Identification of two RAPD markers genetically linked to a recessive allele of a Fusarium wilt resistance gene in pigeonpea (Cajanus cajan L. Millsp.)

Summary Fusarium wilt (Fusarium udum Butler) is a soil borne disease of pigeonpea which causes substantial yield losses. The disease can occur at any stage of plant development, from the young seedling to the pod filling stage. Though resistance is simply inherited, transfer to locally adapted cultivars has been difficult due to linkage drag and difficulty in accurate phenotyping, except in sick plots. An attempt was made to identify RAPD markers associated with wilt phenotype by using F2 populations derived from contrasting parents; GSl (susceptible) ‘ICPL87119 (resistant) and GS1’ ICP8863 (resistant). Parents and F₂s were grown in a national _Fusarium sick-plot at Gulbarga, India and phenotyped as resistant or susceptible during the entire crop growth period. In both the crosses, resistance to wilt segregated as a monogenic dominant character. DNA samples extracted from sick plot grown, early seedling stage plants of parents and 254 F₂ plants of GS1 × ICPL87119 were held separately for marker identification. PCR reactions using 340 random decamer primers with genomic DNA of parents resulted in detection of 45 polymorphic amplicons from 39 primers. PCR testing of bulked DNA from subsets of resistant and susceptible plants revealed the presence of two amplicons at 704 bp and 500 bp (OPM03704 and OPAC11500) with susceptibility. Analysis of individual F₂ plants showed a segregation ratio of 3: 1 for the presence: absence of the amplicon in both crosses. Considering the wilt reaction and susceptibility-linked RAPD marker, it was possible to deduce genotype of every F₂ plant and the genotypic ratio for wilt reaction was 1RR: 2Rr: 1rr, as expected.     

Inheritance of Fusarium resistance in a jointless tomato as affected by the X gametophytic factor

Summary Hybridization between tomato with xx and XX gametophytic genotypes exhibited in segregating generations, deviations from the expected monogenic inheritance of Fusarium wilt resistance conditioned by gene I. These deviations resul ted from gametophytic preference, linkage and the presence of modifying loci associated with the gene I. Although information on the inheritance of Fusarium wilt resistance is based on backcross data, information obtained from the pedigree procedure and testcrosses made with BC₁S₂ plants all supports the presence of modifiers.Michigan Agricultural Experiment Station Journal Number 5457.     

The detection of QTLs controlling bacterial wilt resistance in tobacco (N. tabacum L.)

The bacterial tobacco wilt caused by Ralstonia solanacearum is one of the most destructive soil-borne diseases worldwide. One strategy to improve the resistance to bacterial wilt is to make use of plant varieties expressing wilt resistance genes. To characterize the genetics of wilt resistance and to identify relevant molecular markers for use in plant breeding, quantitative trait loci (QTLs) affecting tobacco bacterial wilt resistance were mapped in the F_2:3 and F_2:4 progeny produced from two crosses between the wilt-resistant breeding lines Enshu and Yanyan97 and the susceptible cultivar TI448A. A linkage map containing 118 loci in 24 linkage groups was constructed for 236 lines from the Enshu×TI448A cross, and a linkage map containing 96 loci in 24 linkage groups was constructed for 264 lines from the Yanyan97×TI448A cross. The wilt resistance of the progeny was examined in field trials conducted in Xuancheng, China, in 2010, 2011, and 2012. The disease severity was assessed on stems using separate rating scales. Mapmaker/EXP 3.0 and Mapmaker/QTL 1.1 were used to identify the qBWR-3a, qBWR-3b, qBWR-5a and qBWR-5b QTLs in linkage group 3 and 5; these four loci were strongly associated with resistance and explain 9.00, 19.70, 17.30, and 17.40 % of the variance in resistance, respectively. The close linkage of the markers PT20275 and PT30229 was detected in both the TI448A×Enshu and TI448A×Yanyan97 crosses, and this linkage group could be used to select individual resistant plants. These findings suggest that one strategy to combat bacterial wilt could be to exploit the resistance genes of the Enshu and Yanyan97 strains.     

Marker assisted backcrossing for introgression of Fusarium wilt resistance gene into melon

Fusarium wilt, caused by Fusarium oxysporum f. sp. melonis is a common vascular wilt fungal disease in melon across the world. The resistance gene to race 1 of this causal agent, Fom-2, has been previously cloned and its sequence is available. The objective of this research was the introgression of Fom-2 from one resistant (Isabelle) genotype into two susceptible cultivars (Garmak and Tile-torogh) via marker assisted backcrossing. First, the leucine-rich repeats (LRR) domain of Fom-2 from resistant and susceptible genotypes was sequenced to develop functional markers. A length of 1274 bp of the 3′ end of this gene was isolated, cloned and sequenced. The difference between resistant and susceptible genotypes in this region was 28 nucleotide substitutions. Two allele specific primer pairs, Fom2-R409 and Fom2-S253, were designed based on nucleotide substitutions to amplify resistant and susceptible alleles, respectively. For introgression of the gene, donor (Isabelle) and recurrent (Garmak and Tile-torogh) parents were crossed. Resistant plants in BC₁F₁ and BC₂F₁ generations were first detected using artificial pathogen inoculation and later the plants were genotyped by functional markers to validate their resistance. The resistant plants were also selected phenotypically in each generation for background genome recovery, which conduced to high similarity of BC₃ generation with the recurrent parents. It was proved the developed markers are more precise and efficient than inoculation trial and could be used as confident tools for screening of resistant melon genotypes to Fusarium wilt.     

User‐friendly markers linked to Fusarium wilt race 1 resistance Fw gene for marker‐assisted selection in pea

Fusarium wilt is one of the most widespread diseases of pea. Resistance to Fusarium wilt race 1 was reported as a single gene, Fw, located on linkage group III. The previously reported AFLP and RAPD markers linked to Fw have limited usage in marker‐assisted selection due to their map distance and linkage phase. Using 80 F₈ recombinant inbred lines (RILs) derived from the cross of Green Arrow × PI 179449, we amplified 72 polymorphic markers between resistant and susceptible lines with the target region amplified polymorphism (TRAP) technique. Marker–trait association analysis revealed a significant association. Five candidate markers were identified and three were converted into user‐friendly dominant SCAR markers. Forty‐eight pea cultivars with known resistant or susceptible phenotypes to Fusarium wilt race 1 verified the marker–trait association. These three markers, Fw_Trap_480, Fw_Trap_340 and Fw_Trap_220, are tightly linked to and only 1.2 cM away from the Fw locus and are therefore ideal for marker‐assisted selection. These newly identified markers are useful to assist in the isolation of the Fusarium wilt race 1 resistance gene in pea.     

Marker-assisted selection of Verticillium wilt resistance in progeny populations of upland cotton derived from mass selection-mass crossing

Two progeny populations of upland cotton derived from mass selection-mass crossing, M₃S₂F₅ and its family lines M₃S₂F_5:6, were generated from 17 hybrid cotton lines derived from regional trials conducted in the Yellow River basin and Yangtze River basin in China. These populations were used to verify 39 reported molecular markers that were related to quantitative trait loci (QTLs) for Verticillium wilt resistance of cotton. Only 12 of 39 markers were polymorphic; 19 had no polymorphisms, and amplification failed for eight markers. The differences in disease grades of aa/AA genotype individuals for five markers, BNL3241, NAU1225, NAU1230, JESPR153, and BNL3031, reached either significant or highly significant levels in at least one population. These markers can thus be effectively used for marker-assisted selection (MAS) of the target trait. Especially for JESPR153 and BNL3031, the differences in disease grades of aa/AA genotype individuals both reached either significant or highly significant levels in the two populations. These two markers should be given preferential consideration when undertaking MAS. The two flanking markers were more effective than the single flanking marker for MAS of single-loci QTL. The selection effect will be greatly enhanced through a reasonable allocation of marker combinations for multi-locus QTL polymerization. When using multi-locus markers for multi-locus QTL-assisted polymerization breeding, the selection effect can be improved progressively by increasing the number of polymerization markers. The possible interaction of different QTLs or genetic backgrounds does not influence the selection effect. A combination of resistant genotypes and disease grade performance enabled final selection of three individuals resistant to Verticillium wilt.     

A second gene for resistance to race 4 of Fusarium wilt in chickpea and linkage with a RAPD marker

Fusarium wilt caused by Fusarium oxysporum Schlechtend.: Fr f. sp. ciceris (Padwick) Matuo & Sato is a devastating disease of chickpea. The current study was conducted to determine the inheritance of the gene(s) for resistance to race 4 of fusarium wilt and to identify linked RAPD markers using an early wilting line, JG-62, as a susceptible parent. Genetic analysis was performed on the F₁s, F₂s and F₃ families from the cross of JG-62 × Surutato-77. The F₃ families were inoculated with a spore suspension of the race 4 wilt pathogen and the results were used to infer the genotypes of the parent F₂ plants. Results indicated that two independent genes controlled resistance to race 4. Linkage analysis of candidate RAPD marker, CS-27₇₀₀, and the inferred F₂ phenotypic data showed that this marker locus is linked to one of the resistance genes. Allelism indicated that the two resistance sources, Surutato-77 and WR-315, shared common alleles for resistance and the two susceptible genotypes, C-104 and JG-62, carried alleles for susceptibility. The PCR-based marker, CS-27₇₀₀, was previously reported to be linked to the gene for resistance to race 1 in a different population which suggested that the genes for resistance to races 1 and 4 are in close proximity in the Cicer genome. This revised version was published online in August 2006 with corrections to the Cover Date.     

Fusarium vascular wilt in lentil: Inheritance and identification of DNA markers for resistance

The inheritance of resistance to lentil (Lens culinaris Medik.) vascular wilt caused by Fusarium oxysporum f.sp. lentis was investigated in a cross between resistant (ILL5588) and susceptible (L692–16-l(s)) lines. F_2:4 progenies and F_6:8, F_6:9 recombinant inbred line (RIL) populations were assessed for their wilt reaction for three seasons in a well-established wilt-sick plot. Resistance to wilt was conditioned by a single dominant gene in the populations studied. The map location of the Fw locus was identified for the first time through linkage to a random amplified polymorphic DNA (RAPD) marker (OPK-15₉₀₀) at 10.8 cM. Two other RAPD markers (OP-BH₈₀₀ and OP-DI5₅₀₀) identified by bulked segregant analysis were associated in the coupling phase with the resistance trait, and another marker (OP-C04₆₅₀) was associated with repulsion. The DNA markers reported here will provide a starting point in marker-assisted selection for vascular wilt resistance in lentil.     

Inheritance of resistance to Fusarium wilt (race 2) in chickpea

The inheritance of resistance to Fusarium wilt (race 2) of chickpea was studied in a set of three crosses, i.e. ‘WR315’בC104’ (resistant × susceptible), ‘WR315’בK850’ (resistant × tolerant) and ‘K850’בGW5/7’ (tolerant × tolerant) in order to investigate the number of genes involved, their complementation and to find out whether resistant segregants are possible in a cross between two tolerant cultivars. Tests of F₁, F₂ and F₃ generations of these crosses under controlled conditions at ICRISAT, Patancheru, India, indicated involvement of three loci (two recessive and one dominant alleles). The homozygous recessive form at the first two loci conferred resistance whereas susceptibility occurred when the first two loci were in the dominant form. A dominant allele at the third locus can complement the dominant alleles at the other two loci to confer tolerance. Occurrence of resistant segregants in a cross between two tolerant cultivars was observed.     

Genetics of resistance to Aphis craccivora in cowpea

Summary Inheritance of aphid resistance and allelic relationships among sources of resistance was studied in the parents, F₁, F₂, F₃, and backcross populations of cowpea crosses. Each 4-day old seedling was infested with five fourthinstar aphids. Seedling reaction was recorded 14–16 days after infestation when the susceptible check was killed. The segregation data from eight crosses between resistant and susceptible cowpea cultivars indicated that aphid resistance was inherited as a monogenic dominant trait. Segregation data from crosses among eight resistant cultivars indicated that one or two loci and modifier(s) were involved in the expression of resistance to aphids. It was suggested that further studies on allelism among sources of resistance needed to be conducted in order to resolve this.     

Inheritance of resistance to Verticillium wilt (Verticillium dahliae) in cotton (Gossypium hirsutum L.)

Verticillium wilt, caused by Verticillium dahliae Kleb., is a major constraint to cotton production in almost all countries where cotton is cultivated. Developing new cotton cultivars resistant to Verticillium wilt is the most effective and feasible way to combat the problem. Little is known about the inheritance of resistance to Verticillium wilt of cotton, especially that caused by the defoliating (D) and nondefoliating (ND) pathotypes of the soil‐borne fungus V. dahliae. The objective of this study was to determine the inheritance of resistance in cotton against both pathotypes of V. dahliae. Crosses were made between the susceptible parent ‘Cukurova 1518’ and each of four resistant parents PAUM 401, PAUM 403, PAUM 405 and PAUM 406 to produce F₂ generations in 2002 and F_2:3 families in 2003. Disease responses of parent and progeny populations to the D and ND pathotypes were scored based on a scale of 0‐4 (0, resistant; 4, susceptible). F₂ populations inoculated with the D pathotype showed a 3 : 1 (resistant : susceptible) plant segregation ratio. Tests of F_2:3 families confirmed that resistance was controlled by a single dominant gene. In contrast, analysis of data from F₂‐ and F₂‐derived F₃ families suggested that resistance to the ND pathotype is controlled by dominant alleles at two loci.     

Molecular mapping of black rot resistance locus Xca1bo on chromosome 3 in Indian cauliflower (Brassica oleracea var. botrytis L.)

Black rot is the most devastating disease of cauliflower worldwide causing severe damage to crop. The identification of markers linked to loci that control resistance can facilitate selection of plants for breeding programmes. In the present investigation, F₂ population derived from a cross between ‘Pusa Himjyoti’, a susceptible genotype, and ‘BR‐161’, a resistant genotype, was phenotyped by artificial inoculation using Xcc race 1. Segregation analysis of F₂ progeny indicated that a single dominant locus governed resistance to Xcc race 1 in ‘BR‐161’. Bulk segregant analysis in resistant and susceptible bulks of F₂ progeny revealed seven differentiating polymorphic markers (three RAPD, two ISSR and two SSR) of 102 markers screened. Subsequently, these markers were used to genotype the entire F₂ population, and a genetic linkage map covering 74.7 cM distance was developed. The major locus Xca1bo was mapped in 1.6‐cM interval flanked by the markers RAPD 04₈₃₃ and ISSR 11₆₃₅. The Xca1bo locus was located on chromosome 3. The linked markers will be useful for marker‐assisted resistance breeding in cauliflower.     

Association of RFLP markers with trait loci affecting clubroot resistance and morphological characters in Brassica oleracea L.

Summary Resistance to Plasmodiophora brassicae Wor. race 7, the causal agent of the disease clubroot, was examined in an F₂ population of a cross between a clubroot resistant broccoli (Brassica oleracea var. italica) and a susceptible cauliflower (B. oleracea var. botrytis). A genetic linkage map was constructed in the same population based on the segregation of 58 dispersed restriction fragment length polymorphism (RFLP) markers. Associations between the inheritance of RFLP marker genotypes and segregation for disease resistance, morphological and maturity characteristics were examined. For each triat examined, several chromosomal regions marked by RFLP probes appeared to contain trait loci, suggesting that each trait was under polygenic control. RFLP marker linkage to a major factor imparting dominance for clubroot resistance from the broccoli parent was observed in this population. Additionally, RFLP marker linkage to an independently segregating factor contributing clubroot resistance from the cauliflower parent was observed, indicating that it should be possible to use RFLP markers to facilitate selection of transgressive segregants having the combined resistance from both parental sources. In some instances, RFLP markers from the same or closely linked chromosomal regions were associated with both clubroot resistance and morphological traits. Analysis of RFLP marker genotypes at linked loci should facilitate the selection of desired disease resistant morphotypes.     

Molecular mapping of two recessive genes controlling resistance to bacterial leaf pustule disease in soybean (Glycine max)

Bacterial leaf pustule (BLP) caused by Xanthomonas axonopodis pv. glycines (Xag) is a serious soybean disease. A BLP resistant genotype ‘TS-3’ was crossed with a BLP susceptible genotype ‘PK472’, and a segregating F₂ mapping population was developed for genetic analysis and mapping. The F₂ population segregation pattern in 15:1 susceptible/resistance ratio against Xag inoculum indicated that the resistance to BLP in ‘TS-3’ was governed by two recessive genes. A total of 12 SSR markers, five SSR markers located on chromosome 2 and seven SSR markers located on chromosome 6 were identified as linked to BLP resistance. One of the resistance loci (r1) was mapped with flanking SSR markers Sat_183 and BARCSOYSSR_02_1613 at a distance of 0.9 and 2.1 cM, respectively. Similarly, SSR markers BARCSOYSSR_06_0024 and BARCSOYSSR_06_0013 flanked the second locus (r2) at distances of 1.5 and 2.1 cM, respectively. The identified two recessive genes imparting resistance to BLP disease and the SSR markers tightly linked to these loci would serve as important genetic and molecular resources to develop BLP resistant genotypes in soybean.     

Identification and characterization of RAPD and SCAR markers linked to anthracnose resistance gene in sorghum [Sorghum bicolor (L.) Moench]

Anthracnose, one of the destructive foliar diseases of sorghum growing in warm humid regions, is incited by the fungus Colletotrichum graminicola.The inheritance of anthracnose resistance was studied using the parental cultivars of Sorghum bicolor (L.) Moench, HC 136 (susceptible to anthracnose) and G 73 (anthracnose resistant). The F₁ and F₂ plants were inoculated with the local isolates of C. graminicola cultures. The F₂ plants showed a segregation ratio of 3 (susceptible): 1(resistant) indicating that the locus for resistance to anthracnose in sorghum accession G 73 segregates as a recessive trait in a cross to susceptible cultivar HC 136. RAPD (random amplified polymorphic DNA) marker OPJ 01₁₄₃₇ was identified as marker closely linked to anthracnose resistance gene in sorghum by bulked segregant analysis of HC 136 × G73 derived recombinant inbred lines (RILs) of sorghum. A total of 84 random decamer primers were used to screen polymorphism among the parental genotypes. Among these, only 24 primers were polymorphic. On bulked segregant analysis, primer OPJ 01 amplified a 1437 bp fragment only in resistant parent G 73 and resistant bulk. The marker OPJ 01₁₄₃₇ was cloned and sequenced. The sequence of RAPD marker OPJ 01₁₄₃₇ was used to generate specific markers called sequence characterized amplified regions (SCARs). A pair of SCAR markers SCJ 01-1 and SCJ 01-2 was developed using Mac Vector program. SCAR amplification of resistant and susceptible parents along with their respective bulks and RILs confirmed that SCAR marker SCJ 01 is at the same loci as that of RAPD marker OPJ 01₁₄₃₇ and hence, is linked to anthracnose resistance gene. Resistant parent G 73 and resistant bulk amplified single specific band on PCR amplification using SCAR primer pairs. The RAPD marker OPJ 01₁₄₃₇ was mapped at a distance of 3.26 cM apart from the locus governing anthracnose resistance on the sorghum genetic map by the segregation analysis of the RILs. Using BLAST program, it was found that the marker showed 100 per cent alignment with the contig{_}3966 located on the longer arm of chromosome 8 of sorghum genome. Therefore, these identified RAPD and SCAR markers can be used in the resistance-breeding program of sorghum anthracnose by marker-assisted selection.An erratum to this article can be found at     

Genetic analysis and identification of DNA markers linked to a novel Phytophthora sojae resistance gene in the Japanese soybean cultivar Waseshiroge

The Glycine max (L.) Merr. cultivar Waseshiroge is highly resistant to several races of Phytophthora sojae in Japan. In order to determine which Rps gene might be present in Waseshiroge, 15 differential cultivars were challenged with 12 P. sojae isolates. None had a reaction pattern identical to that of Waseshiroge, indicating that Waseshiroge may contain a novel Rps gene. In order to characterize the inheritance of Waseshiroge resistance to P. sojae isolates, 98 F₂ progeny and 94 F_7:8 lines were produced from crosses between the susceptible cultivar Tanbakuro and Waseshiroge. Chi-square tests indicated that segregation fit a 3:1 ratio for resistance and susceptibility in two F₂ sub-populations of 42 and 56 seedlings. This and a 46.27:1.46:46.27 (or 63:2:63) ratio for resistance: segregation: susceptibility among the 94 F_7:8 lines indicated that resistance was controlled by a single dominant gene. DNA analyses were carried out on Tanbakuro, Waseshiroge and the 94 F_7:8 lines, and a linkage map was constructed with 17 SSR markers and nine new primer pairs that amplify marker loci linked to Rps1 on soybean chromosome 3 (linkage group N). The closest markers, Satt009 and T000304487l, map to locations 0.9 and 1.6 cM on each side of the estimated position of the Rps gene, respectively. The results showed that the Rps gene in Waseshiroge is either allelic to Rps1, or resides at a tightly linked locus in a gene cluster. A three-way-contingency table analysis indicated that marker-assisted selection with the two flanking markers could be used in the development of new resistant cultivars.     

Effectiveness of combining resistance to Thielaviopsis basicola and Tomato spotted wilt virus in haploid tobacco genotypes

Black root rot (BRR) caused by Thielaviopsis basicola as well as Tomato spotted wilt virus (TSWV) are the most serious problems in tobacco growing regions. We crossed the breeding line WGL 3 carrying BRR resistance derived from N.glauca with the line PW-834 the resistance of which to TSWV was transferred from cultivar Polalta. Anthers obtained from F₁ hybrid plants were cultured to induce haploids combining resistance to Th. basicola and TSWV. Flow cytometry analysis revealed 242 haploids and 2 spontaneous doubled haploids among regenerants. All haploids were cloned and then evaluated for BRR as well as TSWV resistance. The presence of pathogens was detected by microscopic evaluation of roots or using DAS-ELISA test. Microscopic assessment showed that, 132 haploids had no symptoms of Th. basicola which, together with the absence of symptoms in the F₁ hybrids, indicated a dominant monogenic mode of inheritance. At the same time only 30 haploids demonstrated resistance to TSWV. SCAR markers associated with TSWV resistance gene detection was applied. The results indicate that small proportion of TSWV-resistant haploids is probably due to the influence of deleterious genes flanking the resistance factor that reduced vitality of gametophytes. Altogether, 24 haploids showed multiple resistance to Th. basicola and TSWV.     

Segregation distortion in doubled haploid lines of barley (Hordeum vulgare L.) detected by simple sequence repeat (SSR) markers

A total of 147 simple sequence repeat (SSR) markers (including86 barley and 61 wheat microsatellite markers) were tested for their segregation in a doubled haploid (DH) and an F₂ population of barley. The DH population consisted of 71 doubled haploid lines, developed from F₁ plants of a cross between Tadmor and WI2291using isolated microspore culture technique. A genetic linkage map consisting of 43 microsatellite markers was constructed using the DH population. Particularly on chromosome 4H microsatellite markers showed distorted segregation ratios. Segregation of DH lines based on molecular markers were compared with segregation of 92 F₂ lines from the same cross. The proportion of loci deviating from the expected monogenic segregation ratios in the DH population was significantly higher (19/43loci, 44%) than in the F₂ population (7/43 loci, 16%). The deviation was biased towards the WI2291 parent alleles. In line with this observation, WI2291 was found to perform better than Tadmor in regenerating green plantlets with the isolated microspore-culture technique. This revised version was published online in July 2006 with corrections to the Cover Date.     

Inheritance of coffee leaf rust resistance and identification of AFLP markers linked to the resistance gene

The most important disease of Coffea arabica is coffee leaf rust caused by the fungus Hemileia vastatrix. The purpose of this study was to characterize the inheritance of coffee resistance gene(s) to race II of this pathogen and to identify and map molecular markers linked to this trait. Different populations were used: F₂ (160 plants), BCr (20), and BCs (135), derived from a cross between the resistant genotype Híbrido de Timor UFV 427-15 and the susceptible cultivar Catuaí Amarelo UFV 2143-236 (IAC 30). The segregation analysis showed that the resistance of Híbrido de Timor to race II of the H. vastatrix is conferred by a single dominant gene. The amplification of 176 AFLP (Amplified fragment length polymorphism) primer combinations using bulked segregant analysis (BSA) allowed the identification of three molecular markers linked to the resistance gene. Genetic mapping of these three markers in the F₂ population indicated that they are distributed on both sides, flanking the resistance gene. The markers E.CTC/M.TTT405 and E.CGT/M.TGT300 were found linked to the resistance gene at 8.69 cM (LOD 18.91) and 25.10 cM (LOD 5.37), respectively, while E.CCT/M.TTC230 was localized on the other side of the gene, at 20.50 cM (LOD 6.15). These markers are the first rust resistance markers identified in Híbrido de Timor and can be useful for marker assisted selection in coffee breeding programs.     

Identification of microsatellite markers linked to the blast resistance gene <Emphasis Type="Italic">Pi-1(t)</Emphasis> in rice

The present work was conducted to identify microsatellite markers linked to the rice blast resistance gene Pi-1(t) for a marker-assisted selection program. Twenty-four primer pairs corresponding to 19 microsatellite loci were selected from the Gramene database (www. gramene.org) considering their relative proximity to Pi-1(t) gene in the current rice genetic map. Progenitors and DNA bulks of resistant and susceptible families from F₃ segregating populations of a cross between the near-isogenic lines C101LAC (resistant) and C101A51 (susceptible) were used to identify polymorphic microsatellite markers associated to this gene through bulked segregant analysis. Putative molecular markers linked to the blast resistance gene Pi-1(t) were then used on the whole progeny for linkage analysis. Additionally, the diagnostic potential of the microsatellite markers associated to the resistance gene was also evaluated on 17 rice varieties planted in Latin America by amplification of the specific resistant alleles for the gene in each genotype. Comparing with greenhouse phenotypic evaluations for blast resistance, the usefulness of the highly linked microsatellite markers to identify resistant rice genotypes was evaluated. As expected, the phenotypic segregation in the F₃ generation agreed to the expected segregation ratio for a single gene model. Of the 24 microsatellite sequences tested, six resulted polymorphic and linked to the gene. Two markers (RM1233*I and RM224) mapped in the same position (0.0 cM) with the Pi-1(t) gene. Other three markers corresponding to the same genetic locus were located at 18.5 cM above the resistance gene, while another marker was positioned at 23.8 cM below the gene. Microsatellite analysis on elite rice varieties with different genetic background showed that all known sources of blast resistance included in this study carry the specific Pi-1(t) allele. Results are discussed considering the potential utility of the microsatellite markers found, for MAS in rice breeding programs aiming at developing rice varieties with durable blast resistance based on a combination of resistance genes. Centro Internactional de Agricultura Tropical (CIAT) institute where the research was carried out