Short Arm Of Chromosome 5D Research Articles

Variation in Friabilin Composition as Determined by A-PAGE Fractionation and PCR Amplification, and its Relationship to Grain Hardness in Bread Wheat

A-PAGE fractionation of starch granule proteins from 63 bread wheat cultivars with contrasting grain texture characteristics revealed two prominent polypeptides and three minor ones, approximately 15 kDa in size. These proteins were found to be encoded by genes on the short arm of chromosome 5D. The two major friabilin components were assumed to correspond to puroindolines a and b (pinA and pinB), as suggested by PCR amplification of genes coding for pinA, glycine-type or serine-type pinB. Two electrophoretic patterns for pinA (presence vs absence) and three patterns for pinB were obtained by A-PAGE. In cultivars with pinA (allele Pina-D1a), pinB was found to be encoded by wild-type Pinb-D1a, serine-type Pinb-D1b or by the novel glycine-type b1 allele. Cultivars lacking pinA (allele Pina-D1b) were shown to contain eitherPinb-D1a or the novel b2 allele, both alleles coding for glycine-type pinB. The intensity of pinB in A-PAGE gels was found to be associated with grain hardness as determined by the SKCS method. Cultivars lacking pinA had the highest SKCS values, suggesting that both pinA and pinB may affect grain texture. In the presence of pinA, cultivars with wild-type allelePinb-D1a had soft grain texture, whereas those with alleles Pinb-D1b or b1 showed increased grain hardness. It is suggested that allele b1 affects the interaction of pinB with starch granules because of a sequence mutation different from the glycine-to-serine change.

Chromosomal location of a gene for resistance to septoria tritici blotch (Mycosphaerella graminicola)in the hexaploid wheat ’Synthetic 6x’

Septoria tritici blotch, caused by the fungus Mycosphaerella graminicola,is currently the major foliar disease of wheat world-wide, and new sources of resistance and knowledge about the genetics of resistance are needed to improve breeding for resistance to this disease. Sears’s ’Synthetic 6x’ hexaploid wheat, derived from a hybrid of Triticum dicoccoides and Triticum tauschii, was resistant to 12 of 13 isolates of M. graminicola tested. Chromosome 7D of ’Synthetic 6x’ was identified as carrying resistance to all 12 isolates in tests of seedlings of inter-varietal chromosome substitution lines of ’Synthetic 6x’ into ’Chinese Spring’ and to two isolates in tests of adult plants. A septoria tritici blotch resistance gene, named Stb5, was identified using the M. graminicola isolate IPO94269 and mapped on the short arm of chromosome 7D, near the centromere, in a population of single homozygous chromosome-recombinant lines for the 7D chromosome.

Cytogenetic and molecular mapping of the leaf rust resistance gene Lr39 in wheat

Leaf rust, caused by the fungus Puccinia triticina Eriks,is one of the most serious diseases of wheat (Triticum aestivum AABBDD, 2n=6x=42) worldwide. Growing resistant cultivars is an efficient and economical method of reducing losses to leaf rust. Here we report a new leaf rust resistance gene, Lr39, transferred from Aegilops tauschii into common wheat. Lr39 conditions both seedling and adult plant resistance to the leaf rust pathogen. The inter- and intra-chromosomal mapping of the Lr39 gene showed that it is different from all previously described Lr genes. We used monosomic analysis for the inter-chromosomal mapping and wheat microsatellite markers for the intra-chromosomal mapping. The monosomic and ditelosomic analysis indicated that Lr39 is independent of the centromere on the short arm of chromosome 2D. Eight microsatellite markers for 2DS were used for linkage analysis on a population of 57 F2 plants derived from a cross of an Ae. tauschii-derived wheat, cv. Wichita line TA4186 (possessing Lr39), with Wichita monosomics for the D-genome chromosomes. The microsatellite marker analysis confirmed the location of the gene on 2DS. Three markers were polymorphic and linked to the gene. The closest marker Xgwm210 mapped 10.7 cM from Lr39. The location of Lr39 near the telomere of 2DS distinguishes it from the Lr2 and Lr22 loci, which are located on 2DS proximal to Xgwm210.

NBS-LRR sequence family is associated with leaf and stripe rust resistance on the end of homoeologous chromosome group 1S of wheat

A detailed RFLP map was constructed of the distal end of the short arm of chromosome 1D of Aegilops tauschii and wheat. At least two unrelated resistance-gene analogs (RGAs) mapped close to known leaf rust resistance genes (Lr21 and Lr40) located distal to seed storage protein genes on chromosome 1DS. One of the two RGA clones, which was previously shown to be part of a candidate gene for stripe rust resistance (Yr10) located within the homoeologous region on 1BS, identified at least three gene family members on chromosome 1DS of Ae. tauschii. One of the gene members co-segregated with the leaf rust resistance genes, Lr21 and Lr40, in Ae. tauschii and wheat segregating families. Hence, a RGA clone derived from a candidate gene for stripe rust resistance located on chromosome 1BS detected candidate genes for leaf rust resistance located in the corresponding region on 1DS of wheat.

Molecular mapping of the wheat powdery mildew resistance gene Pm24 and marker validation for molecular breeding

Molecular markers were identified in common wheat for the Pm24 locus conferring resistance to different isolates of the powdery mildew pathogen, Erysiphe graminis DM f. sp. tritici (Em. Marchal). Bulked segregant analysis was used to identify amplified fragment length polymorphism (AFLP) markers and microsatellite markers linked to the gene Pm24 in an F2 progeny from the cross Chinese Spring (susceptible)× Chiyacao (resistant). Two AFLP markers XACA/CTA-407 and XACA/CCG-420, and three microsatellite markers Xgwm106, Xgwm337 and Xgwm458, were mapped in coupling phase to the Pm24 locus. The AFLP marker locus XACA/CTA-407 co-segregated with the Pm24 gene, and XACA/CCG-420 mapped 4.5 cM from this gene. Another AFLP marker locus XAAT/CCA-346 co- segregated in repulsion phase with the Pm24 locus. Pm24 was mapped close to the centromere on the short arm of chromosome 1D, contrary to the previously reported location on chromosome 6D. Pm24 segregated independently of gene Pm22, also located on chromosome 1D. An allele of microsatellite locus Xgwm337 located 2.4±1.2 cM from Pm24 was shown to be diagnostic and therefore potentially useful for pyramiding two or more genes for powdery mildew resistance in a single genotype.

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.

A leucine to proline mutation in puroindoline b is frequently present in hard wheats from Northern Europe

Endosperm hardness in wheat (Triticum aes- tivum L.) is determined by one major genetic factor, the Hardness (Ha) gene on the short arm of chromosome 5D. Grain hardness has previously been reported to re- sult from either a failure to express puroindoline a (Pina-D1b ) or a glycine to serine mutation at position 46 in puroindoline b (Pinb-D1b). In this study, which in- volves a large survey of 343 wheat genotypes of mostly Northern European origin, we report two new mutations in puroindoline b associated with hard endosperm. These were characterized as involving a leucine to proline change at position 60 (Pinb-D1c), and a tryptophan to arginine change at position 44 ( Pinb-D1d), respectively. While the former seems to be widely distributed in germplasm around the world, the latter was only found in three winter wheats from Sweden and Netherlands. As discussed in the paper, the three known mutations in pu- roindoline b can be considered "loss-of-function" muta- tions (i.e. soft to hard), and structural analysis may serve to predict that their dramatic effect on wheat grain tex- ture is a result of reduced lipid-binding ability.

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-Am1 and Pina-Am1 were found to be completely linked and lie 0.14 cM distal to Pinb-Am1 in the distal region of the short arm of chromosome 5Am. 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-Am1 is located between Pinb-Am1 and Gsp-Am1. Partial sequences of the T. monococcum genes showed a high degree of similarity with their T. aestivum counterparts (> or =94%). Marker-assisted selection strategies based on the tight linkage among Ha-related genes are discussed.

Chromosome location of the gene for the hulled character in the Tibetan weedrace of common wheat

The hulled character of the Tibetan weedrace ( Triticum aestivum var. tibetanum) was found to be governed by a single dominant gene on the short arm of chromosome 2D. The hulled character gene may be identical to the gene Tg derived from Aegilops tauschii.

Genetic analysis of the dwarfing gene (Rht8) in wheat. Part I. Molecular mapping of Rht8 on the short arm of chromosome 2D of bread wheat (Triticum aestivum L.)

Two sets of single chromosome recombinant lines comparing 2D chromosomes from the wheat varieties ‘Ciano 67’ and ‘Mara’ with the common 2D chromosome of ‘Cappelle-Desprez’ in a ‘Cappelle-Desprez’ background were used to detect a diagnostic wheat microsatellite marker for the dwarfing gene Rht8. The genetic linkage maps place the wheat microsatellite marker WMS 261 0.6 cM distal to Rht8 on the short arm of chromosome 2D. By PCR analysis the WMS 261 alleles of ‘Mara’, ‘Cappelle-Desprez’ and ‘Ciano 67’ could be distinguished by different fragment sizes of 192 bp, 174 bp and 165 bp, respectively. A screen of over 100 international varieties of wheat showed that the three allelic variants were all widespread. It also demonstrated that a limited number of varieties carried novel WMS 261 variants of over 200 bp. Following classification of the individual recombinant lines for allelic variants at the WMS 261 locus it was possible to attribute a 7- to 8-cm reduction in plant height with the WMS 261-192-bp allele compared to the WMS 261-174-bp allele in the set of recombinant lines comparing 2D chromosomes of ‘Mara’ and ‘Cappelle-Desprez’. A height reduction of around 3 cm was detected between the WMS 261-174-bp allele and the WMS 261-165-bp allele in the recombinant lines comparing 2D chromosomes of ‘Cappelle-Desprez’ and ‘Ciano 67’.

Genetic mapping of Russian wheat aphid resistance genesDn2andDn4in wheat

To obtain markers for marker-assisted breeding of Russian wheat aphid resistance in wheat (Triticum aestivum L.), resistance genes Dn2 and Dn4 were mapped with restriction fragment length polymorphism (RFLP) markers, using populations derived from PI 62660 x 'Carson' and PI 372129 x 'Yuma'. PI 262660 and PI 372129 are the donor parents of Dn2 and Dn4, respectively. A locus detected by marker KsuA1 was linked to Dn2 at a distance of 9.8 cM on the long arm of chromosome 7D, and a locus detected by marker ABC156 was 11.6 cM away from Dn4 on the short arm of chromosome 1D.Key words: Russian wheat aphid, RFLP markers, Triticum aestivum.

Chromosome Location of Leaf Rust Resistance Gene Lr43 from Aegilops tauschii in Common Wheat

Wheat leaf rust resistance gene Lr43 was recently transferred from Aegilops tauschii Coss. to common wheat (Triticum aestivum L.) to produce hard red winter wheat germplasm line KS92WGRC16. Monosomic and telocentric analyses were used to determine the chromosomal and chromosome arm location of Lr43. KS92WGRC16 was crossed with each of the seven D‐genome monosomic lines. The F2 families were inoculated with race PBJL of the leaf rust fungus, Puccinia recondita Roberge ex Desmaz. f. sp. tritici (Eriks. &amp; E. Henn.) D.M. Henderson. The number of resistant to susceptible seedlings did not deviate significantly from a 3:1 ratio in crosses involving 1D, 2D, 3D, 4D, 5D, or 6D monosomic lines. However, the 7D monosomic‐derived F2 deviated significantly from a 3:1 ratio (x2 = 32.2, P &lt; 0.001) indicating that Lr43 is located on this chromosome. Twenty resistant F2 plants from the critical cross were progeny tested, and results confirmed the location of Lr43 on chromosome 7D. Telocentric analysis showed that Lr43 is on the short arm of chromosome 7D (7DS) and it is unlinked to the centromere. This information may facilitate development of molecular markers and result in more efficient breeding for resistant cultivars.

A glycine to serine change in puroindoline b is associated with wheat grain hardness and low levels of starch-surface friabilin

The quantitative level of friabilin 15-kDa protein present on the surface of water-washed starch is highly correlated with wheat grain softness. Friabilin is composed primarily, if not exclusively, of the proteins puroindoline a and b. The transcript levels of these two proteins are similar among hard and soft wheat varieties, and the expression of both is controlled by the short arm of chromosome 5D, also the chromosomal location of the Hardness gene. We report here a glycine to serine sequence change in puroindoline b associated with hard grain texture. This amino acid change results from a single nucleotide mutation and resides in a region thought to be important for the lipid-binding properties of puroindolines. No recombination was observed between the serine puroindoline-b mutation, hard grain texture and low levels of starch surface friabilin among a set of 83 homozygous 5D recombinant lines derived from the soft-textured variety ‘Chinese Spring’ and the substitution line ‘Chinese Spring’ containing the 5D chromosome of the hard-textured variety ‘Cheyenne’. The sequence change reported here may adversely affect the lipid-binding properties of puroindoline-b and so effect hard grain texture. The results suggest that grain hardness results from puroindoline-b functionality such that the Hardness gene is a direct manifestation of puroindoline structure. We are suggesting the tentative molecular marker loci designations of Pinb-D1a and Pinb-D1b for the glycine and serine puroindoline-b types, respectively.

Recombination mapping of some chromosome 1A-, 1B-, 1D- and 6B-controlled gliadins and low-molecular-weight glutenin subunits in common wheat

Inheritance of low-molecular-weight glutenin subunits (LMW GS) and gliadins was studied in the segregating progeny from several crosses between common wheat genotypes. The occurrence of a few recombinants in the F2 grains of the cross Skorospelka Uluchshennaya×Kharkovskaya 6 could be accounted for by assuming that the short arm of chromosome 1D contains two tightly linked loci each coding for at least one gliadin plus one C-type LMW GS. These loci were found to recombine at a frequency of about 2%, and to be linked to the Glu-D3 locus coding for B-type LMW GS. Some proteins showing biochemical characteristics of D-type or C-type LMW GS were found to be encoded by the Gli-B1 and Gli-B2 loci, respectively. Strongly stained B-type LMW GS in cvs Skorospelka Uluchshennaya and Richelle were assigned to the Glu-B3 locus, but recombination between this locus and Gli-B1 was not found. Analogously, in the cross Bezostaya 1×Anda, no recombination was found between Gli-A1 and Glu-A3, suggesting the maximum genetic distance between these loci to be 0.97% (P=0.05). A B-type LMW GS in cv Kharkovskaya 6 was assigned to the Glu-B2 locus, with about 25% recombination from the Gli-B1 locus. The present results suggested that alleles at Gli loci may relate to dough quality and serve as genetic markers of certain LMW GS affecting breadmaking quality.

Chromosomal location of genes influencing plant characters and evapotranspiration efficiency in bread wheat

Little is known of genes that influence root development and drought resistance in bread wheat. The evapotranspiration efficiency (ETE = ratio of vegetative dry weight to total water used) of spring bread wheat tall landrace ‘Chinese Spring’ is relatively high. We used 42 ditelosomic and dimonotelosomic lines of Chinese Spring to identify chromosome arms that influence plant characters and ETE. Multiple regression analyses indicated that 96% of the variation observed in ETE was explained by variation in vegetative dry weight and total water used. Variation in plant height, number of spikes (tillers), root dry weight and shoot dry weight (excluding grains) together explained 88% of the variation observed in plant vegetative dry weight. Chromosome arms involved in expression of days to heading and maturity, plant height, number of spikes, root dry weight, shoot dry weight, number of grains, grain weight, and carbon isotope discrimination (Δ) were identified. Specifically, both arms of chromosome 2A, the long arm of chromosome 2B, and the short arm of chromosome 2D might carry genes with positive effects on number of spikes, root dry weight, and shoot dry weight. None of the aneuploids produced grain yield greater than Chinese Spring. The short arms of chromosomes 6A and 4D might carry genes that suppress Δ. Chromosome 1D might carry genes that increase relative water loss. The chromosome arms belonging to homoeologous group 2 might carry genes with positive effects on ETE. The genetic basis of ETE in modern wheats could be broadened by substituting specific chromosome arms of landrace wheats carrying desired characters into their genomes.

GSP-1 genes are linked to the grain hardness locus (Ha) on wheat chromosome 5D.

An important determinant of wheat grain quality is the hardness of the grain. The trait is controlled by a major locus, Ha, on the short arm of chromosome 5D. Purified starch granules from soft-grained wheats have associated with them 15-kDa polypeptides called grain softness proteins (GSPs) or "friabilins." Genes that encode one family of closely related GSP polypeptides - GSP-1 genes - were mapped using chromosome substitution lines to the group 5 chromosomes. An F2 population segregating for hard and soft alleles at the Ha locus on a near-isogenic background was used in a single-seed study of the inheritance of grain softness and of GSP-1 alleles. Grain softness versus grain hardness was inherited in a 3:1 ratio. The presence versus absence of GSPs in single seed starch preparations was coinherited with grain softness versus hardness. This showed that grain softness is primarily determined by seed, and not by maternal, genotype. In addition, no recombination was detected in 44 F2 plants between GSP-1 restriction fragment length polymorphisms and Ha alleles. Differences between hard and soft wheat grains in membrane structure and lipid extractability have been described and, of the three characterized proteins that are part of the mixture of 15-kDa polypeptides called GSPs, at least two, and probably all three, are proteins that bind polar lipids. The data are interpreted to suggest that the Ha locus may encode one or more members of a large family of lipid-binding proteins.

Characterisation of the wheat Mr 15000 ?grain-softness protein? and analysis of the relationship between its accumulation in the whole seed and grain softness

The Mr 15000 protein associated with water-washed wheat starch granules from soft wheats was shown to be heterogeneous: it could be divided into a fraction containing one or moreα-amylase inhibitor subunits and a fraction largely composed of a previously uncharacterised polypeptide(s) referred to as the "grainsoftness protein" (GSP). The major N-terminal sequence and sequences of peptides derived from protease digests of GSP are reported. An antiserum specific for GSP was used to show that GSP accumulated in both hard and soft wheat grains, but the GSP in soft grains associated more strongly with starch granules than the GSP in hard grains. A positive correlation between grain softness and accumulation of GSP in the seed was demonstrated for a range of cultivars. This differs from the qualitative relationship, based on the isolated starch fraction, between GSP and grain softness that has already been reported. Analysis of wholemeal extracts with the antiserum demonstrated that the accumulation of GSP in the seed was dependent on the short arm of chromosome 5D, which also encodes theHa locus. In addition, examination of near-isogenic lines differing in hardness indicated that the gene(s) controlling GSP was (were) linked with theHa locus. The findings indicate that GSP may be the product of theHa locus and thus be the major factor that determines the milling characteristics of bread wheats.

Immunochemical identification of LMW subunits of glutenin associated with bread-making quality of wheat flours

A murine monoclonal antibody (IFRN 0067), one of a library developed against prolamin fractions fromTriticum aestivum, has been characterised using a combination of immunoassay and immunoblotting techniques. The antibody was specific for two glutenin polypeptides which appeared by 2-dimensional electrophoresis to belong to the B group of LMW subunits. From results of antibody-binding studies with material extracted from genetic stocks, it was deduced that the target polypeptides were encoded on the short arm of chromosome 1D. The antibody was used in an immunoassay of bread wheats with a range of anticipated baking scores and for flours of known baking performance. Significant correlations were found between immunoassay and test-bake results. Indeed, correlation of IFRN 0067 binding with loaf volume was equal or better than that provided by alveograph parameters. The results provide evidence that LMW subunits contribute to the bread-making properties of wheat glutenin, as identified by the use of immunological techniques. The use of particular monoclonal antibodies, such as IFRN 0067, in the further development of simple, rapid diagnostic tests for flour quality predictions is discussed.

Restriction fragment analysis of ‘null’ forms at the Gli-1 loci of bread and durum wheats

Wheat accessions lacking some of the ω- and γ-gliadin components encoded by the Gli-1 loci on the short arm of chromosome 1D in bread wheat and chromosome 1A in durum wheat were studied by two-dimensional polyacrylamide gel electrophoresis and restriction fragment analysis. Digested genomic DNAs of 'normal' and 'null' forms were probed with a cDNA clone related to ω-/γ-gliadins and with a genomic clone encoding an LMW subunit of glutenin. The hybridisation patterns with the ω-/γ-gliadin probe were similar to those of cvs 'Chinese Spring' and 'Langdon' used as standards for bread and durum wheats, respectively, but several restriction fragments located on the 1D chromosome of bread wheat and the 1A chromosome of durum wheat were absent in the 'null' forms. In addition, specific LMW glutenin fragments encoded by the same chromosomes were also absent in the 'null' forms, suggesting that simultaneous deletions of blocks of genes for both ω-/γ-gliadins and LMW glutenins had occurred. Comparisons of the protein and RFLP patterns enabled some proteins to be mapped to specific restriction fragments.

The Nor-D3 locus of Triticum tauschii: natural variation and genetic linkage to markers in chromosome 5

Variation in the intergenic spacer region of the ribosomal RNA genes (located at the Nor locus) was assayed in a collection of 411 accessions of Triticum tauschii from Turkey, USSR, Iran, Afghanisan, Pakistan, and China. Twenty rDNA genotypes were identified and it was demonstrated that T. tauschii accessions from the USSR and Iran have the highest diversity at the Nor locus. At least four of the rDNA genotypes were demonstrated to be alleles of a single major locus, in segregating F2 progeny analyses. The TaqI restriction fragment associated with rDNA genotype 7 was shown to be the same as the Nor-D3a allele present in all bread wheats (based on chromosome location and length of the intergenic spacer region). This genotype was significantly associated with T. t. ssp. strangulata, previously argued to be the donor of the D genome to hexaploid wheat. The Nor locus showed a high level of recombination with the 5SDna-2 locus, which was also located on chromosome 5D. The Nor locus is placed distal to the 5SDna-2 locus but proximal to the grain softness protein gene (XGsp) on the short arm of chromosome 5D.Key words: D genome, Nor-D3, rDNA polymorphism, chromosomal location.