Fine mapping and identification of tightly linked DNA markers of blast resistance gene <Emphasis Type="Italic">Pia</Emphasis> by using an introgression line

An introgression line (INL) for a major rice blast resistance gene, Pia, was developed, with the genetic background of a blast susceptible variety, US-2. The reaction pattern of the INL was characterized by using 20 standard blast isolates from the Philippines. The introgression of the Pia gene was confirmed by DNA markers on the short arm of chromosome 11 where Pia was previously mapped. A genome-wide DNA marker survey revealed that most of the chromosomal regions were US-2 type. By using an F2 population derived from a cross between the INL and US-2, the chromosomal location of the Pia locus was mapped between RM26281 and RM3701. For fine mapping of the Pia locus, five additional markers were developed based on the genomic sequence of the corresponding region of a japonica-type variety, Nipponbare. The candidate region of Pia was delimited between two DNA markers, RM26281 and 82N19365, corresponding to a 140 kb region on the Nipponbare genome sequence. We obtained three DNA markers within this region. The developed INL, information on the map position of Pia, and DNA markers developed in the candidate region of the Pia locus are useful tools for blast resistance studies and a marker-aided breeding strategy.     

Genetic and physical mapping of <Emphasis Type="Italic">Pi36</Emphasis>(t), a novel rice blast resistance gene located on rice chromosome 8

Blast resistance in the indica cultivar (cv.) Q61 was inherited as a single dominant gene in two F₂ populations, F₂-1 and F₂-2, derived from crosses between the donor cv. and two susceptible japonica cvs. Aichi Asahi and Lijiangxintuanheigu (LTH), respectively. To rapidly determine the chromosomal location of the resistance (R) gene detected in Q61, random amplified polymorphic DNA (RAPD) analysis was performed in the F₂-1 population using bulked-segregant analysis (BSA) in combination with recessive-class analysis (RCA). One of the three linked markers identified, BA1126₅₅₀, was cloned and sequenced. The R gene locus was roughly mapped on rice chromosome 8 by comparison of the BA1126₅₅₀ sequence with rice sequences in the databases (chromosome landing). To confirm this finding, seven known markers, including four sequence-tagged-site (STS) markers and three simple-sequence repeat (SSR) markers flanking BA1126₅₅₀ on chromosome 8, were subjected to linkage analysis in the two F₂ populations. The locus was mapped to a 5.8 cM interval bounded by RM5647 and RM8018 on the short arm of chromosome 8. This novel R gene is therefore tentatively designated as Pi36(t). For fine mapping of the Pi36(t) locus, five additional markers including one STS marker and four candidate resistance gene (CRG) markers were developed in the target region, based on the genomic sequence of the corresponding region of the reference japonica cv. Nipponbare. The Pi36(t) locus was finally localized to an interval of about 0.6 cM flanked by the markers RM5647 and CRG2, and co-segregated with the markers CRG3 and CRG4. To physically map this locus, the Pi36(t)-linked markers were mapped by electronic hybridization to bacterial artificial chromosome (BAC) or P1 artificial chromosome (PAC) clones of Nipponbare, and a contig map was constructed in silico through Pairwise BLAST analysis. The Pi36(t) locus was physically delimited to an interval of about 17.0 kb, based on the genomic sequence of Nipponbare.     

Identification and fine mapping of <Emphasis Type="Italic">Pi39</Emphasis>(t), a major gene conferring the broad-spectrum resistance to <Emphasis Type="Italic">Magnaporthe oryzae</Emphasis>

Blast, caused by the ascomycete fungus Magnaporthe oryzae, is one of the most devastating diseases of rice worldwide. The Chinese native cultivar (cv.) Q15 expresses the broad-spectrum resistance to most of the isolates collected from China. To effectively utilize the resistance, three rounds of linkage analysis were performed in an F₂ population derived from a cross of Q15 and a susceptible cv. Tsuyuake, which segregated into 3:1 (resistant/susceptible) ratio. The first round of linkage analysis employing simple sequence repeat (SSR) markers was carried out in the F₂ population through bulked-segregant assay. A total of 180 SSR markers selected from each chromosome equally were surveyed. The results revealed that only two polymorphic markers, RM247 and RM463, located on chromosome 12, were linked to the resistance (R) gene. To further define the chromosomal location of the R gene locus, the second round of linkage analysis was performed using additional five SSR markers, which located in the region anchored by markers RM247 and RM463. The locus was further mapped to a 0.27 cM region bounded by markers RM27933 and RM27940 in the pericentromeric region towards the short arm. For fine mapping of the R locus, seven new markers were developed in the smaller region for the third round of linkage analysis, based on the reference sequences. The R locus was further mapped to a 0.18 cM region flanked by marker clusters 39M11 and 39M22, which is closest to, but away from the Pita/Pita ² locus by 0.09 cM. To physically map the locus, all the linked markers were landed on the respective bacterial artificial chromosome clones of the reference cv. Nipponbare. Sequence information of these clones was used to construct a physical map of the locus, in silico, by bioinformatics analysis. The locus was physically defined to an interval of ≈37 kb. To further characterize the R gene, five R genes mapped near the locus, as well as 10 main R genes those might be exploited in the resistance breeding programs, were selected for differential tests with 475 Chinese isolates. The R gene carrier Q15 conveys resistances distinct from those conditioned by the carriers of the 15 R genes. Together, this valuable R gene was, therefore, designated as Pi39(t). The sequence information of the R gene locus could be used for further marker-based selection and cloning. Xinqiong Liu and Qinzhong Yang contributed equally to this work.     

Genetic analysis and fine mapping of a rice brown planthopper (Nilaparvata lugens Stål) resistance gene bph19(t)

Genetic analysis and fine mapping of a resistance gene against brown planthopper (BPH) biotype 2 in rice was performed using two F₂ populations derived from two crosses between a resistant indica cultivar (cv.), AS20-1, and two susceptible japonica cvs., Aichi Asahi and Lijiangxintuanheigu. Insect resistance was evaluated using F₁ plants and the two F₂ populations. The results showed that a single recessive gene, tentatively designated as bph19(t), conditioned the resistance in AS20-1. A linkage analysis, mainly employing microsatellite markers, was carried out in the two F₂ populations through bulked segregant analysis and recessive class analysis (RCA), in combination with bioinformatics analysis (BIA). The resistance gene locus bph19(t) was finely mapped to a region of about 1.0 cM on the short arm of chromosome 3, flanked by markers RM6308 and RM3134, where one known marker RM1022, and four new markers, b1, b2, b3 and b4, developed in the present study were co-segregating with the locus. To physically map this locus, the bph19(t)-linked markers were landed on bacterial artificial chromosome or P1 artificial chromosome clones of the reference cv., Nipponbare, released by the International Rice Genome Sequencing Project. Sequence information of these clones was used to construct a physical map of the bph19(t) locus, in silico, by BIA. The bph19(t) locus was physically defined to an interval of about 60 kb. The detailed genetic and physical maps of the bph19(t) locus will facilitate marker-assisted gene pyramiding and cloning.     

The Pik m gene, conferring stable resistance to isolates of Magnaporthe oryzae , was finely mapped in a crossover-cold region on rice chromosome 11

The Pik ^m gene in rice confers a high and stable resistance to many isolates of Magnaporthe oryzae collected from southern China. This gene locus was roughly mapped to the long arm of rice chromosome 11 with restriction fragment length polymorphic (RFLP) markers in the previous study. To effectively utilize the resistance, a linkage analysis was performed in a mapping population consisting of 659 highly susceptible plants collected from four F₂ populations using the publicly available simple sequence repeat (SSR) markers. The result showed that the locus was linked to the six SSR markers and defined by RM254 and RM144 with ≈13.4 and ≈1.2 cM, respectively. To fine map this locus, additional 10 PCR-based markers were developed in a region flanked by RM254 and RM144 through bioinformatics analysis (BIA) using the reference sequence of cv. Nipponbare. The linkage analysis with these 10 markers showed that the locus was further delimited to a 0.3-cM region flanked by K34 and K10, in which three markers, K27, K28, and K33, completely co-segregated with the locus. To physically map the locus, the Pik ^m -linked markers were anchored to bacterial artificial chromosome clones of the reference cv. Nipponbare by BIA. A physical map spanning ≈278 kb in length was constructed by alignment of sequences of the clones anchored by BIA, in which only six candidate genes having the R gene conserved structure, protein kinase, were further identified in an 84-kb segment.     

Targeting xa13, a recessive gene for bacterial blight resistance in rice

Bacterial blight, caused by Xanthomonas oryzae pv. oryzae (Xoo), is one of the most serious diseases of rice worldwide. Thirty bacterial blight resistance (R) genes (21 dominant genes and 9 recessive genes) in rice have been identified. They are the main sources for the genetic improvement of rice for resistance to Xoo. However, little is known about the recessive R genes. To clone and characterize the recessive R genes, we fine-mapped xa13, a fully recessive gene for Xoo resistance, to a DNA fragment of 14.8 kb using the map-based cloning strategy and a series of sequence-based molecular markers. Sequence analysis of this fragment indicated that this region contains only two apparently intact candidate genes (an extensin-like gene and a homologue of nodulin MtN3) and the 5′ end of a predicted hypothetical gene. These results will greatly facilitate the isolation and characterization of xa13. Four PCR-based markers, E6a, SR6, ST9 and SR11 that were tightly linked to the xa13 locus, were also developed. These markers will be useful tools for the marker-assisted selection of xa13 in breeding programs.     

High-resolution Mapping and Analysis of the Resistance Locus <Emphasis Type="Italic">Rpi-abpt</Emphasis> Against <Emphasis Type="Italic">Phytophthora infestans</Emphasis> in Potato

Introduction of more durable resistance against Phytophthora infestans causing late blight into the cultivated potato is of importance for sustainable agriculture. We identified a new monogenically inherited resistance locus that is localized on chromosome 4. The resistance is derived from an ABPT clone, which is originally a complex quadruple hybrid in which Solanum acaule, S. bulbocastanum, S. phureja and S. tuberosum were involved. Resistance data of the original resistant accessions of the wild species and analysis of mobility of AFLP markers linked to the resistance locus suggest that the resistance locus is originating from S. bulbocastanum. A population of 1383 genotypes was screened with two AFLP markers flanking the Rpi-abpt locus and 98 recombinants were identified. An accurate high-resolution map was constructed and the Rpi-abpt locus was localized in a 0.5 cM interval. One AFLP marker was found to co-segregate with the Rpi-abpt locus. Its DNA sequence was highly similar with sequences found on a tomato BAC containing several resistance gene analogues on chromosome 4 and its translated protein sequence appeared to be homologous to several disease resistance related proteins. The results indicated that the Rpi-abpt gene is a member of an R gene cluster.     

High-resolution genetic mapping of<Emphasis Type="Italic"> Xa27(t)</Emphasis>, a new bacterial blight resistance gene in rice,<Emphasis Type="Italic"> Oryza sativa</Emphasis> L.

Bacterial blight of rice, caused by Xanthomonas oryzae pv. oryzae (Xoo) (Ishyama) Dye, is one of the serious diseases prevalent throughout Asia. In a previous study, a resistance (R) locus was transferred from the tetraploid wild rice Oryza minuta to the cultivated rice species, Oryza sativa L. Here, we report the fine genetic mapping of the R locus, tentatively designated as Xa27(t). We performed disease evaluation with an Xa27(t) near-isogenic line, IRBB27, testing 35 Xoo strains collected from 11 countries. The Xa27(t) locus conferred a high level of resistance to 27 strains and moderate resistance to three strains. Resistance of the Xa27(t) gene was developmentally regulated in IRBB27 and showed semi-dominant or a dosage effect in the cv. CO39 genetic background. As a prelude to cloning Xa27(t), a molecular mapping strategy was employed with a large mapping population consisting of 3,875 gametes. Three molecular markers, M336, M1081, and M1059, closely linked to Xa27(t), were identified to facilitate the mapping of Xa27(t) to the long arm of chromosome 6. The Xa27(t) locus was confirmed by chromosome landing of M1081 and M1095 markers on the rice genome. Markers derived from the genomic sequence of O. sativa cv. Nipponbare were used to further saturate the Xa27(t) genomic region. Xa27(t) was finally located within a genetic interval of 0.052 cM, flanked by markers M964 and M1197, and co-segregated with markers M631, M1230, and M449.Communicated by D.J. Mackill     

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.     

Genetic and physical mapping of Pi37(t), a new gene conferring resistance to rice blast in the famous cultivar St. No. 1

The famous rice cultivar (cv.), St. No. 1, confers complete resistance to many isolates collected from the South China region. To effectively utilize the resistance, a linkage assay using microsatellite markers (SSR) was performed in the three F₂ populations derived from crosses between the donor cv. St. No. 1 and each of the three susceptible cvs. C101PKT, CO39 and AS20-1, which segregated into 3R:1S (resistant/susceptible) ratio, respectively. A total of 180 SSR markers selected from each chromosome equally were screened. The result showed that the two markers RM128 and RM486 located on chromosome 1 were linked to the resistance gene in the respective populations above. This result is not consistent with those previously reported, in which a well-known resistance gene Pif in the St. No. 1 is located on chromosome 11. To confirm this result, additional four SSR markers, which located in the region lanked by RM128 and RM486, were tested. The results showed that markers RM543 and RM319 were closer to, and RM302 and RM212 completely co-segregated with the resistance locus detected in the present study. These results indicated that another resistance gene involved in the St. No. 1, which is located on chromosome 1, and therefore tentatively designated as Pi37(t). To narrow down genomic region of the Pi37(t) locus, eight markers were newly developed in the target region through bioinformatics analysis (BIA) using the publicly available sequences. The linkage analysis with these markers showed that the Pi37(t) locus was mapped to a ≈ 0.8 centimorgans (cM) interval flanked by RM543 and FPSM1, where a total of seven markers co-segregated with it. To physically map the locus, the Pi37(t)-linked markers were landed on the reference sequence of cv. Nipponbare through BIA. A contig map corresponding to the locus was constructed based on the reference sequence aligned by the Pi37(t)-linked markers. Consequently, the Pi37(t) locus was defined to 374 kb interval flanking markers RM543 and FPSM1, where only four candidate genes with the resistance gene conserved structure (NBS-LRR) were further identified to a DNA fragment of 60 kb in length by BIA.     

Characterization and high-resolution mapping of a late blight resistance locus similar to R2 in potato

Identification of resistance (R) genes to Phytophthora infestans is an essential step in molecular breeding of potato. We identified three specific R genes segregating in a diploid mapping population. One of the R genes is located on chromosome 4 and proved phenotypically indistinguishable from the Solanum demissum-derived R2, although S. demissum is not directly involved in the pedigree of the population. By bulked segregant analysis combined with a resistance assay, a genetic linkage map of the R2-like locus was constructed with 30 coupling and 23 repulsion phase AFLP markers. Two markers flanking the R2-like locus were applied to screen an extended population of 1,586 offspring. About 103 recombinants were selected, and an accurate high-resolution map was constructed. The R2-like resistance was localized in a 0.4 cM interval and was found co-segregating with four AFLP markers, which can be used to isolate the R2-like gene by map-based gene cloning. By analyzing race-specificity and R gene-specific molecular markers, we also found that an R1-like gene and an additional unknown R gene are segregating in the population.     

Development of marker sets useful in the early selection of <Emphasis Type="Italic">Ren4</Emphasis> powdery mildew resistance and seedlessness for table and raisin grape breeding

The single, dominant powdery mildew resistance locus Ren4 from Vitis romanetii prevents hyphal growth by Erysiphe necator. Previously, we showed that when introgressed into V. vinifera in the modified BC₂ population 03-3004, Ren4 was linked with the simple sequence repeat marker VMC7f2 on chromosome 18—a marker that is associated with multiple disease resistance and seedlessness. However, in the current study, this marker was monomorphic in related breeding populations 05-3010 and 07-3553. To enhance marker-assisted selection at this locus, we developed multiplexed SNP markers using three approaches: conversion of bulked segregant analysis AFLP markers, sequencing of candidate genes and regions flanking known V. vinifera SNPs, and hybridization to the Vitis9KSNP genotyping array. The Vitis9KSNP array was more cost-efficient than all other approaches tested for marker discovery and genotyping, enabling the genotyping of 1317 informative SNPs within the span of 1 week and at a cost of 11 cents per SNP. From a total of 1,446 high quality, informative markers segregating in 03-3004, we developed a haplotype signature of 15 multiplexed SNP markers linked with Ren4 in 03-3004, 5 of which were linked in 05-3010, and 6 of which were linked in 07-3553. Two of these populations segregated for seedlessness, which was tightly linked with Ren4 in 03-3004 (2 cM) but not in 05-3010 (22 cM). Chromosomal rearrangements were detected among these three populations and the reference genome PN40024. Since this is the first application of the Vitis9KSNP array in a breeding program, some suggestions are provided for application of genotyping arrays. Our results provide novel markers for tracking and pyramiding this unique resistance gene and for further functional characterization of this region on chromosome 18 encoding multiple disease resistance and seedlessness.     

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     

High-resolution genetic mapping of bacterial blight resistance gene <Emphasis Type="Italic">Xa10</Emphasis>

Bacterial blight of rice, caused by Xanthomonas oryzae pv. oryzae (Xoo), is the most devastating disease of rice (Oryza sativa L). Rice lines that carry resistance (R) gene Xa10 confer race-specific resistance to Xoo strains harboring avirulence (Avr) gene avrXa10. Here we report on genetic study, disease evaluation and fine genetic mapping of the Xa10 gene. The inheritance of Xa10-mediated resistance to PXO99^A(pHM1avrXa10) did not follow typical Mendelian inheritance for single dominant gene in F₂ population derived from IR24 × IRBB10. A locus might be present in IRBB10 that caused distorted segregation in F₂ population. To eliminate this locus, an F₃ population (F₃-65) was identified, which showed normal Mendelian segregation ratio of 3:1 for resistance and susceptibility. A new near-isogenic line (F₃-65-1743) of Xa10 in IR24 genetic background was developed and designated as IRBB10A. IRBB10A retained similar resistance specificity as that of IRBB10 and provided complete resistance to PXO99^A(pHM1avrXa10) from seedling to adult stages. Linkage analysis using existing RFLP markers and F₂ mapping population mapped the Xa10 locus to the proximal side of E1981S with genetic distance at 0.93 cM. With five new RFLP markers developed from the genomic sequence of Nipponbare, Xa10 was finely mapped at genetic distance of 0.28 cM between proximal marker M491 and distal marker M419 and co-segregated with markers S723 and M604. The physical distance between M491 and M419 on Nipponbare genome is 74 kb. Seven genes have been annotated from this 74-kb region and six of them are possible Xa10 candidates. The results of this study will be useful in Xa10 cloning and marker-assisted breeding.     

Fine mapping and analyses of <Emphasis Type="Italic">R</Emphasis><Subscript><Emphasis Type="Italic">SC8</Emphasis></Subscript> resistance candidate genes to soybean mosaic virus in soybean

Soybean mosaic virus (SMV) in soybean [Glycine max (L.) Merr.] is a destructive foliar disease in soybean-producing countries worldwide. In this study, F₂, F_2:3, and F_7:11 recombinant inbred lines populations derived from Kefeng No.1 × Nannong 1138-2 were used to study inheritance and linkage mapping of the SMV strain SC8 resistance gene in Kefeng No.1. Results indicated that a single dominant gene (designated R _SC8 ) controls resistance, which is located on chromosome 2 (MLG D1b). A mixed segregating population was developed by selfing two heterozygous plants (RHL153-1 and RHL153-2) at four markers adjacent to the locus and used in fine mapping R _SC8 . In addition, two genomic-simple sequence repeats (SSR) markers BARCSOYSSR_02_0610 and BARCSOYSSR_02_0616 were identified that flank the two sides of R _SC8 . Sequence analysis of the soybean genome indicated that the interval between the two genomic-SSR markers is 200 kb. QRT-PCR analysis of the candidate genes determined that five genes (Glyma02g13310, 13320, 13400, 13460, and 13470) are likely involved in soybean SMV resistance. These results will have utility in cloning, transferring, and pyramiding of the R _SC8 through marker-assisted selection in soybean breeding programs.     

High-resolution mapping and gene prediction of <Emphasis Type="Italic">Xanthomonas Oryzae</Emphasis> pv. <Emphasis Type="Italic">Oryzae</Emphasis> resistance gene <Emphasis Type="Italic">Xa7</Emphasis>

Bacterial blight (BB) caused by Xanthomonas oryzae pv. oryzae (Xoo) is a devastating disease in rice worldwide. The resistance gene Xa7, which provides dominant resistance against the pathogen with avirulence (Avr) gene AvrXa7, has proved to be durably resistant to BB. A set of SSR markers were selected from the “gramene” database based on the Xa7 gene initial mapping region on chromosome 6. These markers were used to construct a high-resolution genetic map of the chromosomal region surrounding the Xa7 gene. An F₂ mapping population with 721 highly susceptible individuals derived from a cross between the near isogenic lines (NILs) IRBB7 and IR24 were constructed to localize the Xa7 gene. In a primary analysis with eleven polymorphic SSR markers, Xa7 was located in approximately the 0.28-cM region. To walk closer to the target gene, recombinant F₂ individuals were tested using newly developed STMS (sequence tagged microsatellite) markers. Finally, the Xa7 gene was mapped to a 0.21-cM interval between the markers GDSSR02 and RM20593. The Xa7-linked markers were landed on the reference sequence of cv. Nipponbare through bioinformatics analysis. A contig map corresponding to the Xa7 gene was constructed. The target gene was assumed to span an interval of approximately 118.5-kb which contained a total of fourteen genes released by the TIGR Genome Annotation Version 5.0. Candidate-gene analysis of Xa7 revealed that the fourteen genes encode novel domains that have no amino acid sequence similar to other cloned Xa(xa) genes. Shen Chen and Zhanghui Huang are contributed equally to this work.     

Localization of the rice stripe disease resistance gene, Stv-bi, by graphical genotyping and linkage analyses with molecular markers

 We used graphical genotyping and linkage analyses with molecular markers to determine the chromosomal location of the rice stripe disease resistance gene, Stv-b ⁱ . The stripe resistance gene from the indica rice (Oryza sativa) cv ‘Modan’ was introgressed into several Japanese rice varieties. We found 4 RFLP markers in ‘Modan’, five susceptible parental rice varieties (‘Norin No. 8’, ‘Sachihikari’, ‘Kanto No. 98’, ‘Hokuriku No.103’ and ‘Koganebare’) and four resistant progeny varieties (‘St. No. 1’, ‘Aichi No. 6’, ‘Aoisora’ and ‘Asanohikari’). Graphical genotyping of the resistant progeny revealed a chromosomal segment ascribable to ‘Modan’ and associated with stripe resistance. The chromosomal segment from ‘Modan’ was located at 35.85 cM on chromosome 11. Linkage analysis using 120 F₂ individuals from a cross between ‘Koshihikari’ (susceptible) and ‘Asanohikari’ (resistant) revealed another 8 RFLP markers in the same chromosome. We performed a bioassay for rice stripe resistance in F₃ lines of the F₂ individuals using infective small brown planthoppers and identified an 1.8-cM segment harboring the rice stripe disease resistance gene, Stv-b ⁱ , between XNpb220 and XNpb257/ XNpb254. Furthermore, Stv-b ⁱ was linked by 0.0 cM to a RFLP marker, ST10, which was developed on the basis of the results of RAPD analysis. These DNA markers near the Stv-b ⁱ locus may be useful in marker-assisted selection and map-based cloning of the Stv-b ⁱ gene. Received: 26 September 1997 / Accepted: 4 November 1997     

Genetics of resistance to ascochyta blight (Ascochyta lentis) of lentil and the identification of closely linked RAPD markers

 Foliar resistance to Ascochyta lentis is controlled at a single major locus by a dominant gene (AbR ₁ ) in the lentil accession ILL5588 (cv ‘Northfield’). Flanking RAPD markers that are closely linked to the resistance locus in coupling phase were identified by bulked segregant analysis. Out of 261 decanucleotide primers screened 7 produced a polymorphic marker that segregated with the resistance locus, and all markers were found to exist within a single linkage group. Five of the seven RAPD markers were within 30 cM of the resistance locus. Log likelihood analysis for detecting QTL associated with the foliar resistance revealed that a single narrow peak accounted for almost 90% of the variance of resistance between the bulks. Preliminary mapping in an F₃ population revealed that the closest flanking markers were approximately 6 and 14 centiMorgans (cM) away from the resistance locus. These markers should be useful for the discrimination of resistant germplasm through marker-assisted selection in future breeding programmes and represent the first essential step towards the map-based cloning of this resistance gene. Received: 18 December 1997 / Accepted: 9 June 1998     

Mapping of a locus for adult plant resistance to downy mildew in broccoli (<Emphasis Type="Italic">Brassica oleracea</Emphasis> convar. <Emphasis Type="Italic">italica</Emphasis>)

The identification of the gene Pp523, conferring downy mildew resistance to adult plants of broccoli (Brassica oleracea convar. italica), led to the construction of a genetic map that included this resistance locus, 301 amplified fragment length polymorphisms, 55 random amplified polymorphic DNAs, 46 inter-simple sequence repeats, three simple sequence repeats, four other PCR markers and a flower colour locus, all gathered into nine major linkage groups. Nineteen additional molecular markers were clustered into one group of four markers, one group of three markers and six pairs of markers. The map spans over 731.9 cM, corresponding to 89.5% of the 818 cM estimated to be the total genome length. A significant number of the mapped markers, 19.3%, showed distorted segregation. The average distance between mapped adjacent markers is 1.64 cM, which places this map among the densest published to date for this species. Using bulked segregant analysis, we identified a group of molecular markers flanking and closely linked in coupling to the resistance gene and included these in the map. Two markers linked in coupling, OPK17_980 and AT.CTA_133/134, are located at 3.1 cM and 3.6 cM, respectively, at each side from the resistance gene. These markers can be used for marker-assisted selection in breeding programs aiming at the introgression of this gene in susceptible B. oleracea genotypes. The fine mapping of the genomic region surrounding the Pp523 resistance gene is currently being carried out, a basic condition for its isolation via positional cloning.     

Molecular mapping of the rust resistance gene <Emphasis Type="Italic">R</Emphasis><Subscript><Emphasis Type="Italic">4</Emphasis></Subscript> to a large NBS-LRR cluster on linkage group 13 of sunflower

Rust is a serious fungal disease in the sunflower growing areas worldwide with increasing importance in North America in recent years. Several genes conferring resistance to rust have been identified in sunflower, but few of them have been genetically mapped and linked to molecular markers. The rust resistance gene R ₄ in the germplasm line HA-R3 was derived from an Argentinean open-pollinated variety and is still one of most effective genes. The objectives of this study were to determine the chromosome location of the R ₄ gene and the allelic relationship of R ₄ with the R _adv rust resistance gene. A total of 63 DNA markers previously mapped to linkage group (LG) 13 were used to screen for polymorphisms between two parental lines HA 89 and HA-R3. A genetic map of LG 13 was constructed with 21 markers, resulting in a total map length of 93.8 cM and an average distance of 4.5 cM between markers. Two markers, ZVG61 and ORS581, flanked the R ₄ gene at 2.1 and 0.8 cM, respectively, and were located on the lower end of LG 13 within a large NBS-LRR cluster identified previously. The PCR pattern generated by primer pair ZVG61 was unique in the HA-R3 line, compared to lines HA-R1, HA-R4, and HA-R5, which carry other R ₄ alleles. A SCAR marker linked to the rust resistance gene R _adv mapped to LG 13 at 13.9 cM from the R ₄ locus, indicating that R _adv is not an allele of the R ₄ locus. The markers tightly linked to the R ₄ gene will facilitate gene pyramiding for rust resistance breeding of sunflower.