Physical mapping of the Vrn-A1 and Fr1 genes on chromosome 5A of wheat using deletion lines

 Homozygous deletion lines of wheat for 5AL, generated in the variety ‘Chinese Spring’, were tested for flowering time without vernalization and for frost resistance after cold hardening. It was found that the Vrn-A1 gene for vernalization requirement mapped between breakpoints 0.68 and 0.78, whilst the frost resistance gene Fr1 was flanked by deletion breakpoints 0.67 and 0.68. This confirms previous evidence that these genes are linked but are not the pleiotropic effect of a single gene. A comparison between the physical and genetic maps for Vrn-A1 and Fr1 shows that the linear order is identical. These results indicate that cytogenetically based physical maps of Vrn-A1 and Fr1 loci, together with genetic maps, could be useful in the further study of genome synteny and in elaborating a gene cloning strategy. Received: 16 November 1998 / Accepted: 28 November 1998     

A comparative genetic and cytogenetic mapping of wheat chromosome 5B using introgression lines

The genetic map of chromosome 5B has been constructed by using microsatellite (SSR) analysis of 381 plants from the F2 population produced by cross of the Chinese Spring (CS) and Renan cultivars. Initially, 180 SSR markers for the common wheat 5B chromosome have been used for analysis of these cultivars. The 32 markers able to detect polymorphism between these cultivars have been located on the genetic map of chromosome 5B. Cytogenetic mapping has involved a set of CS 5B chromosome deletion lines. Totally, 51 SSR markers have been located in ten regions (deletion bins) of this chromosome by SSR analysis of these deletion lines. Five genes—TaCBFIIIc-B10, Vrn-B1, Chi-B1, Skr, and Ph1—have been integrated into the cytogenetic map of chromosome 5B using the markers either specific of or tightly linked to the genes in question. Comparison of the genetic and cytogenetic maps suggests that recombination is suppressed in the pericentromeric region of chromosome 5B, especially in the short arm segment. The 18 markers localized to deletion bins 5BL16-0.79-1.00 and 5BL18-0.66-0.79 have been used to analyze common wheat introgression lines L842, L5366-180, L73/00i, and L21-4, carrying fragments of alien genomes in the terminal region of 5B long arm. L5366-180 and L842 lines carry a fragment of the Triticum timopheevii 5GL chromosome, while L73/00i and L21-4 lines, a fragment of the Aegilops speltoides 5SL chromosome. As has been shown, the translocated fragments in these four lines are of different lengths, allowing bin 5BL18-0.66-0.79 to be divided into three shorter regions. The utility of wheat introgression lines carrying alien translocations for increasing the resolution of cytogenetic mapping is discussed.     

Gamma irradiation induced deletions in an alien chromosome segment of the wheat 'Indis' and their use in gene mapping.

Deletion mutants were produced in a translocated chromosome segment derived from Thinopyrum distichum (Thunb.) L?ve. Spikes of the translocation line 'Indis' were irradiated with gamma rays at dosages of 15, 20, and 25 Gy. The irradiated spikes were pollinated with 'Inia 66' pollen and the F2 and F3 generations screened for translocation mutants, using the genes for leaf rust resistance and yellow endosperm pigmentation as markers. Finally, endopeptidase polymorphisms were utilized to select mutant translocation homozygotes within each of 29 families. An investigation of polymorphisms at the alpha-Amy-D2 and Wsp-D1 loci of chromosome arm 7DL revealed that 'Indis' did not produce an alpha-AMY-D2 product, but it did produce a novel WSP-D1 protein. The mutants were characterized for their leaf and stem rust resistances and the presence of WSP-D1 and yellow flour pigments. The stem rust resistance gene could not be accurately mapped. The linear order of the remaining loci on 7DL was centromere - leaf rust resistance - Wsp-D1 and yellow pigment. The data obtained suggested that the 'Indis' translocation has homo(eo)logy to the Lr19 translocation and homoeology to 7DL of common wheat.     

Molecular characterization of a set of wheat deletion stocks for use in chromosome bin mapping of ESTs

The objective of this study was molecular characterization of a set of deletion stocks and other aneuploids for use in chromosome bin mapping of ESTs in wheat. Wheat aneuploid stocks including 21 nullisomic-tetrasomic (NT), 24 ditelosomic (Dt), and 101 deletion (del) lines were screened with 526 EST clones. A total of 1,951 loci were detected by 493 informative EST clones and tagged 150 of the 159 deletion intervals or chromosome bins. Previously described deletion lines del1AS-4, del6AL-2, del6BS-6, and del7DS-6 were found to have normal chromosome constitution. The short arm deletion in del3AS-3 may be translocated from an unknown chromosome as this stock is nullisomic for the 3AS arm. Thirty-five new deletions were detected in 26 lines. Most of the new deletions occurred in terminal regions of chromosomes and probably resulted from the loss of very small terminal fragments that were difficult to detect cytologically. Eleven chromosome aberrations were also detected in two NT and five Dt lines. Overall, the chromosome bin map provides a resolution of around 28 Mb for an anchor map of a basic set of seven chromosomes of the Triticeae. Any target gene can be allocated to a specific 28-Mb bin and associated ESTs, anchored to the other Triticeae/grass maps including rice and, therefore, amenable to molecular cloning by comparative and wheat-based positional cloning methods. Electronic Publication     

Genetic and physical mapping of homoeologous recombination points involving wheat chromosome 2B and rye chromosome 2R.

Wide hybrids have been used in generating genetic maps of many plant species. In this study, genetic and physical mapping was performed on ph1b-induced recombinants of rye chromosome 2R in wheat (Triticum aestivum L.). All recombinants were single breakpoint translocations. Recombination 2RS-2BS was absent from the terminal and the pericentric regions and was distributed randomly along an intercalary segment covering approximately 65% of the arm's length. Such a distribution probably resulted from structural differences at the telomeres of 2RS and wheat 2BS arm that disrupted telomeric initiation of pairing. Recombination 2RL-2BL was confined to the terminal 25% of the arm's length. A genetic map of homoeologous recombination 2R-2B was generated using relative recombination frequencies and aligned with maps of chromosomes 2B and 2R based on homologous recombination. The alignment of the short arms showed a shift of homoeologous recombination toward the centromere. On the long arms, the distribution of homoeologous recombination was the same as that of homologous recombination in the distal halves of the maps, but the absence of multiple crossovers in homoeologous recombination eliminated the proximal half of the map. The results confirm that homoeologous recombination in wheat is based on single exchanges per arm, indicate that the distribution of these single homoeologous exchanges is similar to the distribution of the first (distal) crossovers in homologues, and suggest that successive crossovers in an arm generate specific portions of genetic maps. A difference in the distribution of recombination between the short and long arms indicates that the distal crossover localization in wheat is not dictated by a restricted distribution of DNA sequences capable of recombination but by the pattern of pairing initiation, and that can be affected by structural differences. Restriction of homoeologous recombination to single crossovers in the distal part of the genetic map complicates chromosome engineering efforts targeting genes in the proximal map regions.     

Rough and fine linkage mapping of the Rhizobium meliloti chromosome.

Summary A circular linkage map of the Rhizobium meliloti chromosome, obtained from R68.45-mediated crosses, has been revised by cotransductional analysis, after general transduction by DF2 phage.Three short chromosomal regions have been mapped by cotransduction. Comparison between conjugal and cotransductional data suggests that R68.45-mediated linkage measures are indeed rough. Cotransduction seems to be a useful tool for improving the linkage map of R. meliloti.     

Genetic mapping of a putative Thinopyrum intermedium-derived stripe rust resistance gene on wheat chromosome 1B

Key message

Stripe rust resistance transferred from Thinopyrum intermedium into common wheat was controlled by a single dominant gene, which mapped to chromosome 1B near Yr26 and was designated YrL693.

Abstract

Stripe rust caused by Puccinia striiformis f. sp. tritici (Pst) is a highly destructive disease of wheat (Triticum aestivum). Stripe rust resistance was transferred from Thinopyrum intermedium to common wheat, and the resulting introgression line (L693) exhibited all-stage resistance to the widely virulent and predominant Chinese pathotypes CYR32 and CYR33 and to the new virulent pathotype V26. There was no cytological evidence that L693 had alien chromosomal segments from Th. intermedium. Genetic analysis of stripe rust resistance was performed by crossing L693 with the susceptible line L661. F₁, F₂, and F_2:3 populations from reciprocal crosses showed that resistance was controlled by a single dominant gene. A total 479 F_2:3 lines and 781 pairs of genomic simple sequence repeat (SSR) primers were employed to determine the chromosomal location of the resistance gene. The gene was linked to six publicly available and three recently developed wheat genomic SSR markers. The linked markers were localized to wheat chromosome 1B using Chinese Spring nulli-tetrasomic lines, and the resistance gene was localized to chromosome 1B based on SSR and wheat genomic information. A high-density genetic map was also produced. The pedigree, molecular marker data, and resistance response indicated that the stripe rust resistance gene in L693 is a novel gene, which was temporarily designated YrL693. The SSR markers that co-segregate with this gene (Xbarc187-1B, Xbarc187-1B-1, Xgwm18-1B, and Xgwm11-1B) have potential application in marker-assisted breeding of wheat, and YrL693 will be useful for broadening the genetic basis of stripe rust resistance in wheat.     

A cytogenetically based physical map of chromosome 1B in common wheat.

We have constructed a cytogenetically based physical map of chromosome 1B in common wheat by utilizing a total of 18 homozygous deletion stocks. It was possible to divide chromosome 1B into 17 subregions. Nineteen genetic markers are physically mapped to nine subregions of chromosome 1B. Comparison of the cytological map of chromosome 1B with an RFLP-based genetic linkage map of Triticum tauschii revealed that the linear order of the genetic markers was maintained between chromosome 1B of hexaploid wheat and 1D of T. tauschii. Striking differences were observed between the physical and genetic maps in relation to the relative distances between the genetic markers. The genetic markers clustered in the middle of the genetic map were physically located in the distal regions of both arms of chromosome 1B. It is unclear whether the increased recombination in the distal regions of chromosome 1B is due to specific regions of increased recombination or a more broadly distributed increase in recombination in the distal regions of Triticeae chromosomes.     

Utilization of deletion bins to anchor and order sequences along the wheat 7B chromosome

Key message

A total of 3,671 sequence contigs and scaffolds were mapped to deletion bins on wheat chromosome 7B providing a foundation for developing high-resolution integrated physical map for this chromosome.

Abstract

Bread wheat (Triticum aestivum L.) has a large, complex and highly repetitive genome which is challenging to assemble into high quality pseudo-chromosomes. As part of the international effort to sequence the hexaploid bread wheat genome by the international wheat genome sequencing consortium (IWGSC) we are focused on assembling a reference sequence for chromosome 7B. The successful completion of the reference chromosome sequence is highly dependent on the integration of genetic and physical maps. To aid the integration of these two types of maps, we have constructed a high-density deletion bin map of chromosome 7B. Using the 270 K Nimblegen comparative genomic hybridization (CGH) array on a set of cv. Chinese spring deletion lines, a total of 3,671 sequence contigs and scaffolds (~7.8 % of chromosome 7B physical length) were mapped into nine deletion bins. Our method of genotyping deletions on chromosome 7B relied on a model-based clustering algorithm (Mclust) to accurately predict the presence or absence of a given genomic sequence in a deletion line. The bin mapping results were validated using three different approaches, viz. (a) PCR-based amplification of randomly selected bin mapped sequences (b) comparison with previously mapped ESTs and (c) comparison with a 7B genetic map developed in the present study. Validation of the bin mapping results suggested a high accuracy of the assignment of 7B sequence contigs and scaffolds to the 7B deletion bins.     

Breakpoint junctions of chromosome 9p deletions in two human glioma cell lines.

Interstitial deletions of the short arm of chromosome 9 are associated with glioma, acute lymphoblastic leukemia, melanoma, mesothelioma, lung cancer, and bladder cancer. The distal breakpoints of the deletions (in relation to the centromere) in 14 glioma and leukemia cell lines have been mapped within the 400 kb IFN gene cluster located at band 9p21. To obtain information about the mechanism of these deletions, we have isolated and analyzed the nucleotide sequences at the breakpoint junctions in two glioma-derived cell lines. The A1235 cell line has a complex rearrangement of chromosome 9, including a deletion and an inversion that results in two breakpoint junctions. Both breakpoints of the distal inversion junction occurred within AT-rich regions. In the A172 cell line, a tandem heptamer repeat was found on either side of the deletion breakpoint junction. The distal breakpoint occurred 5' of IFNA2; the 256 bp sequenced from the proximal side of the breakpoint revealed 95% homology to long interspersed nuclear elements. One- and two-base-pair overlaps were observed at these junctions. The possible role of sequence overlaps, and repetitive sequences, in the rearrangement is discussed.     

Origin, structure, and behavior of a highly rearranged deletion chromosome 1BS-4 in wheat.

Wheat (Triticum aestivum L.) deletion (del) stocks are valuable tools for the physical mapping of molecular markers and genes to chromosome bins delineated by 2 adjacent deletion breakpoints. The wheat deletion stocks were produced by using gametocidal genes derived from related Aegilops species. Here, we report on the origin, structure, and behavior of a highly rearranged chromosome 1BS-4. The cytogenetic and molecular marker analyses suggest that 1BS-4 resulted from 2 breakpoints in the 1BS arm and 1 breakpoint in the 1BL arm. The distal segment from 1BS, except for a small deleted part, is translocated to the long arm. Cytologically, chromosome 1BS-4 is highly stable, but shows a unique meiotic pairing behavior. The short arm of 1BS-4 fails to pair with a normal 1BS arm because of lack of homology at the distal ends. The long arm of 1BS-4 only pairs with a normal 1BS arm within the distal region translocated from 1BS. Therefore, using the 1BS-4 deletion stock for physical mapping will result in the false allocation of molecular markers and genes proximal to the breakpoint of 1BS-4.     

Positive selection of mutants with deletions of the gal-chl region of the Salmonella chromosome as a screening procedure for mutagens that cause deletions.

We have developed a convenient and specific positive selection for long deletions through the gal region of the chromosomes of Salmonella typhimurium and Escherichia coli. Through simultaneous selection for mutations in the two closely linked genes, gal and chlA, a variety of deletions of varying length, some extending through as much as 1 min of the chromosome, could be readily obtained. Many of these deletions resulted in the loss of a gene, which we named dhb, concerned with the ability of the bacterium to synthesize the iron chelating agent enterobactin. The selection was adapted for the screening of mutagens for their ability to generate long deletions in the bacterial deoxyribonucleic acid. Forty agents were screened for this capability. Nitrous acid, previously reported to be an efficient mutagen for this purpose, increased the frequency of deletion mutations 50-fold in our system. Three others, nitrogen mustard, mitomycin C, and fast neutrons, were shown to increase the frequency of long deletions between five- and eightfold. The remainder were found to be incapable of generating these deletions.     

Large-scale mapping and chromosome jumping in the q27 region of the human X chromosome

We have used pulsed field gel electrophoresis for further physical mapping studies in the q27 region of the human X chromosome. We show that the DXS 102 locus and the F9 gene are separated by only 300 kb despite a genetic distance of 1.4 cM; this linkage orients our large-scale map and shows that the mcf.2 transforming sequence is telomeric to F9. A BssHII complete-digest jumping library was used to jump toward the DXS 105 locus; a 130-kb jump was achieved and the corresponding "linking clone" was obtained.     

Multi-environment analysis and improved mapping of a yield-related QTL on chromosome 3B of wheat

Improved mapping, multi-environment quantitative trait loci (QTL) analysis and dissection of allelic effects were used to define a QTL associated with grain yield, thousand grain weight and early vigour on chromosome 3BL of bread wheat (Triticum aestivum L.) under abiotic stresses. The QTL had pleiotropic effects and showed QTL x environment interactions across 21 diverse environments in Australia and Mexico. The occurrence and the severity of water deficit combined with high temperatures during the growing season affected the responsiveness of this QTL, resulting in a reversal in the direction of allelic effects. The influence of this QTL can be substantial, with the allele from one parent (RAC875) increasing grain yield by up to 12.5 % (particularly in environments where both heat and drought stress occurred) and the allele from the other parent (Kukri) increasing grain yield by up to 9 % in favourable environments. With the application of additional markers and the genotyping of additional recombinant inbred lines, the genetic map in the QTL region was refined to provide a basis for future positional cloning.     

The effect of a deficiency and a deletion on recombination in chromosome 1BL in wheat

To test two models of chiasma allocation and the distribution of crossing-over in chromosomes, genetic mapping was performed in normal, deletion and deficiency chromosome arms 1BL of wheat, Triticum aestivum L. Shortening of the chromosome arm, either by a deletion of the proximal half of the arm or by a deficiency of the terminal quarter of the arm's length, significantly reduced the frequency of multiple crossovers but did not affect the distribution of the distal, presumably the first, crossover in the arm. In the deficiency chromosome, the recombination rate in the terminal segment was much higher than that in the same segment of the complete arm. This suggests that recombination frequency is not an inherent characteristic of a segment but depends on the segment's position on the centromere-telomere axis. These observations support the classical model of chiasma distribution along the chromosome based on the point of pairing initiation, chromosome length and the positive chiasma interference. The study also demonstrates that the distribution and frequency of recombination in a chromosome segment can be manipulated. Therefore, even the segments with very low recombination frequencies could be saturated with large numbers of crossover events to produce high-density genetic maps.     

Fine mapping of a preharvest sprouting QTL interval on chromosome 2B in white wheat

Key message

Fine mapping by recombinant backcross populations revealed that a preharvest sprouting QTL on 2B contained two QTLs linked in coupling with different effects on the phenotype.

Abstract

Wheat preharvest sprouting (PHS) occurs when grain germinates on the plant before harvest, resulting in reduced grain quality. Previous mapping of quantitative trait locus (QTL) revealed a major PHS QTL, QPhs.cnl-2B.1, located on chromosome 2B significant in 16 environments that explained from 5 to 31 % of the phenotypic variation. The objective of this project was to fine map the QPhs.cnl-2B.1 interval. Fine mapping was carried out in recombinant backcross populations (BC₁F₄ and BC₁F₅) that were developed by backcrossing selected doubled haploids to a recurrent parent and self-pollinating the BC₁F₄ and BC₁F₅ generations. In each generation, three markers in the QPhs.cnl-2B.1 interval were used to screen for recombinants. Fine mapping revealed that the QPhs.cnl-2B.1 interval contained two PHS QTLs linked in coupling. The distal PHS QTL, located between Wmc453c and Barc55, contributed 8 % of the phenotypic variation and also co-located with a major seed dormancy QTL determined by germination index. The proximal PHS QTL, between Wmc474 and CNL415-rCDPK, contributed 16 % of the variation. Several candidate genes including Mg-chelatase H subunit family protein, GTP-binding protein and calmodulin/Ca²⁺-dependent protein kinase were linked to the PHS QTL. Although many recombinant lines were identified, the lack of polymorphism for markers in the QTL interval prevented the localization of the recombination breakpoints and identification of the gene underlying the phenotype.