RFLP markers linked to powdery mildew resistance genes Pm1, Pm2, Pm3, and Pm4 in wheat.

Near-isogenic lines (NILs) and their recurrent parent Chancellor (Cc) were used to identify restriction fragment length polymorphic markers linked to powdery mildew (Blumeria graminis (DC.) E.O. Speer f.sp. tritici) resistance genes Pm1, Pm2, Pm3, and Pm4 in wheat (Triticum aestivum L. em. Thell). By mapping these polymorphic markers in F2 progenies from crosses of the NILs with Cc, it was found that Pm1 cosegregated with a polymorphic locus detected by DNA probe CDO347; Pm2 was linked to a locus detected by probe BCD1871 with a distance of 3.5 cM; Pm3b was linked to a locus detected by probe BCD1434 with a distance of 1.3 cM; Pm4a cosegregated with Xbcd1231-2A(2) and Xcdo678-2A, and was closely flanked by Xbcd1231-2A(1) and Xbcd292-2A both with a distance of 1.5 cM. Aneuploid mapping of these markers indicated that locus Xcdo347-7A is on 7AL, Xbcd1871-5D on 5DS, Xbcd1434-1A on 1AS, and loci Xbcd292-2A and Xcdo678-2A are on 2AL. The same polymorphic fragments detected in the Pm3b NIL by Xbcd1434-1A were found in Pm3a NIL using several enzyme digestions.     

Physical mapping of the barley stem rust resistance gene rpg4

The barley stem rust resistance gene rpg4 was physically and genetically localized on two overlapping BAC clones covering an estimated 300-kb region of the long arm of barley chromosome 7(5H). Initially, our target was mapped within a 6.0-cM region between the previously described flanking markers MWG740 and ABG391. This region was then saturated by integrating new markers from several existing barley and rice maps and by using BAC libraries of barley cv. Morex and rice cv. Nipponbare. Physical/genetic distances in the vicinity of rpg4 were found to be 1.0 Mb/cM, which is lower than the average for barley (4 Mb/cM) and lower than that determined by translocation breakpoint mapping (1.8 Mb/cM). Synteny at high resolution levels has been established between the region of barley chromosome 7(5H) containing the rpg4 locus and the subtelomeric region of rice chromosome 3 between markers S16474 and E10757. This 1.7-cM segment of the rice genome was covered by two overlapping BAC clones, about 250 kb of total length. In barley the markers S16474 and E10757 genetically delimit rpg4, lying 0.6 cM distal and 0.4 cM proximal to the locus, respectively.     

Progress towards elucidating the mechanisms of self-incompatibility in the grasses: further insights from studies in Lolium

BACKGROUND AND SCOPE: Self-incompatibility (SI) in flowering plants ensures the maintenance of genetic diversity by ensuring outbreeding. Different genetic and mechanistic systems of SI among flowering plants suggest either multiple origins of SI or considerable evolutionary diversification. In the grasses, SI is based on two loci, S and Z, which are both polyallelic: an incompatible reaction occurs only if both S and Z alleles are matched in individual pollen with alleles of the pistil on which they alight. Such incompatibility is referred to as gametophytic SI (GSI). The mechanics of grass GSI is poorly understood relative to the well-characterized S-RNase-based single-locus GSI systems (Solanaceae, Rosaceae, Plantaginaceae), or the Papaver recognition system that triggers a calcium-dependent signalling network culminating in programmed cell death. There is every reason to suggest that the grass SI system represents yet another mechanism of SI. S and Z loci have been mapped using isozymes to linkage groups C1 and C2 of the Triticeae consensus maps in Secale, Phalaris and Lolium. Recently, in Lolium perenne, in order to finely map and identify S and Z, more closely spaced markers have been developed based on cDNA and repeat DNA sequences, in part from genomic regions syntenic between the grasses. Several genes tightly linked to the S and Z loci were identified, but so far no convincing candidate has emerged. RESEARCH AND PROGRESS: From subtracted Lolium immature stigma cDNA libraries derived from S and Z genotyped individuals enriched for SI potential component genes, kinase enzyme domains, a calmodulin-dependent kinase and a peptide with several calcium (Ca(2+)) binding domains were identified. Preliminary findings suggest that Ca(2+) signalling and phosphorylation may be involved in Lolium GSI. This is supported by the inhibition of Lolium SI by Ca(2+) channel blockers lanthanum (La(3+)) and verapamil, and by findings of increased phosphorylation activity during an SI response.     

Genetic Analysis and Molecular Mapping of a Rolling Leaf Mutation Gene in Rice

A rice mutant with rolling leaf, namely γ-rl, was obtained from M2 progenies of a native indica rice stable strain Qinghuazhan （QHZ） from mutagenesis of dry seeds by γ-rays. Genetic analysis using the F2 population from a cross between this mutant and QHZ indicated the mutation was controlled by a single recessive gene. In order to map the locus for this mutation, another F2 population with 601 rolling leaf plants was constructed from a cross between y-rl and a japonica cultivar 02428. After primary mapping with SSR （simple sequence repeats） markers, the mutated locus was located at the short arm of chromosome 3, flanked by RM6829 and RM3126. A number of SSR, InDel （insertion/deletion） and SNP （single nucleotide polymorphism） markers within this region were further developed for fine mapping. Finally, two markers, SNP121679 and InDe1422395, were identified to be flanked to this locus with genetic distances of 0.08 cM and 0.17 cM respectively, and two SNP markers, SNP75346 and SNPl10263, were found to be co-segregated with this locus. These results suggested that this locus was distinguished from all loci for the rolling leaf mutation in rice reported so far, and thus renamed rl10（t）. By searching the rice genome database with closely linked markers using BLAST programs, an e-physical map covering rl10（t） locus spanning about a 50 kb region was constructed. Expression analysis of the genes predicted in this region showed that a gene encoding putative flavin-containing monooxygenase （FMO） was silenced in γ-rl, thus this is the most likely candidate responsible for the rolling leaf mutation.     

Fine mapping and SNP analysis of positional candidates at the preeclampsia susceptibility locus (PREG1) on chromosome 2

Genome scans in Icelandic, Australian and New Zealand, and Finnish families have localized putative susceptibility loci for preeclampsia/ eclampsia to chromosome 2. The locus mapped in the Australian and New Zealand study (designated PREG1) was thought to be the same locus as that identified in the Icelandic study. In both these studies, two distinct quantitative trait locus (QTL) regions were evident on chromosome 2. Here, we describe our fine mapping of the PREG1 locus and a genetic analysis of two positional candidate genes. Twenty-five additional microsatellite markers were genotyped within the 74-cM linkage region defined by the combined Icelandic and Australian and New Zealand genome scans. The overall position and shape of the localization evidence obtained using nonparametric multipoint analysis did not change from that seen previously in our 10-cM resolution genome scan; two peaks were displayed, one on chromosome 2p at marker D2S388 (107.46 cM) and the other on chromosome 2q at 151.5 cM at marker D2S2313. Using the robust two-point linkage analysis implemented in the Analyze program, all 25 markers gave positive LOD scores with significant evidence of linkage being seen at marker D2S2313 (151.5 cM), achieving a LOD score of 3.37 under a strict diagnostic model. Suggestive evidence of linkage was seen at marker D2S388 (107.46 cM) with a LOD score of 2.22 under the general diagnostic model. Two candidate genes beneath the peak on chromosome 2p were selected for further analysis using public single nucleotide polymorphisms (SNPs) within these genes. Maximum LOD scores were obtained for an SNP in TACR1 (LOD = 3.5) and for an SNP in TCF7L1 (LOD = 3.33), both achieving genome-wide significance. However, no evidence of association was seen with any of the markers tested. These data strongly support the presence of a susceptibility gene on chromosome 2p11-12 and substantiate the possibility of a second locus on chromosome 2q23.     

Identification of RAPD markers linked to a major blast resistance gene in rice

Rice blast, caused byPyricularia grisea, is a major production constraint in many parts of the world. The Korean rice variety Tongil showed high levels of resistance for about six years when widely planted under highly disease-conducive conditions, before becoming susceptible. Tongil was found to carry a single dominant gene, designatedPi-10t, conferring resistance to isolate 106 of the blast pathogen from the Philippines. We report here the use of bulked segregant RAPD analysis for rapid identification of DNA markers linked toPi-10t. Pooled DNA extracts from five homozygous blast-resistant (RR) and five susceptible (rr) BC₃F₂ plants, derived from a CO39 × Tongil cross, were analyzed by RFLP using 83 polymorphic probes and by RAPD using 468 random oligomers. We identified two RAPD markers linked to thePi-10t locus: RRF6 (3.8 ± 1.2 cM) and RRH18 (2.9 ± 0.9 cM). Linkage of these markers withPi-10t was verified using an F2 population segregating forPi-10t. The two linked RAPD markers mapped 7 cM apart on chromosome 5. Chromosomal regions surrounding thePi-10t gene were examined with additional RFLP markers to define the segment introgressed from the donor genome.Pi-10t is likely to be a new blast-resistance locus, because no other known resistance gene has been mapped on chromosome 5. These tightly linked RAPD markers could facilitate early selection of thePi-10t locus in rice breeding programmes.     

RFLP mapping of the genes controlling hybrid breakdown in rice (Oryza sativa L.)

 Complementary recessive genes hwd1 and hwd2 controlling hybrid breakdown (weakness of F₂ and later generations) were mapped in rice using RFLP markers. These genes produce a plant that is shorter and has fewer tillers than normal plants when the two loci have only one or no dominant allele at both loci. A cultivar with two dominant alleles at the hwd1 locus and a cultivar with two dominant alleles at the hwd2 locus were crossed with a double recessive tester line. Linkage analysis was carried out for each gene independently in two F₂ populations derived from these crosses. hwd1 was mapped on the distal region of rice genetic linkage map for chromosome 10, flanked by RFLP markers C701 and R2309 at a distance of 0.9 centiMorgans (cM) and 0.6 cM, respectively. hwd2 was mapped in the central region of rice genetic linkage map for chromosome 7, tightly linked with 4 RFLP markers without detectable recombination. The usefulness of RFLP mapping and map information for the genes controlling reproductive barriers are discussed in the context of breeding using diverse rice germplasm, especially gene introduction by marker-aided selection.     

矮泰引-3中半矮秆基因的分子定位

矮泰引-3的矮生性状受两对独立遗传的半矮秆基因控制,利用SSR标记将这两个矮秆基因分别定位到第1和第4染色体上。等位性测交的结果表明,位于第1染色体上的矮秆基因与sd1是等位的,所以仍然称其为sd1;而位于第4染色体上的矮秆基因是一个新基因,暂命名为sdt2。利用SSR标记将sd1定位于RM297、RM302和RM212的同一侧,而与OSR3共分离,它们之间的位置关系可能是RM297-RM302-RM212-OSR3-sd1,遗传距离分别为4．7cM、0cM、0．8cM和0cM,这与sd1在第1染色体长臂上的确切位置是基本一致的。利用已有的SSR标记和拓展的SSR标记将sdt2定位于SSR332、RM1305和RM5633、RM307、RM401之间,它们的排列位置可能是SSR332-RM1305-sdt2-RM5633-RM307-RM401,它们之间的遗传距离分别为11．6cM、3．8cM、0．4cM、0cM和0．4cM。     

Genetic and physical mapping of a rice blast resistance locus,Pi-CO39(t), that corresponds to the avirulence gene AVR1-CO39 of Magnaporthe grisea

We have identified, genetically mapped and physically delineated the chromosomal location of a new rice blast resistance locus, designated Pi-CO39(t). This locus confers resistance to Magnaporthe grisea isolates carrying the AVR1-CO39 avirulence locus. The AVR1-CO39 locus is conserved in non-rice (cereals and grasses)-infecting isolates of M. grisea, making Pi-CO39(t) useful for engineering M. grisea resistance in rice and other cereals. The resistance in the rice line CO39 was inherited as a single dominant locus in segregating populations derived from F(2) and F(3) crosses between disease-resistant (CO39) and susceptible (51583) rice genotypes. Microsatellite, RFLP and resistance gene analog (RGA) markers were used to map the Pi-CO39(t) locus to a 1.2-cM interval between the probenazole-responsive ( RPR1) gene (0.2 cM) and RFLP marker S2712 (1.0 cM) on the short arm of rice chromosome 11. RFLP markers G320 and F5003, and resistance gene analogs RGA8, RGA38 and RGACO39 were tightly linked to the Pi-CO39(t) locus (no recombination detected in a sample of ~2400 gametes). A large-insert genomic library of CO39 was constructed in the binary plant transformation vector pCLD04541. A library screen using RGA8, RGA38 and probes derived from the ends of CO39 clones, as well as BAC end probes from the corresponding locus in the rice cv. Nipponbare, resulted in the assembly of three CO39 contigs of 180 kb, 110 kb and 145 kb linked to the Pi-CO39(t) locus. A 650-kb contig was also constructed representing the susceptible locus, pi-CO39(t), in the Nipponbare genome. The two genomes are highly divergent with respect to additions, deletions and translocations at the Pi-CO39(t) locus, as revealed by the presence or absence of mapping markers.     

The scurs locus in cattle maps to bovine chromosome 19

Polled, or the absence of horns, is a desirable trait for many cattle breeders. However, the presence of scurs, which are small horn-like structures that are not attached to the skull, can lower the value of an animal. The scurs trait has been reported as sex influenced. Using a genome scan with 162 autosomal microsatellite markers genotyped across three full-sib families, the scurs locus was mapped near BMS2142 on cattle chromosome 19 (LOD = 4.21). To more precisely map scurs, the families from the initial analysis and three additional families were genotyped for 16 microsatellite markers and SNPs in three genes on chromosome 19. In this subsequent analysis, the scurs locus was mapped 4 cM distal of BMS2142 (LOD = 4.46) and 6 cM proximal to IDVGA46 (LOD = 2.56). ALOX12 and MFAP4 were the closest genes proximal and distal, respectively, to the scurs locus. Three microsatellite markers on the X chromosome were genotyped across these six families but were not linked to scurs, further demonstrating that this trait was not sex linked. Because the polled locus has been mapped to the centromeric end of chromosome 1 and scurs has now been mapped to chromosome 19, these two traits are not linked in Bos taurus.     

Regional mapping of the Batten disease locus (CLN3) to human chromosome 16p12

The gene for Batten disease (CLN3) has been mapped to human chromosome 16 by demonstration of linkage to the haptoglobin locus, and its localization has been further refined using a panel of DNA markers. The aim of this work was to refine the genetic and physical mapping of this disease locus. Genetic linkage analysis was carried out in a larger group of families by using markers for five linked loci. Multipoint analysis indicated a most likely location for CLN3 in the interval between D16S67 and D16S148 (Z = 12.5). Physical mapping of linked markers was carried out using somatic cell hybrid analysis and in situ hybridization. A mouse/human hybrid cell panel containing various segments of chromosome 16 has been constructed. The relative order and physical location of breakpoints in the proximal portion of 16p were determined. Physical mapping in this panel of the markers for the loci flanking CLN3 positioned them to the bands 16p12.1----16p12.3. Fluorescent in situ hybridization of metaphase chromosomes by using these markers positioned them to the region 16p11.2-16p12.1. These results localize CLN3 to an interval of about 2 cM in the region 16p12.     

A map of barley chromosome 2 using isozymic and morphological markers

The F₂ progeny of a cross between a chromosome 2 multiple marker stock and an adapted cultivar of barley were analyzed for four morphological markers and electrophoretic patterns of eight leaf isozymes. TheIdh-2 locus was linked to thePer-5 locus (27.96±5.07 cM) and to thee locus (10.26±3.13 cM). Also, thePer-5 ande loci were located on the short arm of chromosome 2. In additionIdh-2 was also located on barley chromosome 2 and was linked to thev locus (13.18±3.56 cM), which is located on the long arm of chromosome 2. Two other marker genes,li andwst,,B, were linked (26.50±5.24 cM) on chromosome 2 but segregate independently of the other loci evaluated. This project was supported by funds from the U.S.-Spain Joint Committee for Scientific and Technological Cooperation.     

Molecular mapping of two semidwarf genes in an indica rice variety Aitaiyin3 (Oryza sativa L.)

Genetic analysis established that Aitaiyin3,a dwarf rice variety derived from a semidwarf cultivar Taiyin1,carries two recessive semidwarf genes.By using simple sequence repeat(SSR)markers,we mapped the two semidwarf genes,sd-1 and sd-t2 on chromosomes 1 and 4,respectively.Sd-t2 was thus named because the semidrawf gene sd-t has already been identified from Aitaiyin 2 whose origin could be traced back to Taivin1.The result of the molecular mappingof sd-1 gene revealed it is linked to four SSR markers found on chromosome 1.These markers are:RM297,RM302,RM212,and OSR3 spaced at 4.7 cM,0 cM,0.8cM and 0 cM,respectively.Sd-t2 was found to be located on chromosome 4 using five SSR markers:two markers,SSR332 and RM1305 located proximal to sd-t2 are spaced 11.6 cM,3.8 cM,respectively,while the three distally located primers,RM5633,RM307,and RM401 are separated by distances of 0.4 cM,0.0 cM,and 0.4 cM,respectively.     

RFLP mapping of loci controlling self-incompatibility in <Emphasis Type="Italic">Brassica campestris</Emphasis> and their comparative mapping with <Emphasis Type="Italic">B. napus</Emphasis> and <Emphasis Type="Italic">B. oleracea</Emphasis>

RFLP analysis of a cDNA probe SLG6, governing self incompatibility (SI) in Brassica oleracea, using a recombinant inbred population of Brassica campestris followed by genetic linkage analysis led to the detection of two marker loci, SLG6a and SLG6b controlling SI. SLG6a was mapped in linkage group (LG) 9 and was flanked by the RFLP markers ec4f10 (6.4 cM) and wg5b9 (4.2 cM). SLG6b positioned in LG 2 and was flanked by the RFLP markers wg2d11 (9.9 cM) and ec4e7 (26.9 cM). These results indicated the scope of marker-aided introgression of these genes into self-compatible genotypes for production of SI lines suitable for hybridization in B. campestris. Comparative mapping of LG 9 containing SLG6b with corresponding linkage groups of B. oleracea (BO 2) and B. napus (BN 16) led to the detection of small homologous regions with SLG6 locus linked with another RFLP locus. This evidenced for homology of the SLG genes across Brassica species and possibility of using any single cloned SLG gene for development of SI lines in any Brassica species.     

Genetic analysis and molecular mapping of a novel recessive gene xa34(t) for resistance against Xanthomonas oryzae pv. oryzae

A new bacterial blight recessive resistance gene xa34(t) was identified from the descendant of somatic hybridization between an aus rice cultivar (cv.) BG1222 and susceptible cv. IR24 against Chinese race V (isolate 5226). The isolate was used to test the resistance or susceptibility of F₁ progenies and reciprocal crosses of the parents. The results showed that F₁ progenies appeared susceptibility there were 128R (resistant):378S (susceptible) and 119R:375S plants in F₂ populations derived from two crosses of BG1222/IR24 and IR24/BG1222, respectively, which both calculates into a 1R:3S ratio. 320 pairs of stochastically selected SSR primers were used for genes?? initial mapping. The screened results showed that two SSR markers, RM493 and RM446, found on rice chromosome 1 linked to xa34(t). Linkage analysis showed that these two markers were on both sides of xa34(t) with the genetic distances 4.29 and 3.05?cM, respectively. The other 50 SSR markers in this region were used for genes?? fine mapping. The further results indicated that xa34(t) was mapped to a 1.42?cM genetic region between RM10927 and RM10591. In order to further narrow down the genomic region of xa34(t), 43 of insertion/deletion (Indel) markers (BGID1-43) were designed according to the sequences comparison between japonica and indica rice. Parents?? polymorphic detection and linkage assay showed that the Indel marker BGID25 came closer to the target gene with a 0.4?cM genetic distance. A contig map corresponding to the locus was constructed based on the reference sequences aligned by the xa34(t) linked markers. Consequently, the locus of xa34(t) was defined to a 204?kb interval flanked by markers RM10929 and BGID25.     

Construction of a 1.2-Mb contig including the citrus tristeza virus resistance gene locus using a bacterial artificial chromosome library of Poncirus trifoliata (L.) Raf.

The citrus tristeza virus resistance gene (Ctv) is a single dominant gene in Poncirus trifoliata, a sexually compatible relative of citrus. To clone this gene, a bacterial artificial chromosome (BAC) library has been constructed from an individual plant that was homozygous for Ctv. This library contains 45,696 clones with an average insert size of 80 kb, corresponding to 9.6 genome equivalents. Screening of the BAC library with five chloroplast DNA probes indicated that 0.58% of the BAC clones contained chloroplast-derived inserts. The chromosome walk across the Ctv locus was initiated using three closely linked genetic markers: C19, AD8, and Z16. The walk has been completed and a contig of ca. 1.2 Mb was constructed. Based on new data, the genetic map in the Ctv region was revised, with Ctv being located between AD8-Z16 and C19 at distances of 1.2 and 0.6 cM, respectively. Utilizing DNA fragments isolated from the contig as RFLP markers, the Ctv locus was further mapped to a region of ca. 300 kb. This contig contains several putative disease-resistance genes similar to the rice Xa21 gene, the tomato Cf-2 gene, and the Arabidopsis thaliana RPS2 gene. This library will therefore allow cloning of Ctv and other putative disease-resistance genes.     

Genetic mapping and molecular characterization of the self-incompatibility (S) locus in Petunia inflata

Gametophytic self-incompatibility (SI) possessed by the Solanaceae is controlled by a highly polymorphic locus called the S locus. The S locus contains two linked genes, S-RNase, which determines female specificity, and the as yet unidentified pollen S gene, which determines male specificity in SI interactions. To identify the pollen S gene of Petunia inflata, we had previously used mRNA differential display and subtractive hybridization to identify 13 pollen-expressed genes that showed S -haplotype-specific RFLP. Here, we carried out recombination analysis of 1205 F2 plants to determine the genetic distance between each of these S -linked genes and S-RNase. Recombination was observed between four of the genes (3.16, G211, G212, and G221) and S-RNase, whereas no recombination was observed for the other nine genes (3.2, 3.15, A113, A134, A181, A301, G261, X9, and X11). A genetic map of the S locus was constructed, with 3.16 and G221 delimiting the outer limits. None of the observed crossovers disrupted SI, suggesting that all the genes required for SI are contained in the chromosomal region defined by 3.16 and G221. These results and our preliminary chromosome walking results suggest that the S locus is a huge multi-gene complex. Allelic sequence diversity of G221 and 3.16, as well as of 3.2, 3.15, A113, A134 and G261, was determined by comparing two or three alleles of their cDNA and/or genomic sequences. In contrast to S-RNase, all these genes showed very low degrees of allelic sequence diversity in the coding regions, introns, and flanking regions.     

First-generation linkage map of the warningly colored butterfly Heliconius erato

We report the first genetic linkage map of Heliconius erato, a species that shows remarkable variation in its warningly colored wing patterns. We use crosses between H. erato and its sister species, H. himera, to place two major color pattern genes, D and Cr, on a linkage map containing AFLP, allozyme, microsatellite and single-copy nuclear loci. We identified all 21 linkage groups in an initial genetic screen of 22 progeny from an F1 female x male H. himera family. Of the 229 markers, 87 used to identify linkage groups were also informative in 35 progeny from a sibling backcross (H. himera female x F1 male). With these, and an additional 33 markers informative in the second family, we constructed recombinational maps for 19 of the 21 linkage groups. These maps varied in length from 18.1 to 431.1 centimorgans (cM) and yielded an estimated total length of 2400 cM. The average distance between markers was 23 cM, and eight of the 19 linkage groups, including the sex chromosome (Z) and the chromosome containing the Cr locus, contained two or more codominant anchor loci. Of the three potential candidate genes mapped here, Cubitus interruptus (Ci), Decapentaplegic (Dpp) and Wingless (Wg), only Ci was linked, although loosely, to a known Heliconius color pattern locus. This work is an important first step for constructing a denser genetic map of the H. erato color pattern radiation and for a comparative genomic study of the architecture of mimicry in Heliconius butterflies.     

The cream dilution gene, responsible for the palomino and buckskin coat colours, maps to horse chromosome 21

The colour locus historically referred to as C in the horse is linked to microsatellites markers on horse chromosome 21. Preliminary results demonstrated linkage of Ccr, thought to be the cream dilution variant of the C locus, to HTG10. An analysis of horse chromosome 21 using additional families confirmed and established a group of markers linked to Ccr. This work also improved the resolution of previously reported linkage maps for this chromosome. Linkage analysis unambiguously produced the map order: SGCV16-(19.1 cM)-HTG10-(3.8 cM)-LEX60/COR73-(1.3 cM)-COR68-(4.5 cM)- Ccr-(11.9 cM)-LEX31. Comparative and synteny data suggested that the horse C locus is not tyrosinase (TYR).