Development of SNP assays for genotyping the puroindoline b gene for grain hardness in wheat using pyrosequencing

Grain hardness is one of the most important quality characteristics of cultivated bread wheat (Triticum aestivum L.) and has been reported to result from either a failure to express puroindoline a (Pina) or single-nucleotide mutations in puroindoline b (Pinb). Up to now, seven alleles from Pinb-D1a to Pinb-D1g were identified in bread wheat. Compared to the DNA coding region of Pinb-D1a (allele for softness), six single-nucleotide polymorphisms (SNPs) were detected in six alleles for Pinb-D1. In this study, we used pyrosequencing technology to develop two SNP assays for identification of the seven Pinb alleles and characterized SNP variations in the Pinb of 493 European wheat varieties. Of the three hardness alleles Pinb-D1b, Pinb-D1c, and Pinb-D1d detected in this study, Pinb-D1b was the most predominant hardness allele in European hard wheats. The hardness genotypes of partial German wheat varieties available confirmed the reliability and validation of the SNP assays developed for the Pinb locus. Therefore, pyrosequencing technology offers an efficient, precise, and reliable concept for high-throughout genotyping to assist selection of grain hardness genes in wheat quality breeding programs.     

A Study of Puroindoline b Gene Involvement in the Milling Behavior of Hard‐Type Common Wheats

Differences in milling behavior among hard‐type common wheat (Triticum aestivum) cultivars are well known to millers. Among them, the French cultivar Soissons, which contains the Pinb‐D1d allelic form of the puroindoline b gene, is particularly distinguished for its high milling value. Near‐isogenic lines (NILs) differing by the allelic forms of the puroindoline b gene, Pinb‐D1d or Pinb‐D1b (one of the most frequent alleles found in the European wheat population), were constructed. Grain characteristics obtained after wheat cultivation in distinct environmental conditions were compared between NILs and the cultivar Soissons, as was their fractionation behavior. Results showed that NILs containing the Pinb‐D1d allele displayed lower values of grain hardness and vitreousness than did the corresponding lines containing the Pinb‐D1b allelic form under the same cultivation conditions. Both genetic background and environmental conditions appeared to affect grain texture. Measured single‐kernel characterization system hardness index values of the samples under study were found to be correlated with the vitreousness values. Studies of the milling behavior helped to point out that grain vitreousness is an important factor acting on endosperm breakage ability, whatever the genetic background of the wheat. Our results also demonstrated that, at similar levels of vitreousness, the endosperm of Soissons could more easily be reduced than that of other wheat lines.     

Molecular characterization of a novel waxy allele (Wx-A u 1a) from Triticum urartu Thum. ex Gandil.

Granule Bound Starch Synthase I, or waxy protein, is the sole enzyme responsible for the accumulation of amylose during the development of starch granules in wheat. The full coding region of the waxy (Wx) gene was sequenced in Triticum urartu, (a wild diploid species) and is related to the A genome of polyploid wheats. The Wx gene of T. urartu (Wx-A ^u 1) showed a homology of ~88.0?% with Wx-A1 from polyploid wheats. A greater homology was found with Wx-A ^m 1 from the diploid cultivated wheat einkorn. Most of the differences were found in introns although several changes were also detected in exons that led to amino acid changes in the transit peptide and mature protein. These results show the potential of T. urartu as a source of new alleles that could be used in the breeding of durum and common wheat in order to synthesize starches with different properties.     

Cloning and phylogenetic analysis of polyphenol oxidase genes in common wheat and related species

Cloning and phylogenetic analysis of polyphenol oxidase (PPO) genes in common wheat and its relatives would greatly advance the understanding of molecular mechanisms of grain PPO activity. In the present study, six wheat relative species, including T. urartu, T. boeoticum, T. monococcum, T. dicoccoides, T. durum and Ae. tauschii, were sampled to isolate new alleles at Ppo-A1 and Ppo-D1 loci corresponding to common wheat PPO genes, and seven new alleles were identified from these species, which were designated as Ppo-A1c (from T. urartu), Ppo-A1d (T. boeoticum), Ppo-A1e (T. monococcum and T. durum), Ppo-A1f (T. dicoccoides), Ppo-A1g (T. durum), Ppo-D1c (Ae. tauschii) and Ppo-D1d (Ae. tauschii), respectively. Five out of the seven alleles detected in the wheat relatives contained an open reading frame (ORF) of 1,731 bp, encoding a polypeptide of 577 residues, which is the same as those of Ppo-A1 and Ppo-D1 genes in common wheat, whereas, the full-length ORF of the allele Ppo-A1g from T. durum was not obtained, and a 73-bp deletion occurred in the third exon of Ppo-D1d, an allele from Ae. tauschii, resulting in a shorter polypeptide of 466 amino acids. The 191-bp insertion in the first intron reported previously in common wheat was also found in T. dicoccoides lines, implying that more than one tetraploid wheat lines may be involved in the origination of common wheat. Phylogenetic trees were constructed using the genomic DNA sequences of the seven alleles, together with four from common wheat and four partial PPO gene sequences deposited in GenBank. The genome tribe A was divided into two clusters, one of which contained Ppo-A1d and Ppo-A1e, and the other included the remaining five alleles at Ppo-A1 locus. The alleles from different clusters showed high sequence divergences, indicated by dozens of SNPs and five to six InDels. The genome tribe D comprised the alleles Ppo-D1a, Ppo-D1c, Ppo-D1d and Ppo-D1b, and the former three were clustered together, showing significant sequence divergence from Ppo-D1b. In addition, the relationships between these allelic variants and grain PPO activities were also discussed. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Novel and ancient HMW glutenin genes from Aegilops tauschii and their phylogenetic positions

A pair of novel high-molecular-weight glutenin subunits (HMW-GS) 1Dx3.1^t and 1Dy11*^t were revealed and characterized from Aegilops tauschii Coss. subspecies tauschii accession AS60. SDS-PAGE band of 1Dx3.1^t was between those of 1Dx2 and 1Dx3, while 1Dy11*^t was between 1Dy11 and 1Dy12. The lengths of 1Dx3.1 ^t and 1Dy11* ^t were 2,514?bp and 1,968?bp, encoding 836 and 654 amino acid residues, respectively. Their authenticity was confirmed by successful expression of the coding regions in Escherichia coli. Network analysis indicated that 1Dx3.1 ^t together with other five rare alleles only detected in Asia common wheat populations represented the ancestral sequences in Glu-D1 locus. Neighbor-joining tree analysis of previously cloned x-type and y-type alleles in the Glu-D1 locus supported the hypothesis that more than one Ae. tauschii genotypes were involved in the origin of hexaploid wheat and that different Ae. tauschii accessions contributed the D genome to common wheat and Ae. cylindrical Host, respectively. An Ae. tauschii accession with 1Dx3.1 ^t or a closely related allele probably have involved in the origin of common wheat. Since accession AS60 used in this study belonged to typical ssp. tauschii, present results suggested the possibility that ssp. tauschii was involved in the evolution of common wheat.     

Occurrence of Puroindoline Alleles in Chinese Winter Wheats

Grain hardness is one of the most important characters that determine the end‐use quality of bread wheat (Triticum aestivum L.). Mutations in genes encoding either puroindoline a (Pina) or b (Pinb) have been associated with hard grain texture, i.e., Pina null at Pina‐D1 or seven mutations at Pinb‐D1. In this study, the diversity of puroindoline alleles in 251 Chinese winter wheat cultivars and advanced lines from four major autumn‐planted wheat regions were investigated. Among the examined cultivars, 79 were classified as soft, while 53 were mixed in hardness, and 119 were uniformly hard. Of these hard winter wheats, three of the seven reported mutation types were observed, with Pina‐D1a/Pinb‐D1b being the dominant type for hard texture; 91 genotypes carried this allele. Sixteen genotypes had the Pina‐D1b allele, and two genotypes had the Pinb‐D1d allele. A new mutation, designated as Pinb‐D1p, was detected in 10 hard genotypes, with a single nucleotide (A) deletion corresponding to position 42 in the amino acid sequence of puroindoline b, involving a lysine (K) to asparagine (N) change, and leading to a shift in the open reading frame (ORF). This deletion disrupts the last part of the tryptophanrich domain, changing it from KWWK to NGGR, which is considered essential for the lipid‐binding activity of this protein, and results in a stop codon corresponding to position Pro‐60 in the amino acid sequence. The characterization of different hardness alleles provides useful information in understanding the mechanism underlying the formation of endosperm hardness while providing breeders the means of manipulating this important trait.     

Novel Ha Locus,Pina‐D1c/Pinb‐D1h,Affects Soft White Spring Wheat Milling and Baking

Wheat (Triticum aestivum L.) grain hardness is controlled by the Hardness locus on chromosome 5D which consists of the linked genes Puroindoline a and b (Pina and Pinb, respectively). The Ha locus haplotype, Pina‐D1a/Pinb‐D1a, is found in all soft hexaploid wheats. While Pin diversity is low among soft wheats, several novel Ha haplotypes were reported among synthetic hexaploid wheats created using the D genome donor, Aegilops tauschii. One haplotype, Pina‐D1c/Pinb‐D1h, confers a soft phenotype with increased grain hardness over Pina‐D1a/Pinb‐D1a wheats. Here, the Pina‐D1c/Pinb‐D1h haplotype was backcrossed into the soft white spring wheat cultivars ‘Vanna’ and ‘Alpowa’. Then the effect of the two haplotypes on soft wheat milling and baking quality was compared. The effects of the Pina‐D1c/Pinb‐D1h Ha locus haplotype were similar in both the Vanna and Alpowa backgrounds. The Pina‐D1c/Pinb‐D1h lines had significantly more large and fewer small flour particles in both backgrounds and 1.51% higher flour yield in the Alpowa background. The Pina‐D1c/Pinb‐D1h haplotype group was not associated with any consistent differences in solvent retention capacities or sugar snap cookie quality parameters. The results indicate that the Pina‐D1c/Pinb‐D1h haplotype could be used to modify soft wheat milling properties without substantial effects on baking quality.     

Mutagenesis‐Derived Puroindoline Alleles in Triticum aestivum and Their Impacts on Milling and Bread Quality

Wheat (Triticum aestivum) end‐product quality is impacted by grain hardness, which is determined by the Hardness locus consisting of the Puroindoline a and Puroindoline b genes, Pina and Pinb, respectively. Hard wheats commonly contain just one of two Pin mutations. We previously demonstrated the creation and preliminary hardness testing of 46 Pin missense alleles. In this study we examine the degree that individual Pin missense alleles confer unique milling and bread quality traits. Three Pina (PINA‐R103K, ‐G47S, and ‐P35S) and four Pinb (PINB‐D34N, ‐T38I, ‐G46D, and ‐E51K) missense alleles were chosen because they impart variable grain hardness levels, with one allele conferring soft seed texture, three conferring intermediate hardness (single‐kernel characterization system [SKCS] hardness approximately 50), and three conferring hard grain texture (SKCS hardness greater than 60). All but two of the alleles (PINA‐R103K and PINA‐G47S) resulted in higher total flour yield when compared with wild‐type controls. All hard and intermediate hardness alleles had decreased break flour yield, but intermediate hardness allele PINA‐P35S had higher break flour yield than common hard allele Pinb‐D1b. Intermediate and hard alleles resulted in increased abundance of larger and reduced levels of smaller flour particles. None of the missense alleles differed from their controls for loaf volume. The seven selected Pin alleles imparted defined levels of grain hardness and milling properties not previously available that may prove useful in wheat improvement.     

Characterization of Genes Encoding Wheat Grain Hardness from Chinese Cultivar GaoCheng 8901

The puroindoline‐b (Pinb‐D1) gene from Chinese hard wheat cultivar GaoCheng 8901 (Triticum aestivum L.) was obtained using two pairs of primers designed based on the known Pinb‐D1 gene sequence and polymerase chain reaction (PCR) amplification. The PCR amplification was made using the genomic DNA of the wheat as a template and the specific fragment ≈450 bp in size was screened. The results indicated that the Pinb‐D1 gene in GaoCheng 8901 shared 99.78% and 99.32% homology in nucleotide acid sequence and amino acid sequence, respectively, compared with the Pinb‐D1 gene from hard wheat cultivars Wanser and Cheyenne. A new mutation in this Pinb‐D1 gene, different from the six known mutations in the Pinb‐D1 gene, was characterized with a change of a lysine to glutamic acid at position 45 in its protein sequence. This mutation, designated as Pinb‐D1l in this study, might contribute to the formation of grain hardness in GaoCheng 8901. The characterization of Pinb‐D1 gene would be helpful in manipulating grain hardness of wheat through genetic engineering.     

Characterization of the Wx gene in diploid Aegilops species and its potential use in wheat breeding

Wx gene encodes for the granule-bound starch synthase I or waxy protein, which is the sole enzyme responsible for amylose synthesis in wheat seeds. The Aegilops species, which are related to wheat, could be important sources of variation in this gene. In addition to its role in starch quality, this gene has been used in phylogenetic studies of wheat. The current study evaluated the variability of Wx gene in seven diploid species of Aegilops genus and compared their nucleotide sequences with the wheat homeologous genes. Nineteen new Wx alleles were found in the seven species evaluated. The alleles detected in two species of the Sitopsis section, Ae. searsii and Ae. speltoides, were related to the Wx-B1 gene of wheat. Two more of the Sitopsis species did not appear to be associated with this genome, whereas the remaining species were related to the Wx-D1 gene of wheat. The results showed an important variation of the Wx gene present in the Aegilops genus, and the 19 new Wx alleles detected could enlarge the genetic pool of wheat.     

Influence of Soft Kernel Texture on the Flour,Water Absorption,Rheology, and Baking Quality of Durum Wheat

Durum wheat (Triticum turgidum subsp. durum ) production worldwide is substantially less than that of common wheat (T. aestivum ). Durum kernels are extremely hard; thus, most durum wheat is milled into semolina, which has limited utilization. Soft kernel durum wheat was created by introgression of the puroindoline genes via homoeologous recombination. The objective of this study was to determine the effects of the puroindoline genes and soft kernel texture on flour, water absorption, rheology, and baking quality of durum wheat. Soft Svevo and Soft Alzada, back‐cross derivatives of the durum varieties Svevo and Alzada, were compared with Svevo, a hard durum wheat, Xerpha, a soft white winter wheat, and Expresso, a hard red spring wheat. Soft Svevo and Soft Alzada exhibited soft kernel texture; low water, sodium carbonate, and sucrose solvent retention capacities (SRCs); and reduced dough water absorptions similar to soft wheat. These results indicate a pronounced effect of the puroindolines. Conversely, SDS flour sedimentation volume and lactic acid SRC of the soft durum samples were more similar to the Svevo hard durum and Expresso samples, indicating much less effect of kernel softness on protein strength measurements. Alveograph results were influenced by the inherent differences in water absorption properties of the different flours and their genetic background (e.g., W and P were markedly reduced in the Soft Svevo samples compared with Svevo, whereas the puroindolines appeared to have little effect on L ). However, Soft Svevo and Soft Alzada differed markedly for W and L . Soft durum samples produced bread loaf volumes between the soft and hard common wheat samples but larger sugar‐snap cookie diameters than all comparison samples. The soft durum varieties exhibited new and unique flour and baking attributes as well as retaining the color and protein characteristics of their durum parents.     

Effect of Soft Kernel Texture on the Milling Properties of Soft Durum Wheat

Worldwide, nearly 20 times more common wheat (Triticum aestivum) is produced than durum wheat (T. turgidum subsp. durum). Durum wheat is predominately milled into coarse semolina owing to the extreme hardness of the kernels. Semolina, lacking the versatility of traditional flour, is used primarily in the production of pasta. The puroindoline genes, responsible for kernel softness in wheat, have been introduced into durum via homoeologous recombination. The objective of this study was to determine what impact the introgression of the puroindoline genes, and subsequent expression of the soft kernel phenotype, had on the milling properties and flour characteristics of durum wheat. Three grain lots of Soft Svevo and one of Soft Alzada, two soft‐kernel back‐cross derived durum varieties, were milled into flour on the modified Quadrumat Senior laboratory mill at 13, 14, and 16% temper levels. Samples of Svevo (a durum wheat and recurrent parent of Soft Svevo), Xerpha (a soft white winter wheat), and Expresso (a hard red spring wheat) were included as comparisons. Soft Svevo and Soft Alzada exhibited dramatically lower single‐kernel characterization system kernel hardness than the other samples. Soft Svevo and Soft Alzada had high break flour yields, similar to the common wheat samples, especially the soft hexaploid wheat, and markedly greater than the durum samples. Overall, Soft Svevo and Soft Alzada exhibited milling properties and flour quality comparable, if not superior, to those of common wheat.     

Effects of Allelic Variations in Wx‐1, Glu‐D1, Glu‐B3, and Pinb‐D1 Loci on Flour Characteristics and White Salted Noodle‐Making Quality of Wheat Flour

Doubled haploid wheat lines developed from a cross between a hard white winter wheat variety of normal starch endosperm and a waxy wheat variety were used to determine the effects of allelic variation in Wx‐1, Glu‐D1, Glu‐B3, and Pinb‐D1 loci on physiochemical properties of flour, noodle dough properties, and textural quality of cooked noodles. Milling yield, damaged starch content, protein content, and SDS sedimentation volume of flour were influenced the most by allelic composition of Pinb‐D1 loci, less by Wx‐1 loci, and least by Glu‐B3. Wheat lines carrying Pinb‐D1b or Glu‐B3h alleles exhibited higher milling yield and damaged starch content of flour than those with Pinb‐D1a and Glu‐B3d alleles. Wheat lines carrying the Pinb‐D1b allele were higher in protein content and SDS sedimentation volume than those carrying Pinb‐D1a. Mixograph water absorption was largely influenced by allelic composition of Wx‐1 loci, whereas mixograph mixing time and mixing tolerance were predominantly determined by allelic composition of Glu‐D1 loci. Amylose content and pasting properties of starch were mainly determined by allelic composition of Wx‐1 loci with little influence by allelic compositions of Glu‐D1, Glu‐B3, and Pinb‐D1 loci. Allelic composition of Wx‐1 loci contributed 53.4% of the variation in optimum water absorption of noodle dough and 26.7% of the variation in thickness of the noodle dough sheet. The variation of 7.8% in optimum water absorption of noodle dough was contributed by the allelic composition of Pinb‐D1 loci. Allelic composition of Wx‐1 loci was responsible for 73.2, 74.4, and 59.6% in the variation of hardness, springiness, and cohesiveness of cooked noodles, respectively. Cohesiveness of cooked noodles was also influenced by the allelic compositions of Glu‐B3 and Pinb‐D1 loci to a smaller extent.     

Genetic relationship of diploid wheat (<Emphasis Type="Italic">Triticum</Emphasis> spp.) species assessed by SSR markers

Genetic diversity of 139 accessions of diploid Triticum species including Triticum urartu, Triticum boeoticum and Triticum monococcum was studied using 11 SSR (simple sequence repeats) markers. A total of 111 alleles with an average of 10 alleles per locus were detected. The polymorphism information content (PIC) of each SSR marker ranged from 0.30 to 0.90 with an average value of 0.62. Among the three Triticum species T. urartu had the highest number of total alleles (Na?=?81), private alleles (Npa?=?15) and showed higher genetic diversity (Hex?=?0.58; PIC?=?0.54). The genotypes from Turkey exhibited the highest genetic diversity (PIC?=?0.6), while the least diversity was observed among 4 Georgian accessions (PIC?=?0.11). Cluster analysis was able to distinguish 139 wheat accessions at the species level. The highest genetic similarity (GS) was noted between T. boeticum and T. monococcum (GS?=?0.84), and the lowest between T. urartu and T. monococcum (GS?=?0.46). The grouping pattern of the PCoA analysis corresponded with cluster analysis. No significant differences were found in clustering of T. urartu and T. monococcum accessions with respect to their geographic regions, while within T. boeoticum species, accessions from Iran were somewhat associated with their geographical origin and clustered as a close and separate group. The results from our study demonstrated that SSR markers were good enough for further genetic diversity analysis in einkorn wheat species.     

Genetic diversity of HMW glutenin subunit in Chinese common wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) landraces from Hubei province

The high molecular weight (HMW) glutenin subunit composition of 111 common landraces of bread wheat collected from Hubei province, China has been determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Ninety six of the accessions were homogeneous for HMW glutenin subunit composition and 15 were heterogeneous. For the Glu-1 loci, 16 alleles were detected, 3 at the Glu-A1locus, 9 at the Glu-B1and 4 at the Glu-D1. Three novel alleles were identified, two at the Glu-B1 and one at the Glu-D1locus. Combination of these 16 alleles resulted in 14 different HMW subunit patterns. The distribution of HMW glutenin subunit alleles in a subset of 105 of the 111 accessions representing six populations was assessed both at the individual population and whole population levels. The results demonstrated that the distribution of allelic patterns varied among populations. Taken together, 62.5% of the alleles detected were considered to be rare alleles while the Glu-A1c (null), Glu-B1b (1Bx7 + 1By8) and Glu-D1a (1Dx2 + 1Dy12) alleles were found most frequently in the six populations. The subset exhibited relatively high genetic diversity (A = 5.33, P = 1.00, Ae = 1.352 and He = 0.238) with 81.5% of the diversity being within populations and 18.5% between populations.     

Polymorphisms in two homeologous γ-gliadin genes and the evolution of cultivated wheat

The polymorphisms in two -gliadin genes GAG56D and GAG56B on the D- and B-genomes of polyploid wheat, respectively, were investigated by sequencing PCR products and by PCR-RFLP. Of GAG56D, two alleles fo and ok were previously known to occur in hexaploid wheat. Here, we found that 16 sequenced fragments of GAG56D from six recognized subspecies of Triticum aestivum, including 13 contributed by this study, were identical to either the fo or the ok allele. Considering published evidence, it was concluded that the investigated alleles of GAG56D stemmed from two different Aegilops tauschii plants and thus two independent origins of hexaploid wheat. Compared to GAG56D-sequences obtained from 10 accessions of Ae. tauschii, the fo and ok alleles clustered with fragments from three accessions collected in the Caspian region. By sequencing fragments of GAG56B, four distinct allelic groups were found among cultivated wheats, typical of bread wheat (p-aes), durum wheat of gliadin 45-type (a), durum wheat of gliadin 42-type (p-dur) and Timopheev's wheat (p-tim), respectively. Interestingly, the a allele found in gliadin 45-type durum wheat was shared by European spelt cultivars, which strongly supported the hypothesis that European spelt originated from a hybridization event between a tetra- and hexaploid wheat. The data also suggested that emmer might have been domesticated more than once. Phylogenetic analysis of GAG56-fragments obtained from putative B/G-genome donors excluded all candidate species as immediate donors of the B/G-genome, but instead indicated a monophyletic origin of all GAG56B alleles found in polyploid wheat, i.e. including T. timopheevii.     

Re-sequencing of vrs1 and int-c loci shows that labile barleys (Hordeum vulgare convar. labile) have a six-rowed genetic background

Labile-barleys (Hordeum vulgare convar. labile (Schiem.) Mansf.) are found in the highlands of Ethiopia, Eretria and North India-Pakistan districts. They represent a distinct spike form showing row-type alterations even within individual spikes of the same genotypes. Variation at the six-rowed spike 1 (vrs1) locus is sufficient to control barley lateral spikelet fertility, which is also modified by alleles at the intermedium-c (int-c) locus. This study aimed at re-sequencing these two loci to investigate whether labile-barleys have a two-rowed genetic background, resulting in increased lateral spikelet fertility, or show reduced lateral spikelet fertility if they possess a six-rowed genetic background. The Vrs1 re-sequencing results of 221 supposedly labile-barley accessions from Ethiopia revealed 13 accessions with two novel vrs1.a1 haplotypes. Following the current nomenclature of vrs1 haplotypes, the new haplotypes were named as haplotypes 66 and 67. Re-sequencing at the int-c locus showed that 118 of the labile-barleys possessed the previously described Int-c.a allele but only one accession was found having a novel Int-c.a haplotype in the homozygous state (termed Int-c.a haplotype1; Hap_1). Interestingly, 101 labile-barleys carried the Int-c.a allele and Int-c.a haplotype1 simultaneously, suggesting maintained heterozygosity or recent gene duplication at this locus. Only one accession had a two-rowed haplotype (Vrs1.b3, int-c.b1) and one accession possessed the Vrs1.t (deficiens) and Int-c.a alleles (six-rowed). These two accessions were considered as misclassified labile genotypes and not included in further analysis. Thus, these results confirmed that all of the 219 labile accessions studied in this work showed six-rowed alleles at vrs1 but reduced lateral spikelet fertility. This reduction is most likely caused by the recessive labile (lab) locus which we are in the process to characterize further.     

Molecular diversity of restriction enzyme sites, Indels and upstream open reading frames (uORFs) of 5′ untransalted regions (UTRs) of Waxy genes in Triticum L. and Aegilops L. species

5′ Untransalted regions (UTR) sequences of Waxy genes were amplified from all 81 Triticum L. and Aegilops L. species by PCR with specific primers. It was found that the sequence length at 7D loci was longer than that 7B and 7A. These sequences contained 170 singleton variable sites and 484 polymorphic sites and that the average length of Indels was 8.5?bp. There were abundant regions of restriction enzyme sites and two regions of simple sequence repeat, “GAA” and “CTGA”, in all sequences. A total of 65 uORFs were detected and classed into 37 types, with the variation in uORFs mainly due to single nucleotide polymorphisms (SNPs), and also to the presence of Indels. All sequences in tetraploids and hexaploids could be grouped into Types A, B, I, II, GI and GII based on sequence variation. Restriction enzyme sites, Indel polymorphisms and the classes of uORFs present together indicated that Type I was more similar to Ae. tauschii whereas Type II was more similar to Type B, and both more similar to Ae. longissima; Type A was more similar to Ae. speltoides. Population analysis was performed and Neighbour-joining trees derived from different species, types and accessions further confirmed that the ancestors of T. urartu, Ae. speltoides, Ae. longissima and Ae. tauschii were involved to the evolution of common wheat, and also implied that Ae. longissima might have participated later than Ae. speltoides. 5′ UTRs of Waxy genes in tetraploid and hexaploid species conserved characters from their respective progenitors when compared with diploid. For the first time, we are able to conclude there is abundant variation in SNPs, Indels and uORFs between 5′ UTRs of Waxy genes from different species related to common wheat, and suggest that further research could help to understand Waxy gene function more deeply and hence improve wheat breeding. Our results also show that three hexaploid species in China have unique diversity in the 5′ UTR of their Waxy gene.     

Evaluation of geographic distribution of high-molecular-weight glutenin subunits (HMW-GS) in wild and cultivated Triticum species

Variation of high-molecular-weight glutenin subunit (HMW-GS) in 632 wild and cultivated Triticum accessions was investigated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. A total of 11 alleles of HMW-GS in diploid species, 22 in tetraploid species, and 15 in hexaploid species were detected. Diploid species on Glu-1A locus and tetraploid species Glu-1B locus showed the highest diversity, respectively. Tetraploid species had the highest level of diversity on three Glu-1 loci, followed by hexaploid and diploid, based on Shannon’s information index, Nei’s genetic diversity, and percentage of polymorphic loci. Molecular variance analysis confirmed main variance of HMW-GS within species, regions, and locations, respectively. Variance among species and regions was enhanced gradually with the increase of ploidy. Significant non-random distributions between the phylogenic trees of HMW-GS and the locations of accessions were tested by GenGIS software, indicated that geographic factors played an important role along the different orientations in the spread of Triticum species. We found one original diversified center in diploid what located around Elazig, Malatya, Gaziantep, Urfa, and Kiziltepe in Turkey, and three diversified centers in tetraploid wheat, including Turkey–Armenia–Georgia–Iran, Portugal–Spain, and Ethiopia, respectively, and two diversified adjacent areas between Turkey and Switzerland and around Turkey, Georgia, and Armenia. The original center of diploid species located in southeast Turkey, where the unexpressed 1Ay subunit was mainly distributed in T. urartu, could be one of the candidate regions of polyploidization of Triticum L. The regional distribution of HMW-GS and species also provided geographic evidences for the existence of founder effect on the spread of Triticum species. The present study suggests that integrating genetic diversity with geographic characterization in Triticum could very useful for collection, conservation, and utilization, as well as for research microevolution and domestication.     

Genetic diversity assessment of Bulgarian durum wheat (<Emphasis Type="Italic">Triticum durum</Emphasis> Desf.) landraces and modern cultivars using microsatellite markers

The genetic diversity in a Triticum durum Desf. collection, consisting of 102 Bulgarian landraces, nine Bulgarian and 25 introduced cultivars was studied using 14 highly polymorphic microsatellite markers. A total of 100 alleles were identified, with an average of 7.14 alleles per marker. The gene diversity values (He) of the markers for the total samples ranged from 0.23 (WMS357 and WMS631) to 0.77 (WMS46), with an average of 0.52. Within the landraces that were collected from 18 sites in Southern Bulgaria showed 2–11 alleles per locus with an average of 6.07. The microsatellite analysis suggests that the genetic diversity among landraces is lower compared to the diversity levels for durum wheat in countries close to the main centers of wheat domestication. Breeding activities have caused significant reduction of the allelic polymorphism, elimination of rare alleles, and increase in the number of common alleles and the frequency of dominating alleles.