The Ha locus of wheat: identification of a polymorphic region for tracing grain hardness in crosses.

The grain softness proteins or friabilins are known to be composed of three main components: puroindoline a, puroindoline b, and GSP-1. cDNAs for GSP-1 have previously been mapped to group-5 chromosomes and their location on chromosome 5D is closely linked to the grain hardness (Ha) locus of hexaploid wheat. A genomic DNA clone containing the GSP-1 gene (wGSP1-A1) from hexaploid wheat has been identified by fluorescent in situ hybridization as having originated from the distal end of the short arm of chromosome 5A. A genomic clone containing the gene (wGSP1-D1) was also isolated from Aegilops tauschii, the donor of the D genome to bread wheat. There are no introns in the GSP-1 genes, and there is high sequence identity between wGSP1-A1 and wGSP1-D1 up to 1 kb 5' and 300 bp 3' to wGSP1-D1. However, regions further upstream and downstream of wGSP1-D1 share no significant sequence identity to corresponding sequences in wGSP1-A1. These regions therefore identified potentially valuable sequences for tracing the Ha locus through assaying polymorphic DNA sequences. The sequence from 300 to 500 bp 3' to wGSP1-D1 (wGSP1-D13) was mapped to the Ha locus in a mapping population. wGSP1-D13 was also tightly linked to genes for puroindoline a and puroindoline b which have been previously mapped to be at the Ha locus. In addition wGSP1-D13 was used to detect RFLPs between near isogenic soft and hard Falcon lines and in a random selection of soft and hard wheats.     

Mapping QTLs for grain hardness and puroindoline content in wheat ( Triticum aestivum L.)

Genes for puroindoline-a (Pin-a), puroindoline-b (Pin-b) and grain-softness proteins (GSP) have been shown to be linked to the dominant Ha locus responsible for the soft texture of the grain. Though linkage has been demonstrated of the puroindoline genes to the Ha locus, there is no clear evidence that puroindoline content is the product of the gene Ha. A segregating population of 115 recombinant inbred lines (RILs) originating from a cross between the hexaploid Synthetic wheat ( Triticum durum x Aegilops tauschii, W 7984) and the cultivar 'Opata' (M 85) was studied in two different experimental years to detect Quantitative Trait Loci (QTLs) for three traits: grain hardness (Hard), puroindoline-a (Pin-a) and puroindoline-b (Pin-b) contents. The detection of QTLs was performed using marker linear regression. Negative correlation coefficients (-0.86 and -0.80) were identified between grain hardness and puroindoline content (a and b, respectively) on data obtained in 1996. Results obtained in 1999 confirmed the negative correlation between Hard and Pin-a (-0.73); however a positive correlation coefficient was found with Pin-b content (0.41). Total phenotypic variation explained by each QTL was calculated (R2). For each of the Hard, Pin-a and Pin-b traits one major QTL was detected on the short arm of chromosome 5D, located close to the mta9 allele (puroindoline-a). For the first year (1996) the QTL in this region explained around 63% of the phenotypic variability in grain hardness, 77% in Pin-a and 45% in Pin-b contents. These values were confirmed in trials carried out in 1999 with a R2 value of 0.71, 0.72 and 0.25 for Hard, Pin-a and Pin-b, respectively. In 1996 and 1999 a second major QTL was detected for grain hardness on the long arm of the same chromosome. Present results indicate that it cannot be definitely concluded that puroindoline content represents a linear explanation for variations in grain hardness.     

Genome organisation and retrotransposon driven molecular evolution of the endosperm Hardness (Ha) locus in Triticum aestivum cv Glenlea

Wheat endosperm texture is controlled primarily by a locus (Ha), which comprises Gsp-1, Pina and Pinb genes encoding the so-called grain softness protein, puroindoline-a and puroindoline-b, respectively. Pina and Pinb were detected only on the D-genome of hexaploid wheat and its diploid progenitors while Gsp-1 was on all three homoeologous loci. Hexaploid cultivar Glenlea has a hard phenotype due to a null Pina genotype (D-genome) but the sequence organization is not reported. This study aimed at understanding the evolution of homoeologous Ha loci. Sequencing of three BAC clones from cv Glenlea was performed and sequence analyses delimited the Ha loci which spanned 3,925, 5,330 and 31,607 bp in the A-, B- and D-genomes, respectively. A solo LTR of Angela retroelement, downstream to Gsp-A1 and a fragment of Sabrina retroelement, downstream of Gsp-B1, were discovered. We propose that the insertion of these elements into the intergenic regions have driven the deletions of genomic segments harbouring Pina and Pinb genes in the A- and B-genomes of hexaploid wheat. Similarly, fragments of Romani and Vagabond retroelements were identified between truncated Pina and Pinb genes, indicating their role in the deletion of Pina in Glenlea, leading to its hard texture. Structural differences of the Ha locus region of the A-genome between two hexaploid wheat varieties namely Glenlea and Renan (CR626929), suggested the presence of more than one tetraploid ancestor in the origin of hexaploid wheat.     

Transfer of grain softness from 5U-5A wheat-Aegilops triuncialis substitution line to bread wheat through induced homeologous pairing

Journal of Plant Biochemistry and Biotechnology - Bread wheat sustains genes for grain softness on “Ha” locus of short arm of 5D chromosome. Pina-D1 and Pinb-D1 alleles of...     

Highly recombinogenic regions at seed storage protein loci on chromosome 1DS of Aegilops tauschii, the D-genome donor of wheat

A detailed RFLP map was constructed of the distal end of the short arm of chromosome 1D of Aegilops tauschii, the diploid D-genome donor species of hexaploid wheat. Ae. tauschii was used to overcome some of the limitations commonly associated with molecular studies of wheat such as low levels of DNA polymorphism. Detection of multiple loci by most RFLP probes suggests that gene duplication events have occurred throughout this chromosomal region. Large DNA fragments isolated from a BAC library of Ae. tauschii were used to determine the relationship between physical and genetic distance at seed storage protein loci located at the distal end of chromosome 1DS. Highly recombinogenic regions were identified where the ratio of physical to genetic distance was estimated to be <20 kb/cM. These results are discussed in relation to the genome-wide estimate of the relationship between physical and genetic distance.     

The sugary-type isoamylase gene from rice and Aegilops tauschii: characterization and comparison with maize and arabidopsis.

Genes for an isoamylase-like debranching enzyme have been isolated from rice and Aegilops tauschii, the donor of the D genome to wheat. The structures of the genes are very similar to each other and to the maize SU1 isoamylase gene and consist of 18 exons spread over approximately 7.5 kb. Southern analysis and fluorescent in situ hybridization showed the Ae. tauschii gene to be located in the proximal region of the short arm of chromosome 7D, thus showing synteny with the localization of the rice isoamylase gene on rice chromosome 8. Analysis of the expression pattern of wheat sugary isoamylase genes indicates that they are strongly expressed in the developing endosperm 6 days after flowering. Three distinct Sugary-type cDNA sequences were isolated from the wheat endosperm that are likely to correspond to the products of the three genomes. The deduced amino acid sequence of rice and wheat Sugary-type isoamylase is compared with other sequences available in the database and the results demonstrate that there are three types of isoamylase sequences in plants: those containing 18 exons (the Sugary-type isoamylase gene), those containing 21 exons, and those containing only 1 exon. It is possible that different combinations of isoamylase genes are expressed in different tissues.     

High-resolution mapping of the leaf rust disease resistance gene <Emphasis Type="Italic">Lr1</Emphasis> in wheat and characterization of BAC clones from the <Emphasis Type="Italic">Lr1</Emphasis> locus

Leaf rust is the most common disease in wheat production. There are more than 45 specific resistance genes described and used in wheat breeding to control epidemics of leaf rust, but none of them has been cloned. The leaf rust disease resistance gene 1 ( Lr1) is a good model gene for isolation by map-based cloning because it is a single, dominant gene which is located in the distal region of chromosome 5DL of wheat. As the first step towards the isolation of this gene we constructed a high-resolution genetic map in the region of the Lr1 locus by saturation mapping of two large segregating F(2) populations (Thatcher Lr1 x Thatcher, Thatcher Lr1 x Frisal). The resistance gene Lr1 was delimited in a 0.16-cM region between the RFLP markers ABC718 and PSR567 (0.12 cM from ABC718 and 0.04 cM from PSR567). A genomic BAC library of Aegilops tauschii (D genome) was screened using the RFLP markers ABC718 and PSR567. Five positive BAC clones were identified by ABC718 and four clones by PSR567. Two NBS-LRR type of resistance gene analogs, which encode proteins highly homologous to the bacterial blight disease resistance protein Xa1 of rice, were identified on BAC clones isolated with PSR567. Polymorphic BAC end probes were isolated from both ends of a 105-kb large BAC clone identified by ABC718. The end probes were mapped at the same locus as ABC718, and no recombination event was found within 105 kb around ABC718 in our analysis of more than 4,000 gametes.     

Simultaneous painting of three genomes in hexaploid wheat by BAC-FISH.

Fluorescence in situ hybridization (FISH) is widely used in the physical mapping of genes and chromosome landmarks in plants and animals. Bacterial artificial chromosomes (BACs) contain large inserts, making them amenable for FISH mapping. In our BAC-FISH experiments, we selected 56 restriction fragment length polymorphism (RFLP)-locus-specific BAC clones from the libraries of Triticum monococcum and Aegilops tauschii, which are the A- and D-genome donors of wheat (Triticum aestivum, 2n = 6x = 42), respectively. The BAC clone 676D4 from the T. monococcum library contains a dispersed repeat that preferentially hybridizes to A-genome chromosomes, and two BAC clones, 9I10 and 9M13, from the Ae. tauschii library contain a dispersed repeat that preferentially hybridizes to the D-genome chromosomes. These repeats are useful in simultaneously discriminating the three different genomes in hexaploid wheat, and in identifying intergenomic translocations in wheat or between wheat and alien chromosomes. Sequencing results show that both of these repeats are transposable elements, indicating the importance of transposable elements, especially retrotransposons, in the genome evolution of wheat.     

Genetic and physical characterization of grain texture-related loci in diploid wheat

Endosperm texture, i.e. the hardness or softness of the grain, is an important quality criterion in cereals because it determines many grain end-use properties. Grain softness is the dominant trait and is mainly controlled by the Ha locus on the short arm of chromosome 5D in hexaploid bread wheat. Genes for puroindoline a (Pina-D1), puroindoline b (Pinb-D1), and grain softness related protein (Gsp-D1) have been shown to be linked to the Ha locus in different mapping populations and have been associated with the expression of grain softness. The study of the linkage relationships among these genes has been limited by the low level of polymorphism in the D genome of hexaploid Triticum aestivum. In the present study, a highly polymorphic Triticum monococcum mapping population was used to analyze linkage relationships among these three genes. Gsp-A ^m 1 and Pina-A ^m 1 were found to be completely linked and lie 0.14 cM distal to Pinb-A ^m 1 in the distal region of the short arm of chromosome 5A^m. The tight genetic linkage among these three genes was paralleled by their physical proximity within a single 105-kb clone isolated from a T. monococcum bacterial artificial chromosome (BAC) library. A restriction map of this BAC clone showed that Pina-A ^m 1 is located between Pinb-A ^m 1 and Gsp-A ^m 1. Partial sequences of the T. monococcum genes showed a high degree of similarity with their T. aestivum counterparts (≥ 94%). Marker-assisted selection strategies based on the tight linkage among Ha-related genes are discussed. Received: 27 June 1999 / Accepted: 18 August 1999     

Characterization of wheat puroindoline proteins

Puroindoline proteins were purified from selected UK-grown hexaploid wheats. Their identities were confirmed on the basis of capillary electrophoresis mobilities, relative molecular mass and N-terminal amino acid sequencing. Only one form of puroindoline-a protein was found in those varieties, regardless of endosperm texture. Three allelic forms of puroindoline-b protein were identified. Nucleotide sequencing of cDNA produced by RT-PCR of isolated mRNA indicated that these were the 'wild-type', found in soft wheats, puroindoline-b containing a Gly-->Ser amino acid substitution (position 46) and puroindoline-b containing a Trp-->Arg substitution (position 44). The latter two were found in hard wheats. Microheterogeneity, due to short extensions and/or truncations at the N-terminus and C-terminus, was detected for both puroindoline-a and puroindoline-b. The type of microheterogeneity observed was more consistent for puroindoline-a than for puroindoline-b, and may arise through slightly different post-translational processing pathways. A puroindoline-b allele corresponding to a Leu-->Pro substitution (position 60) was identified from the cDNA sequence of the hard variety Chablis, but no mature puroindoline-b protein was found in this or two other European varieties known to possess this puroindoline-b allele. Wheats possessing the puroindoline-b proteins with point mutations appeared to contain lower amounts of puroindoline protein. Such wheats have a hard endosperm texture, as do wheats from which puroindoline-a or puroindoline-b are absent. Our results suggest that point mutations in puroindoline-b genes may confer hard endosperm texture through accumulation of allelic forms of puroindoline-b proteins with altered functional properties and/or through lower amounts of puroindoline proteins.     

Molecular-genetic maps for group 1 chromosomes of Triticeae species and their relation to chromosomes in rice and oat

Group 1 chromosomes of the Triticeae tribe have been studied extensively because many important genes have been assigned to them. In this paper, chromosome 1 linkage maps of Triticum aestivum, T. tauschii, and T. monococcum are compared with existing barley and rye maps to develop a consensus map for Triticeae species and thus facilitate the mapping of agronomic genes in this tribe. The consensus map that was developed consists of 14 agronomically important genes, 17 DNA markers that were derived from known-function clones, and 76 DNA markers derived from anonymous clones. There are 12 inconsistencies in the order of markers among seven wheat, four barley, and two rye maps. A comparison of the Triticeae group 1 chromosome consensus map with linkage maps of homoeologous chromosomes in rice indicates that the linkage maps for the long arm and the proximal portion of the short arm of group 1 chromosomes are conserved among these species. Similarly, gene order is conserved between Triticeae chromosome 1 and its homoeologous chromosome in oat. The location of the centromere in rice and oat chromosomes is estimated from its position in homoeologous group 1 chromosomes of Triticeae.     

A ‘Chinese Spring’ wheat (Triticum aestivum L.) bacterial artificial chromosome library and its use in the isolation of SSR markers for targeted genome regions

A bacterial artificial chromosome (BAC) library was constructed from the bread wheat (Triticum aestivum L.) genotype ‘Chinese Spring’ (‘CS’). The library consists of 395,136 clones with an estimated average insert size of 157 kb. This library provides an estimated 3.4-fold genome coverage for this hexaploid species. The genome coverage was confirmed by RFLP analysis of single-copy RFLP clones. The CS BAC library was used to develop simple sequence repeat (SSR) markers for targeted genome regions using five sequence-tagged-site (STS) markers designed from the chromosome arm of 3BS. The SSR markers for the targeted genome region were successfully obtained. However, similar numbers of new SSR markers were also generated for the other two homoeologous group 3 chromosomes. This data suggests that BAC clones belonging to all three chromosomes of homoeologous group 3 were isolated using the five STS primers. The potential impacts of these results on marker isolation in wheat and on library screening in general are discussed.     

Resistance to wheat leaf rust and stem rust in Triticum tauschii and inheritance in hexaploid wheat of resistance transferred from T. tauschii.

Twelve accessions of Triticum tauschii (Coss.) Schmal. were genetically analyzed for resistance to leaf rust (Puccinia recondita Rob. ex Desm.) and stem rust (Puccinia graminis Pers. f.sp. tritici Eriks. and E. Henn.) of common wheat (Triticum aestivum L.). Four genes conferring seedling resistance to leaf rust, one gene conferring seedling resistance to stem rust, and one gene conferring adult-plant resistance to stem rust were identified. These genes were genetically distinct from genes previously transferred to common wheat from T. tauschii and conferred resistance to a broad spectrum of pathogen races. Two of the four seedling leaf rust resistance genes were not expressed in synthetic hexaploids, produced by combining tetraploid wheat with the resistant T. tauschii accessions, probably owing to the action of one or more intergenomic suppressor loci on the A or B genome. The other two seedling leaf rust resistance genes were expressed at the hexaploid level as effectively as in the source diploids. One gene was mapped to the short arm of chromosome 2D more than 50 cM from the centromere and the other was mapped to chromosome 5D. Suppression of seedling resistance to leaf rust in synthetic hexaploids derived from three accessions of T. tauschii allowed the detection of three different genes conferring adult-plant resistance to a broad spectrum of leaf rust races. The gene for seedling resistance to stem rust was mapped to chromosome ID. The degree of expression of this gene at the hexaploid level was dependent on the genetic background in which it occurred and on environmental conditions. The expression of the adult-plant gene for resistance to stem rust was slightly diminished in hexaploids. The production of synthetic hexaploids was determined to be the most efficient and flexible method for transferring genes from T. tauschii to T. aestivum, but crossing success was determined by the genotypes of both parents. Although more laborious, the direct introgression method of crossing hexaploid wheat with T. tauschii has the advantages of enabling selection for maximum expression of resistance in the background hexaploid genotype and gene transfer into an agronomically superior cultivar.     

Linkage between RFLP markers and genes affecting kernel hardness in wheat

A molecular-marker linkage map of wheat (Triticum aestivum L. em. Thell) provides a powerful tool for identifying genomic regions influencing breadmaking quality. A variance analysis for kernel hardness was conducted using 114 recombinant inbred lines (F₇) from a cross between a synthetic and a cultivated wheat. The major gene involved in kernel hardness, ha (hard), known to be on chromosome arm 5DS, was found to be closely linked with the locus Xmta9 corresponding to the gene of puroindoline-a. This locus explained around 63% of the phenotypic variability but there was no evidence that puroindoline-a is the product of Ha (soft). Four additional regions located on chromosomes 2A, 2D, 5B, and 6D were shown to have single-factor effects on hardness, while three others situated on chromosomes 5A, 6D and 7A had interaction effects. Positive alleles were contributed by both parents. A three-marker model explains about 75% of the variation for this trait.     

SPC-P1: a pathogenicity-associated prophage of Salmonella paratyphi C

Background

The presence of closely related genomes in polyploid species makes the assembly of total genomic sequence from shotgun sequence reads produced by the current sequencing platforms exceedingly difficult, if not impossible. Genomes of polyploid species could be sequenced following the ordered-clone sequencing approach employing contigs of bacterial artificial chromosome (BAC) clones and BAC-based physical maps. Although BAC contigs can currently be constructed for virtually any diploid organism with the SNaPshot high-information-content-fingerprinting (HICF) technology, it is currently unknown if this is also true for polyploid species. It is possible that BAC clones from orthologous regions of homoeologous chromosomes would share numerous restriction fragments and be therefore included into common contigs. Because of this and other concerns, physical mapping utilizing the SNaPshot HICF of BAC libraries of polyploid species has not been pursued and the possibility of doing so has not been assessed. The sole exception has been in common wheat, an allohexaploid in which it is possible to construct single-chromosome or single-chromosome-arm BAC libraries from DNA of flow-sorted chromosomes and bypass the obstacles created by polyploidy.

Results

The potential of the SNaPshot HICF technology for physical mapping of polyploid plants utilizing global BAC libraries was evaluated by assembling contigs of fingerprinted clones in an in silico merged BAC library composed of single-chromosome libraries of two wheat homoeologous chromosome arms, 3AS and 3DS, and complete chromosome 3B. Because the chromosome arm origin of each clone was known, it was possible to estimate the fidelity of contig assembly. On average 97.78% or more clones, depending on the library, were from a single chromosome arm. A large portion of the remaining clones was shown to be library contamination from other chromosomes, a feature that is unavoidable during the construction of single-chromosome BAC libraries.

Conclusions

The negligibly low level of incorporation of clones from homoeologous chromosome arms into a contig during contig assembly suggested that it is feasible to construct contigs and physical maps using global BAC libraries of wheat and almost certainly also of other plant polyploid species with genome sizes comparable to that of wheat. Because of the high purity of the resulting assembled contigs, they can be directly used for genome sequencing. It is currently unknown but possible that equally good BAC contigs can be also constructed for polyploid species containing smaller, more gene-rich genomes.     

Construction and characterization of a bacterial artificial chromosome library of thermo-sensitive genie male-sterile rice 5460S

In order to develop a detailed physical map of the thermo-sensitive genie male-sterile (TGMS) gene-encompassing region and finally clone the TGMS gene, a high-quality rice bacterial artificial chromosome (BAC) library from TGMS rice 5460S was constructed. The method of constructing BAC library was examined and optimized. The 5460S library consists of 19 584 BAC clones with an average insert size of 110 kb, which represents about 5 times rice haploid genome equivalents. Rice inserts of up to 140 kb and 250 kb were isolated and appeared stable after 100 generations of serial growth. Hybridization of BAC clones with mitochondrial and chloroplastic genes as probes demonstrated that this library has no organellar contamination. The 5460S library was screened with 3 molecular markers linked to tmsl gene as probes and at least 1 BAC clone was identified with each probe. The insert ends of positive clones were successfully isolated using thermal asymmetric interlaced PCR (TAIL-PCR) technique.     

野生一粒小麦BAC文库的构建和鉴定

以细菌人工染色体pECBAC1为载体 ,构建了野生一粒小麦 (TriticumboeoticumBoiss)的基因组BAC文库。该文库共包含约 17万个克隆 ,平均插入片段长度为 10 4kb ,按野生一粒小麦基因组为 5 6 0 0Mb计算 ,文库覆盖了约 3倍的该物种基因组。用大麦叶绿体psbA基因和玉米线粒体atp6基因作混合探针 ,检测发现该文库中含细胞器基因组同源序列的克隆数小于 1%。该文库的建成 ,为小麦基因的克隆及基因组学研究提供了技术平台