with Gm8 locus. Unlike AR257, the PCR using SCAR markers based on region flanking AS 168 produced a monomorphic band (276 bp) across all individuals which upon double digestion with Pst I and Mse I, distinguished between the susceptible and resistant phenotypes (Fig. 4). Thus, with the use of restriction enzymes, a monomorphic PCR product could be converted to a useful co-dominant marker. Again, the two lines, 198R and 215S, showed the presence of both, resistant phenotype-specific as well as susceptible phenotype-specific bands upon restriction, thereby, indicating the heterozygous nature of these lines. In addition, two more susceptible lines, 99S and 186S, revealed the presence of both, 225 and 168 bp fragments—a result which does not correspond to the fact that they are susceptible individuals. This could happen if these two individuals are heterozygous for this SCAR-marker but not for the Gm8 locus. Further, restricting the SCAR amplified monomorphic fragment singly with Pst I or Mse I revealed that the polymorphism was actually due a modification of the Pst I site in the resistant individuals (data not shown). This also highlights a strategy for developing SCARs i.e. when SCARs developed from phenotype-specific fragments fail to generate a phenotype-specific amplification, then one could choose to design primers from regions flanking this fragment. This would allow greater success rates for primers for use in MAS based on phenotype-specific fragments. In the present study, SCAR markers developed from the AFLP markers are also found to be more robust in terms of both, their specificity as well as greater reliability and is ideally suited as a tool for marker-aided selection in breeding programmes involving the gall midge resistance gene Gm8. The conversion of both the tightly-linked, phenotype-specific dominant AFLP markers into co-dominant, allele- specific SCAR markers is, thus, advantageous from the point of view of marker- assisted selection as they can detect the presence of both the alleles in a single PCR reaction using only one set of SCAR primer pairs. This translates to a lot of savings for a breeder in terms of time, manpower and test-plot area. Markers specific to Gm8^' along with markers specific to Gm2 (Nair et al. 1995), Gm4(t) (Nair et al. 1996) and Gm7 Sardesai et al. 2002) will be used in pyramiding these genes in different combinations for developing durable resistance against different biotypes of gall midge prevalent in India, in elite cultivars of rice. Previous studies have mapped putative resistance genes in the region between Gm4(t) and Gm8 (Mohan et al. 1997b; Berruyer et al. 2003) and therefore, possibility exists that these could be potential candidates for the gall midge resistance gene Gm8. Interestingly, the resistant phenotype-specific fragment, AR257, has strong homology to an EST clone from Oryza sativa var japonica leaf cDNA library. This EST is known to be involved in the interaction between rice and its fungal pathogen, Magnaporthe grisea. It is therefore possible that the map-position of Gm8, as identified on the short arm of chromosome 8, would help in the isolation of this gene through map-based cloning strategy. In this invention, the SCAR markers have been developed with two major aims; (a) for marker-aided selection of Gm8, and; (b) to localize these markers on the rice genetic map to identify the chromosomal location of Gm8. We have also screened the genomic region between markers Gm4(t) and R 727 (Fig. 2). This covers a region of -26 cM. We have developed a large number of primer pairs (more than 150, every 50 kb on an average) for this region. The primers are based on the sequence of this region available at TIGR and RGP databases. Primers were selected so as to PCR amplify 1-2 kb regions. After screening 79 pairs of primers we were able to identify only three pairs that amplified fragments in a phenotype-specific manner. On screening 40 F₄ individuals with these primers it was found that none of these SCAR markers were more closely associated than the ones already mentioned earlier in this study (data not included). Identifying markers closer to Gm8 than the present ones will also help in the map-based gene cloning of Gm8.

References Behura SK, Nair S, Sahu SC, Mohan M (2000) An AFLP marker that differentiates biotypes of the asian rice gall midge (Orseolia oryzae, Wood-Mason) is sex-linked and also linked to avirulence. Mol Gen Genet 263:328-334 Bentur JS, Pasalu IC, Sarma NP, Prasada Rao U, Mishra B (2003) Gall midge resistance in rice. DRR Research paper series 01/ 2003, 20 pp. Directorate of Rice Research, Hyderabad, India Berruyer R, Adreit H, Milazzo J, Gaillard S, Berger A, Dioh W, Lebrun M-H, Tharreau D (2003) Identification and fine mapping of Pi33, the rice resistance gene corresponding to Magnaporthe grisea avirulence gene ACE1. Theor Appl Genet 107:1139-1147 Bonnema G, van den Berg P, Lindhout P (2002) AFLPs mark different genomic regions compared with FLPs: a case study in tomato. Genome 45:217- 225 Ellis RP, Mc Nicol JW, Baird E, Booth A, Lawrence P, Thomas B, Powell W (1997) The use of AFLPs to examine genetic relatedness in barley. Mol Breed 3:359- 369 Harris MO, Stuart JJ, Mohan M, Nair S, Lamb RJ, Rohfritsch O (2003) Grasses and gall midges: Plant defence and insect adaptation. Annu Rev Entomol 48:549- 577 Hill M, Witsenboer H, Zabaeu M, Nos P, Kesseli R, Michelmore R (1996)

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35:70- 73 Mackill DJ, Zhang Z, Redona ED, Colowit PM (1996) Level of polymorphism and genetic mapping of AFLP markers in rice. Genome 39:969-977 Maughan PJ, Saghai Maroof MA, Buss GR, Huestis GM (1996) Amplified Fragment Length Polymorphism (AFLP) in soybean: species diversity, inheritance and near isogenic line analysis. Theor Appl Genet 93:392-401 Michelmore RW, Paran I, Kesseli RN (1991) Identification of markers linked to disease- resistance genes by bulked segregant analysis: a rapid method to detect markers in specific genomic regions by using segregating populations. Proc Νatl Acad Sci USA 88:9828-9832 Mohan M, Νair S, Bentur JS, Prasada Rao U, Bennett J (1994) RFLP and

RAPD mapping of the rice Gm2 gene that confers resistance to biotype 1 of gall midge (Orseolia oryzae). Theor Appl Genet 87:782-788 Mohan M, Νair S, Bhagwat A, Krishna TG, Yano M, Bhatia CR, Sasaki T (1997a) Genome mapping, molecular markers and marker- assisted selection in crop plants. Mol Breed 3 : 87- 103 Mohan M, Sathyanarayanan PN, Kumar A, Srivastava MΝ, Νair S (1997b) Molecular mapping of a resistance-specific PCR-based marker linked to a gall midge resistance gene (Gm4t) in rice. Theor Appl Genet 95:777-782 Murray MG, Thompson WF (1980) Rapid isolation of high-molecular-weight plant DΝA. Nucleic Acids Res 8:4321-4325 Nair S, Bentur JS, Prasada Rao U, Mohan M (1995) DNA markers tightly linked to a gall-midge resistance gene (Gm2) are potentially useful for marker- aided selection in rice breeding. Theor Appl Genet 91:68-73 Nair S, Kumar A, Srivastava MN, Mohan M (1996) PCR-based DNA markers linked to a gall-midge resistant gene, Gm4(t), has potential for marker-aided selection in rice. Theor Appl Genet 92:660- 665 Sanger F, Nicklen S, Coulson A (1977) DNA sequencing with chain terminating inhibitors. Proc Natl Acad Sci USA 74:5463-5467 Sardesai N, Kumar A, Rajyashri KR, Nair S, Mohan (2002) 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. Theor Appl Genet 105:691-698 Sardesai N, Rajyashri KR, Behura SK, Nair S, Mohan M (2001) Genetic, physiological and molecular interactions of rice and its major dipteran pest, gall midge. Plant Cell Tiss Organ Cult 64:115-131 Singh A, Negi MS, Rajagopal J, Bhatia S, Tomar UK, Srivastava PS, Lakshmikumaran M (1999) Assessment of genetic diversity in Azadirachta indica using AFLP markers. Theor Appl Genet 99:272-279 Nos P, Hogers R, Bleeker M, Reijans M, van de Lee T, Hoenes M, Frijters A, Pot J, Peleman J, Kuiper M, Zabeau M (1995) AFLP: a new technique for DΝA fingerprinting. Nucleic Acids Res 23:4407-4414 Williams JGK, Kubelik AR, Livak KJ, Rafalski JA, Tingey SA (1990) DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Res 18:6531-6535 Williams MNV, Pande N, Nair S, Mohan M, Bennett J (1991) Restriction fragment length polymorphism analysis of polymerase chain reaction products amplified from mapped loci of rice (Oiyza sativa L.) genomic DNA. Theor Appl Genet 82:489-498 Zhu J, Gale MD, Quarrie S, Jackson MT, Bryan GJ (1998) AFLP markers for study of rice biodiversity. Theor Appl Genet 96:602-611

Claims

Claim:

1. A combination of sequence characterized amplified region (SCAR) primers for use in the marker aided selection of rice varieties which are resistant to attack by gall midge, said primers having the sequence: 5' GACTGCGTACATGCAAT 3'....SeqID # 1 and 5' GATGAGTCCTGAGTAACGT 3'....SeqID # 2

2. A combination of sequence characterized amplified region (SCAR) primers for use in the marker aided selection of rice varieties which are susceptible to attack by gall midge, said primers having the sequence: 5' GACTGCGTACATGCAAA 3' ....Seq ID # 3 and 5' GATGAGTCCTGAGTAACAC 3'. . ..Seq ID #

3. A combination of sequence characterized amplified region (SCAR) primers for use in the marker aided selection of rice varieties which are resistant or susceptible to attack by gall midge, said primers being selected from the primer pairs having the sequences as shown in the following table:

Primer Sequence

5 '-ATC GAA GGA GGA GCC TTT GC-3 ' .... Seq ID # 5 (F)

5 '-AAC GTA TCA TAC CTT ACC CAT AAA CCA-3 ' .... Seq ID # 6 (R)

5 '-ATA TTT ACT TGA ATT TAC AGA TG-3 ' .... Seq ID # 7 (F) 5 '-AAT AGG GCT TAG CTT GAT GAT G-3 ' .... Seq ID # 8 (R)

5 '-ACA AAA TCA AAT GTG AAA CTA GG-3 ' .... Seq ID # 9 (F) 5'-AGT CCG CTT CGT CCG TCG TT-3' Seq ID # 10 (R)

5'-TGA TGT TTC CCT TGC TTT TCT T-3' .... Seq ID # 11 (F)

5'-TAC GGA CGG AGA TGAACT GT-3' ... Seq ID # 12 (R)

F= forward; R= reverse

4. A method for screening a rice variety to determine whether it is resistant or susceptible to gall midge biotypes which comprises extracting DNA from said rice variety, subjecting said rice variety to polymerase chain amplification reaction in the presence of a combination of primers said primers being selected from the primer pairs having the sequences as shown in the following table:

Primer Sequence 5'-ATC GAAGGA GGAGCC TTT GC-3' .... Seq ID # 5 (F) 5'-AAC GTA TCA TAC CTT ACC CAT AAA CCA-3' .... Seq ID # 6 (R)

5'-ATA TTT ACT TGA ATT TAC AGA TG-3' .... Seq ID # 7 (F) 5'-AAT AGG GCT TAG CTT GAT GAT G-3' .... Seq ID # 8 (R)

5'-ACA AAA TCA AAT GTG AAA CTA GG-3' .... Seq ID # 9 (F) 5'-AGT CCG CTT CGT CCG TCG TT-3' Seq ID # 10 (R)

5'-TGA TGT TTC CCT TGC TTT TCT T-3' .... Seq ID # 11 (F) ^• 5'-TAC GGA CGG AGATG ACT GT-3' ... Seq H> # 12 (R) and determining the extent of amplification, amplification of two bands indicating that said variety is resistant to said gall midge phenotypes due to the presence of the gene Gm8 and amplification of only a single band indicating that said variety is susceptible to said gall midge phenotypes due to the absence of the gene Gm8.