Chromosome landing at the tomato Bs4 locus

The tomato (Lycopersicon esculentum) Bs4 gene confers resistance to strains of Xanthomonas campestris pathovar vesicatoria that express the avirulence protein AvrBs4. As part of a map-based cloning strategy for the isolation of Bs4, we converted Bs4-linked amplified fragment length polymorphism (AFLP) and restriction fragment length polymorphism (RFLP) markers into locus-specific sequence-tagged-site (STS) markers. The use of these markers for the analysis of 1972 meiotic events allowed high-resolution genetic mapping within a 1.2-cM interval containing the target gene. Two tomato yeast artificial chromosome (YAC) clones, each harboring inserts of approximately 250 kb, were identified using the marker most closely linked to Bs4. YAC end-specific markers were established and employed to construct a local YAC contig. The ratio of physical to genetic distance at Bs4 was calculated to be 280 kb/cM, revealing that recombination rates in this region are about three times higher than the genome-wide average. Mapping of YAC end-derived markers demonstrated that the Bs4 locus maps within a region of 250 kb, corresponding to a genetic interval of 0.9 cM.     

Identification of AFLP markers closely linked to the <Emphasis Type="Italic"> rhm </Emphasis>gene for resistance to Southern Corn Leaf Blight in maize by using bulked segregant analysis

Southern Corn Leaf Blight (SCLB) is an important disease in warm-temperate and tropical corn-producing areas throughout the world. We applied a combination of the amplified fragment length polymorphism (AFLP) technique and bulked segregant analysis (BSA) to a large F2 population in order to identify molecular markers linked to the rhm gene for resistance to SCLB. One co-dominant AFLP marker, p7m36, was mapped to a position 1.0 cM from rhm, and we converted this marker to an STS (sequence-tagged site) marker. Combined with the previously identified agrP144, this new marker may be useful for map-based cloning of the rhm gene and marker-assisted selection for rhm.     

A genetic map of Blumeria graminis based on functional genes, avirulence genes, and molecular markers

A genetic map of the powdery mildew fungus, Blumeria graminis f. sp. hordei, an obligate biotrophic pathogen of barley, is presented. The linkage analysis was conducted on 81 segregating haploid progeny isolates from a cross between 2 isolates differing in seven avirulence genes. A total of 359 loci were mapped, comprising 182 amplified fragment length polymorphism markers, 168 restriction fragment length polymorphism markers including 42 LTR-retrotransposon loci and 99 expressed sequence tags (ESTs), all the seven avirulence genes, and a marker closely linked to the mating type gene. The markers are distributed over 34 linkage groups covering a total of 2114 cM. Five avirulence genes were found to be linked and mapped in clusters of three and two, and two were unlinked. The Avr(a6) gene was found to be closely linked to markers suitable for a map-based cloning approach. A linkage between ESTs allowed us to demonstrate examples of synteny between genes in B. graminis and Neurospora crassa.     

Construction of a genetic linkage map based on amplified fragment length polymorphism markers and development of sequence-tagged site markers for marker-assisted selection of the sporeless trait in the oyster mushroom (Pleurotus eryngii)

A large number of spores from fruiting bodies can lead to allergic reactions and other problems during the cultivation of edible mushrooms, including Pleurotus eryngii (DC.) Quél. A cultivar harboring a sporulation-deficient (sporeless) mutation would be useful for preventing these problems, but traditional breeding requires extensive time and labor. In this study, using a sporeless P. eryngii strain, we constructed a genetic linkage map to introduce a molecular breeding program like marker-assisted selection. Based on the segregation of 294 amplified fragment length polymorphism markers, two mating type factors, and the sporeless trait, the linkage map consisted of 11 linkage groups with a total length of 837.2 centimorgans (cM). The gene region responsible for the sporeless trait was located in linkage group IX with 32 amplified fragment length polymorphism markers and the B mating type factor. We also identified eight markers closely linked (within 1.2 cM) to the sporeless locus using bulked-segregant analysis-based amplified fragment length polymorphism. One such amplified fragment length polymorphism marker was converted into two sequence-tagged site markers, SD488-I and SD488-II. Using 14 wild isolates, sequence-tagged site analysis indicated the potential usefulness of the combination of two sequence-tagged site markers in cross-breeding of the sporeless strain. It also suggested that a map constructed for P. eryngii has adequate accuracy for marker-assisted selection.     

Molecular mapping of the Oregon Wolfe Barleys: a phenotypically polymorphic doubled-haploid population

A phenotypically polymorphic barley (Hordeum vulgare L.) mapping population was developed using morphological marker stocks as parents. Ninety-four doubled-haploid lines were derived for genetic mapping from an F₁ using the Hordeum bulbosum system. A linkage map was constructed using 12 morphological markers, 87 restriction fragment length polymorphism (RFLP), five random amplified polymorphic DNA (RAPD), one sequence-tagged site (STS), one intron fragment length polymorphism (IFLP), 33 simple sequence repeat (SSR), and 586 amplified fragment length polymorphism (AFLP) markers. The genetic map spanned 1,387 cM with an average density of one marker every 1.9 cM. AFLP markers tended to cluster on centromeric regions and were more abundant on chromosome 1 (7H). RAPD markers showed a high level of segregation distortion, 54% compared with the 26% observed for AFLP markers, 27% for SSR markers, and 18% for RFLP markers. Three major regions of segregation distortion, based on RFLP and morphological markers, were located on chromosomes 2 (2H), 3 (3H), and 7 (5H). Segregation distortion may indicate that preferential gametic selection occurred during the development of the doubled-haploid lines. This may be due to the extreme phenotypes determined by alleles at morphological trait loci of the dominant and recessive parental stocks. Several molecular markers were found to be closely linked to morphological loci. The linkage map reported herein will be useful in integrating data on quantitative traits with morphological variants and should aid in map-based cloning of genes controlling morphological traits. Received: 23 August 2000 / Accepted: 15 December 2000     

Development and characterization of SCAR markers linked to the citrus tristeza virus resistance gene from Poncirus trifoliata.

Twelve new dominant randomly amplified polymorphic DNA (RAPD) fragments associated with a single dominant gene for resistance to citrus tristeza virus (CTV) were identified using bulked segregant analysis of an intergeneric backcross family. These and eight previously reported RAPDs were mapped in the resistance gene (Ctv) region; the resulting localized linkage map spans about 32 cM, with nine close flanking markers within 2.5 cM of Ctv. Seven of 20 RAPD fragments linked with the resistance gene were cloned and sequenced, and their sequences were used to design longer primers to develop sequence characterized amplified region (SCAR) markers that can be utilized reliably in marker-assisted selection, high-resolution mapping, and map-based cloning of the resistance gene. All seven cloned RAPDs were converted successfully into SCARs by redesigning primers, optimizing PCR parameters (especially the annealing temperature), or digesting amplification products with restriction enzymes. Four of the seven remained dominant markers, displaying presence-absence polymorphism patterns; the other three detected restriction site changes or length variations and thus were transformed into codominant markers. Two genomic regions rich in variability were also detected by two codominant SCAR markers.     

Fine-mapping of the BaMMV, BaYMV-1 and BaYMV-2 resistance of barley (Hordeum vulgare) accession PI1963

Barley yellow mosaic disease caused by the bymoviruses barley mild mosaic virus (BaMMV) and barley yellow mosaic virus (BaYMV) is one of the economically most important diseases of winter barley in Europe. In European barley breeding programmes, resistance is currently due to only two genes—rym4, which is effective against viruses BaMMV and BaYMV-1, and rym5, which is effective against BaYMV-2. Diversification of resistance is therefore an important task. Because the accession PI1963 confers immunity against all European strains of barley yellow mosaic disease and is not allelic to rym5, we have attempted to develop closely linked markers in order to facilitate the efficient introgression of this resistance into adapted germplasm. By means of restriction fragment length polymorphism analysis, we located a gene locus for resistance to BaMMV, BaYMV-1 and BaYMV-2 of PI1963 on chromosome 4HL using a mapping population (W757) comprising 57 doubled haploid (DH) lines. Subsequent tests for allelism indicated that the BaMMV resistance gene in PI1963 is allelic to rym11. Two DH populations, IPK1 and IPK2, comprising 191 and 161 DH lines, respectively, were derived from the initial mapping population W757 and used for further analysis. As random amplified polymorphic DNA development did not facilitate the identification of more closely linked markers, simple sequence repeat (SSR) analyses were conducted. For population IPK1, the closest SSRs detected were Bmac181 and Bmag353, which flank the gene at 2.1 cM and 2.7 cM, respectively. For the IPK2 population, the SSR markers HVM3 and Bmag353 are located proximally at 2.5 cM and distally at 8.2 cM, respectively. In order to develop markers more tightly linked to rym11, a targeted amplified fragment length polymorphism (AFLP) marker identification approach was adopted using bulks comprising lines carrying recombination events proximal and distal to the target interval. Using this approach we identified six AFLP markers closely linked to rym11, with the two markers, E56M32 and E49M33, co-segregating with rym11 in both populations. The SSRs and AFLPs identified in this study represent useful tools for marker-assisted selection.     

DNA markers for downy mildew resistance genes in sorghum.

The random amplified polymorphic DNA technique was used to find markers for a downy mildew resistance gene in sorghum. Of the 674 random primers screened for polymorphism, 2 amplified fragments were linked to a downy mildew resistance gene in sorghum line SC414. Utilization of an existing restriction fragment length polymorphism mapping population (IS3620C x BTx623) also revealed two markers that are linked to a different resistance gene in another sorghum line, BTx623.     

Construction of a high-density linkage map of Italian ryegrass (Lolium multiflorum Lam) using restriction fragment length polymorphism, amplified fragment length polymorphism, and telomeric repeat associated sequence markers.

To construct a high-density molecular linkage map of Italian ryegrass (Lolium multiflorum Lam), we used a two-way pseudo-testcross F1 population consisting of 82 individuals to analyze three types of markers: restriction fragment length polymorphism markers, which we detected by using genomic probes from Italian ryegrass as well as heterologous anchor probes from other species belonging to the Poaceae family, amplified fragment length polymorphism markers, which we detected by using PstI/MseI primer combinations, and telomeric repeat associated sequence markers. Of the restriction fragment length polymorphism probes that we generated from a PstI genomic library, 74% (239 of 323) of randomly selected probes detected hybridization patterns consistent with single-copy or low-copy genetic locus status in the screening. The 385 (mostly restriction fragment length polymorphism) markers that we selected from the 1226 original markers were grouped into seven linkage groups. The maps cover 1244.4 cM, with an average of 3.7 cM between markers. This information will prove useful for gene targeting, quantitative trait loci mapping, and marker-assisted selection in Italian ryegrass.     

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 reverse thermo-sensitive genic male-sterile gene (rtms1) in rice

TGMS (thermo-sensitive genic male-sterile) rice is widely used in hybrid rice production. Because of a specific temperature requirement, it can be used only in a narrow rice-growing zone in Asia. A newly discovered reverse thermo-sensitive genic male-sterile line, J207S, has an opposite phynotype compared to the normal TGMS lines. J207S is completely sterile when the temperature is lower than 31°C. Thus, it can be widely used in a larger area. Genetic analysis indicated that the sterility of J207S was controlled by a single recessive gene which was first named as rtms1. An F₂ population from the cross between J207S and E921 was developed and used for molecular mapping of the rtms1 gene. The AFLP (amplified fragment length polymorphism) technique, combined with BSA (bulked segregant analysis), was used to screen markers linked to the target gene, and eight polymorphic AFLP loci were identified. Co-segregating analysis using the F₂ population showed that two of them, Rev1 and Rev7, were closely linked to the target gene with a recombinant rate of 3.8% and 7.7%, respectively. Both Rev1 and Rev7 were found to be single-copy sequences through Southern analysis. Rev1 was subsequently mapped on chromosome 10 with a doubled-haploid mapping populations derived from the cross CT9993 × IR62266 available at Texas Tech University. RM222 and RG257 were linked to Rev1 at a distance of 11.8 cM and 4.6 cM, respectively. Additional SSR markers from the rice map of Cornell University, RFLP markers from the map of RGP in Japan and the map of Texas Tech University were selected from the region surrounding Rev1 on chromosome 10 to conduct the fine-mapping of the rtms1 gene. Presently, rtms1 was mapped between RM239 and RG257 with genetic distance of 3.6 cM and 4.0 cM, respectively. The most-closely linked AFLP marker, Rev1, 4.2 cM from the rtms1 gene, was sequenced and converted into a SCAR (sequence characterized amplified region) marker which could facilitate marker-assisted selection of the rtms1 gene. Received: 2 November 2000 / Accepted: 21 November 2000     

Primary genetic linkage maps of the ascidian, Ciona intestinalis

For whole-genome analysis in a basal chordate (protochordate), we used F1 pseudo-testcross mapping strategy and amplified fragment length polymorphism (AFLP) markers to construct primary linkage maps of the ascidian tunicate Ciona intestinalis. Two genetic maps consisted of 14 linkage groups, in agreement with the haploid chromosome number, and contained 276 and 125 AFLP loci derived from crosses between British and Neapolitan individuals. The two maps covered 4218.9 and 2086.9 cM, respectively, with an average marker interval of 16.1 and 18.9 cM. We observed a high recombinant ratio, ranging from 25 to 49 kb/cM, which can explain the high degree of polymorphism in this species. Some AFLP markers were converted to sequence tagged sites (STSs) by sequence determination, in order to create anchor markers for the fragmental physical map. Our recombination tools provide basic knowledge of genetic status and whole genome organization, and genetic markers to assist positional cloning in C. intestinalis.     

Mapping of avirulence genes in the rice blast fungus, Magnaporthe grisea, with RFLP and RAPD markers

Three genetically independent avirulence genes, AVR1-Irat7, AVRI-MedNoi; and AVR1-Ku86, were identified in a cross involving isolates Guy11 and 2/0/3 of the rice blast fungus, Magnaporthe grisea. Using 76 random progeny, we constructed a partial genetic map with restriction fragment length polymorphism (RFLP) markers revealed by probes such as the repeated sequences MGL/MGR583 and Pot3/MGR586, cosmids from the M. grisea genetic map, and a telomere sequence oligonucleotide. Avirulence genes AVR1-MedNoi and AVR1-Ku86 were closely linked to telomere RFLPs such as marker TelG (6 cM from AVR1-MedNoi) and TelF (4.5 cM from AVR1-Ku86). Avirulence gene AVR1-Irat7 was linked to a cosmid RFLP located on chromosome 1 and mapped at 20 cM from the avirulence gene AVR1-CO39. Using bulked segregant analysis, we identified 11 random amplified polymorphic DNA (RAPD) markers closely linked (0 to 10 cM) to the avirulence genes segregating in this cross. Most of these RAPD markers corresponded to junction fragments between known or new transposons and a single-copy sequence. Such junctions or the whole sequences of single-copy RAPD markers were frequently absent in one parental isolate. Single-copy sequences from RAPD markers tightly linked to avirulence genes will be used for positional cloning.     

An improved genetic linkage map for cowpea (Vigna unguiculata L.) combining AFLP, RFLP, RAPD, biochemical markers, and biological resistance traits.

An improved genetic linkage map has been constructed for cowpea (Vigna unguiculata L. Walp.) based on the segregation of various molecular markers and biological resistance traits in a population of 94 recombinant inbred lines (RILs) derived from the cross between 'IT84S-2049' and '524B'. A set of 242 molecular markers, mostly amplified fragment length polymorphism (AFLP), linked to 17 biological resistance traits, resistance genes, and resistance gene analogs (RGAs) were scored for segregation within the parental and recombinant inbred lines. These data were used in conjunction with the 181 random amplified polymorphic DNA (RAPD), restriction fragment length polymorphism (RFLP), AFLP, and biochemical markers previously mapped to construct an integrated linkage map for cowpea. The new genetic map of cowpea consists of 11 linkage groups (LGs) spanning a total of 2670 cM, with an average distance of 6.43 cM between markers. Astonishingly, a large, contiguous portion of LG1 that had been undetected in previous mapping work was discovered. This region, spanning about 580 cM, is composed entirely of AFLP markers (54 in total). In addition to the construction of a new map, molecular markers associated with various biological resistance and (or) tolerance traits, resistance genes, and RGAs were also placed on the map, including markers for resistance to Striga gesnerioides races 1 and 3, CPMV, CPSMV, B1CMV, SBMV, Fusarium wilt, and root-knot nematodes. These markers will be useful for the development of tools for marker-assisted selection in cowpea breeding, as well as for subsequent map-based cloning of the various resistance genes.     

Development of sequence-characterized amplified regions (SCARs) from amplified fragment length polymorphism (AFLP) markers tightly linked to the Vf gene in apple.

Amplified fragment length polymorphism (AFLP) markers have become widely used in saturating the region of a gene of interest for the ultimate goal of map-based cloning of the gene or for marker-assisted selection. However, conversion of AFLP markers into restriction fragment length polymorphism (RFLP) or polymerase chain reaction (PCR)-based markers will greatly expand their usefulness in genetic applications. Previously, we have identified 15 AFLP markers tightly linked to the Vf gene conferring scab resistance in apple. In this study, we have successfully converted 11 of these AFLPs into sequence-characterized amplified region (SCAR) markers. Of the remaining four nonconverted AFLP markers, one, ET2MC8-1, has been found to be very short (83 base pairs) and is an A/T rich (90%) marker; a second, EA2MG11-1, has shown identical sequences between Malus floribunda 821 (the original source of the Vf gene) and scab-susceptible apple cultivars; while the other two, EA12MG16-1 and ET8MG1-1, have not been cloned. Using the 11 converted SCAR markers along with 5 previously identified SCAR markers, a high-resolution linkage map around the Vf gene has been constructed, and found to be consistent with its corresponding AFLP map. Three converted SCAR markers (ACS-3, -7, and -9) are inseparable from the Vf gene; whereas one (ACS-6) is located left of, and the remaining seven (ACS-1, -2, -4, -5, -8, -10, and -11) are located right of the Vf gene at genetic distances of 0.4 and 0.2 cM, respectively. A reliable and robust procedure for development of SCAR markers from AFLP markers is presented.     

SCAR markers linked to the common bean rust resistance gene Ur-13

Rust in common bean (Phaseolus vulgaris L.) is caused by Uromyces appendiculatus Pers.:Pers. (Unger) which exhibits a high level of pathogenic diversity. Resistance to this disease is conditioned by a considerable number of genes. Pyramiding resistance genes is desirable and could be simplified by the use of molecular markers closely linked to the genes. The resistance gene Ur-13, present in the South African large seeded cultivar Kranskop, has been used extensively in the local breeding program. The purpose of this study was the development of a molecular marker linked to Ur-13. An F₂ population derived from a cross between Kranskop and a susceptible (South African) cultivar Bonus was used in combination with bulked segregant analysis utilizing the amplified fragment length polymorphism (AFLP) technique. Seven AFLP fragments linked significantly to the rust resistance and five were successfully converted to sequence characterized amplified region (SCAR) markers. The co-dominant SCAR markers derived from a 405 bp EAACMACC fragment, KB126, was located 1.6 cM from the gene. Two additional SCAR markers and one cleaved amplified polymorphic sequence marker were located further from the gene. The gene was mapped to linkage group B8 on the BAT 93/Jalo EEP 558 core map (chromosome 3).     

Fine mapping of the yellow seed locus in Brassica juncea L

The yellow mustard plant in Northern Shaanxi is a precious germplasm, and the yellow seed trait is controlled by a single recessive gene. In this report, amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) techniques were used to identify markers linked to the brown seed locus in an F(2) population consisting of 1258 plants. After screening 256 AFLP primer combinations and 456 pairs of SSR primers, we found 14 AFLP and 2 SSR markers that were closely linked to the brown seed locus. Among these markers, the SSR marker CB1022 showed codominant inheritance. By integrating markers previously found to be linked to the brown seed locus into the genetic map of the F(2) population, 23 markers were linked to the brown seed locus. The two closest markers, EA02MC08 and P03MC08, were located on either side of the brown seed locus at a distance of 0.3 and 0.5 cM, respectively. To use the markers for the breeding of yellow-seeded mustard plants, two AFLP markers (EA06MC11 and EA08MC13) were converted into sequence-characterized amplified region (SCAR) markers, SC1 and SC2, with the latter as the codominant marker. The two SSR markers were subsequently mapped to the A9/N9 linkage group of Brassica napus L. by comparing common SSR markers with the published genetic map of B. napus. A BLAST analysis indicated that the sequences of seven markers showed good colinearity with those of Arabidopsis chromosome 3 and that the homolog of the brown seed locus might exist between At3g14120 and At3g29615 on this same chromosome. To develop closer markers, we could make use of the sequence information of this region to design primers for future studies. Regardless, the close markers obtained in the present study will lay a solid foundation for cloning the yellow seed gene using a map-based cloning strategy.     

Identification and mapping of a leaf rust resistance gene in barley line Q21861.

Barley line Q21861 possesses an incompletely dominant gene (RphQ) for resistance to leaf rust caused by Puccinia hordei. To investigate the allelic and linkage relations between RphQ and other known Rph genes, F2 populations from crosses between Q21861 and donors of Rph1 to Rph14 (except for Rph8) were evaluated for leaf rust reaction at the seedling stage. Results indicate that RphQ is either allelic with or closely linked to the Rph2 locus. A doubled haploid population derived from a cross between Q21861 and SM89010 (a leaf rust susceptible line) was used for molecular mapping of the resistance locus. Bulked segregant analysis was used to identify markers linked to RphQ, using random amplified polymorphic DNAs (RAPDs), restriction fragment length polymorphisms (RFLPs), and sequence tagged sites (STSs). Of 600 decamer primers screened, amplified fragments generated by 9 primers were found to be linked to the RphQ locus; however, only 4 of them were within 10 cM of the target. The RphQ locus was mapped to the centromeric region of chromosome 7, with a linkage distance of 3.5 cM from the RFLP marker CDO749. Rrn2, an RFLP clone from the ribosomal RNA intergenic spacer region, was found to be very closely linked with RphQ, based on bulked segregant analysis. An STS marker, ITS1, derived from Rrn2, was also closely linked (1.6 cM) to RphQ.     

SSR and SCAR mapping of a multiple-allele male-sterile gene in Chinese cabbage (Brassica rapa L.)

The genic multiple-allele inherited male-sterile gene Ms in Chinese cabbage (Brassica rapa L.) was identified as a spontaneous mutation. Applying this gene to hybrid seed production, several B. rapa cultivars have been successfully bred in China. A BC₁ population (244 plants) was constructed for mapping the Ms gene. Screening 268 simple sequence repeat (SSR) markers which cover the entire genome of Chinese cabbage was performed with bulked segregant analysis (BSA). On the basis of linkage analysis, the Ms gene was located on linkage group R07. In addition, through the amplified fragment length polymorphism (AFLP) and the sequence-characterized amplified region (SCAR) techniques combining BSA, two SCAR markers which were converted from corresponding AFLP markers flanked the Ms gene. Finally, a genetic map of the Ms gene was constructed covering a total interval of 9.0 cM. Two SCAR markers, syau_scr01 and syau_scr04, flanked the Ms gene at distances of 0.8 and 2.5 cM, respectively. All the SSR markers (cnu_m273, cnu_m030, cnu_m295, and syau_m13) were mapped on the same side of the gene as syau_scr04, the nearest one of which, syau_m13, was mapped at a distance of 3.3 cM. These SSR and SCAR markers may be useful in marker-assisted selection and map-based cloning. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Map-based cloning in crop plants. Tomato as a model system: I. Genetic and physical mapping of jointless

A map-based cloning scheme is being used to isolate the jointless (j) gene of tomato. The jointless locus is defined by a single recessive mutation that completely suppresses the formation of the fruit and flower pedicel and peduncle abscission zone. jointless was mapped in an F₂ population of an interspecific cross between Lycopersicon esculentum and Lycopersicon pennellii to a 7.1 cM interval between two restriction fragment length polymorphism (RFLP) markers TG523 and TG194. Isogenic DNA pools were then constructed from a subset of the mapping population and screened with 800 random decamers for random amplification of polymorphic DNA (RAPD) polymorphisms. Five new RAPD markers were isolated and mapped to chromosome 11, two of which were mapped within the targeted interval. One marker, RPD158, was mapped 1.5 cM to the opposite side of jointless relative to TG523 and thus narrowed the interval between the closest flanking markers to 3.0 cM. Physical mapping by pulse-field gel electrophoresis using TG523 and RPD158 as probes demonstrated that both markers hybridize to a common 600 kb SmaI restriction fragment. This provided an estimate of 200 kb/cM for the relationship between physical and genetic distances in the region of chromosome 11 containing the j locus. The combined results provide evidence for the feasibility of the next step toward isolation of the jointless gene by map-based cloning — a chromosome walk or jump to jointless.