Homoeologous Group Research Articles

A high-density genetic map for P genome of Agropyron Gaertn. based on specific-locus amplified fragment sequencing (SLAF-seq).

This genetic map for Agropyron Gaertn. contained 1023 markers on seven linkage groups, with a total of 907.8 cM and an average distance of 1.5 cM between adjacent loci. Many wheat- Agropyron cristatum derivative lines exhibit superior agronomic traits, and part of them are valuable for future wheat breeding. To date, no high-density genetic map for Agropyron Gaertn. has been published. Specific-locus amplified fragment sequencing (SLAF-seq), a recently developed strategy for large scale de novo discovery and genotyping of single nucleotide polymorphisms (SNPs), was employed in this study to develop sufficient markers for a segregating Agropyron F1 population derived from an interspecific cross between two cross-pollinated diploid collections A. cristatum (L.) Beauv. 'Z1842' and A. mongolicum Keng 'Z2098'. In total, we obtained raw data consisting of 128,932,358 pair-end reads of ~80 bp long after sequencing. Then 69,325 high-quality SLAFs were detected, of which 26,248 SLAFs were polymorphic and 1752 of the polymorphic markers were used for the genetic map construction. The final map contained 1023 markers on the seven linkage groups (LGs), which spanned a total of 907.8 cM with an average number of 146 markers and 89 loci per LG and an average distance of 1.5 cM between adjacent loci. To our knowledge, this map is the densest genetic linkage map for Agropyron so far. Through BLAT alignment of Agropyron SLAF marker sequences with the draft genome assemblies of wheat and barley, the Agropyron LGs were assigned as LG1-7 according to their corresponding homoeologous chromosomal groups of wheat. Results of this study will not only provide a platform for gene/QTL fine mapping, but also serve as a reference to assist the assembling of the P genome sequence in future.

Identification of Novel SNP in Promoter Sequence of TaGW2-6A Associated with Grain Weight and Other Agronomic Traits in Wheat (Triticum aestivum L.).

TaGW2 is an orthologue of rice gene OsGW2, which encodes E3 RING ubiquitin ligase and controls the grain size in rice. In wheat, three copies of TaGW2 have been identified and mapped on wheat homoeologous group 6 viz. TaGW2-6A, TaGW2-6B and TaGW2-6D. In the present study, using as many as 207 Indian wheat genotypes, we identified four SNPs including two novel SNPs (SNP-988 and SNP-494) in the promoter sequence of TaGW2-6A. All the four SNPs were G/A or A/G substitutions (transitions). Out of the four SNPs, SNP-494 was causal, since it was found associated with grain weight. The mean TGW (41.1 g) of genotypes with the allele SNP-494_A was significantly higher than mean TGW (38.6 g) of genotypes with the allele SNP-494_G. SNP-494 also regulates the expression of TaGW2-6A so that the wheat genotypes with SNP-494_G have higher expression and lower TGW and the genotypes with SNP-494_A have lower expression but higher TGW. Besides, SNP-494 was also found associated with grain length-width ratio, awn length, spike length, grain protein content, peduncle length and plant height. This suggested that gene TaGW2-6A not only controls grain size, but also controls other agronomic traits. In the promoter region, SNP-494 was present in ‘CGCG’ motif that plays an important role in Ca2+/calmodulin mediated regulation of genes. A user-friendly CAPS marker was also developed to identify the desirable allele of causal SNP (SNP-494) for use in marker-assisted selection for improvement of grain weight in wheat. Using four SNPs, five haplotypes were identified; of these, Hap_5 (G_A_G_A) was found to be a desirable haplotype having significantly higher grain weight (41.13g) relative to other four haplotypes (36.33-39.16 g).

Flow sorting of C-genome chromosomes from wild relatives of wheat Aegilops markgrafii, Ae. triuncialis and Ae. cylindrica, and their molecular organization.

Aegilops markgrafii (CC) and its natural hybrids Ae. triuncialis (U(t)U(t)C(t)C(t)) and Ae. cylindrica (D(c)D(c)C(c)C(c)) represent a rich reservoir of useful genes for improvement of bread wheat (Triticum aestivum), but the limited information available on their genome structure and the shortage of molecular (cyto-) genetic tools hamper the utilization of the extant genetic diversity. This study provides the complete karyotypes in the three species obtained after fluorescent in situ hybridization (FISH) with repetitive DNA probes, and evaluates the potential of flow cytometric chromosome sorting. The flow karyotypes obtained after the analysis of 4',6-diamidino-2-phenylindole (DAPI)-stained chromosomes were characterized and the chromosome content of the peaks on the flow karyotypes was determined by FISH. Twenty-nine conserved orthologous set (COS) markers covering all seven wheat homoeologous chromosome groups were used for PCR with DNA amplified from flow-sorted chromosomes and genomic DNA. FISH with repetitive DNA probes revealed that chromosomes 4C, 5C, 7C(t), T6U(t)S.6U(t)L-5C(t)L, 1C(c) and 5D(c) could be sorted with purities ranging from 66 to 91%, while the remaining chromosomes could be sorted in groups of 2-5. This identified a partial wheat-C-genome homology for group 4 and 5 chromosomes. In addition, 1C chromosomes were homologous with group 1 of wheat; a small segment from group 2 indicated 1C-2C rearrangement. An extensively rearranged structure of chromosome 7C relative to wheat was also detected. The possibility of purifying Aegilops chromosomes provides an attractive opportunity to investigate the structure and evolution of the Aegilops C genome and to develop molecular tools to facilitate the identification of alien chromatin and support alien introgression breeding in bread wheat.

Metabolomic profiling and genomic analysis of wheat aneuploid lines to identify genes controlling biochemical pathways in mature grain

Metabolomics is becoming an increasingly important tool in plant genomics to decipher the function of genes controlling biochemical pathways responsible for trait variation. Although theoretical models can integrate genes and metabolites for trait variation, biological networks require validation using appropriate experimental genetic systems. In this study, we applied an untargeted metabolite analysis to mature grain of wheat homoeologous group 3 ditelosomic lines, selected compounds that showed significant variation between wheat lines Chinese Spring and at least one ditelosomic line, tracked the genes encoding enzymes of their biochemical pathway using the wheat genome survey sequence and determined the genetic components underlying metabolite variation. A total of 412 analytes were resolved in the wheat grain metabolome, and principal component analysis indicated significant differences in metabolite profiles between Chinese Spring and each ditelosomic lines. The grain metabolome identified 55 compounds positively matched against a mass spectral library where the majority showed significant differences between Chinese Spring and at least one ditelosomic line. Trehalose and branched-chain amino acids were selected for detailed investigation, and it was expected that if genes encoding enzymes directly related to their biochemical pathways were located on homoeologous group 3 chromosomes, then corresponding ditelosomic lines would have a significant reduction in metabolites compared with Chinese Spring. Although a proportion showed a reduction, some lines showed significant increases in metabolites, indicating that genes directly and indirectly involved in biosynthetic pathways likely regulate the metabolome. Therefore, this study demonstrated that wheat aneuploid lines are suitable experimental genetic system to validate metabolomics-genomics networks.

Microsatellite analysis of chromosomes from the fifth homoeologous group in the introgressive Triticum aestivum/Amblyopyrum muticum wheat lines

Introgressive hybridization of common wheat has been and remains an efficient way for enriching its gene pool with resistance genes against environmental stress factors. In this aspect, T genome of the diploid species Amblyopyrum muticum (Boiss.) Eig. is believed to be prospective. The genome substitution amphidiploid Aurotica (AABBTT) with the A and B subgenomes from the common wheat cultivar Aurora and with the T subgenome from the indicated species is characterized by a high level of winter resistance. This trait is associated in wheat with the chromosomes of the fifth homoeologous group. Using a comparative microsatellite analysis of Aurora and Aurotica DNAs, we identified nine SSR loci specific for the wheat D genome, whose primers produced different amplicons from Aurotica DNA. The latter were used in screening F5 plants (Aurora × Aurotica) for the search of the specimens that carried the 5T chromosome or its fragments in their genomes. Among 234 plants, we detected 24 plants with disomic 5D/5T substitutions, which were the progeny of five F4 plants grown from two F2 plants. A significant number of plants contained the recombinant 5D/5T chromosome. The recombination event could occur in any generation starting with F2.

Molecular Characterization of a New Wheat-Thinopyrum intermedium Translocation Line with Resistance to Powdery Mildew and Stripe Rust

A new wheat-Thinopyrum translocation line CH13-21 was selected from the progenies derived from a cross between wheat-Th. intermedium partial amphiploid TAI7047 and wheat line Mianyang11. CH13-21 was characterized by using genomic in situ hybridization (GISH), multicolor-GISH (mc-GISH), multicolor-fluorescence in situ hybridization (mc-FISH) and chromosome-specific molecular markers. When inoculated with stripe rust and powdery mildew isolates, CH13-21 displayed novel resistance to powdery mildew and stripe rust which inherited from its Thinopyrum parent. The chromosomal counting analyses indicated that CH13-21 has 42 chromosomes, with normal bivalent pairing at metaphase I of meiosis. GISH probed by Th. intermedium genomic DNA showed that CH13-21 contained a pair of wheat-Th. intermedium translocated chromosomes. Sequential mc-FISH analyses probed by pSc119.2 and pAs1 clearly revealed that chromosome arm 6BS of CH13-21 was replaced by Thinopyrum chromatin in the translocation chromosome. The molecular markers analysis further confirmed that the introduced Th. intermedium chromatin in CH13-21 belonged to the long arm of homoeologous group 6 chromosome. Therefore, CH13-21 was a new T6BS.6Ai#1L compensating Robertsonian translocation line. It concludes that CH13-21 is a new genetic resource for wheat breeding programs providing novel variation for disease resistances.

Isolation of candidate genes for the barley Ant1 and wheat Rc genes controlling anthocyanin pigmentation in different vegetative tissues.

MYB transcription factors exist in a large copy number and control various plant phenotypes. We cloned R2R3 MYB-type transcription factors that determine the coloration of basal sheaths in barley and wheat coleoptiles. These genes are highly homologous to maize C1 and rice OsC1, regulators for anthocyanin biosynthesis, but they control seed pigmentation in maize and rice. On the basis of high homology, barley and wheat counterparts are designated HvC1 and TaC1, respectively. HvC1 gene is located on the short arm of chromosome 7H, and TaC1 genes are located on the short arms of chromosomes 7A, 7B, and 7D (TaC1-A1, B1, and D1, respectively). HvC1 is a strong candidate for Ant1 because of (1) complete co-segregation of anthocyanin pigmentation phenotype of the basal sheath with the HvC1 genotype in genetic mapping, and (2) complete deletion of the HvCl gene in two anthocyanin-decreased allelic mutants (ant1.1 and ant1.2) that were induced by irradiation. In contrast, colorless coleoptile wheat lines had lesions in all three genomes consisting of a single-nucleotide substitution or a 1-bp deletion of TaC1-A1, a 1.7-kb insertion of TaC1-B1, and a 2.0-kb insertion of TaC1-D1. At least one normal TaC1 gene appears to be sufficient to produce anthocyanin pigments in wheat coleoptiles. Previous crossing experiments localized Rc (red coleoptile) genes to homoeologous group 7 chromosomes and deduced Rc genotypes of several wheat lines. Their TaC1 gene sequence variation coincided with deduced Rc genotypes; therefore, the present molecular genetic study demonstrates that TaC1 is a strong candidate for Rc in wheat.

Lutein esterification in wheat endosperm is controlled by the homoeologous group 7, and is increased by the simultaneous presence of chromosomes 7D and 7Hch from Hordeum chilense

The high carotenoid content in tritordeum (×Tritordeum Ascherson et Graebner) grains is derived from its wild parent, Hordeum chilense Roem. et Schulz. Phytoene synthase 1 (Psy1) is located on chromosome 7HchS and plays a major role in this trait. This study investigates the impact of the introgression of chromosome 7Hch into common wheat background on carotenoid composition, including xanthophylls esterified with fatty acids (monoesters and diesters). All of the genetic stocks carrying Psy1 from H. chilense increased their carotenoid content relative to common wheat. In addition, significant changes in the carotenoid profile were detected in different genetic stocks. The most relevant was the increase in content of lutein diesters when both 7Hch and 7D were present, which indicates the existence of genes involved in the esterification of xanthophylls in both chromosomes. Furthermore, our results suggest that 7Hch genes preferentially esterify lutein with palmitic acid, whereas 7D is either indifferent to the fatty acid or it prefers linoleic acid for lutein esterification. The involvement and complementarity of 7Hch and 7D are highly significant considering the scarcity of previous results on lutein esterification in wheat.

Molecular-cytogenetic analysis of Aegilops triuncialis and identification of its chromosomes in the background of wheat.

BackgroundSpecies belonging to the genus Aegilops L. are an important source of genetic material for expanding genetic variability of wheat. Ae. triuncialis is an allotetraploid in this genus which was originated from hybridization of Ae. umbellulata and Ae. markgrafii (Greuter) Hammer. Although the Ae. triuncialis karyotype was thoroughly examined by conventional chromosome staining and Giemsa C-banding, it is still poorly characterized using FISH markers. The objective of this study was to test the fluorescence in situ hybridization (FISH) patterns of Ae. triuncialis (2n = 4x = 28, CtCtUtUt) chromosomes using different repetitive sequences and to compare the produced patterns to the chromosomes of its diploid ancestors, with the aim of establishing a generalized Ae. triuncialis idiogram and detection of Aegilops chromosomes in the background of wheat.ResultsThe probes pSc119.2-1, pTa535-1, pAs1-1, (CTT)10 and the 45S rDNA clone from wheat (pTa71) were hybridized to chromosomes of Ae. triuncialis and compared with its diploid progenitors (Ae. umbellulata Zhuk., 2n = 2x = 14, UU and Ae. markgrafii (Greuter) Hammer, 2n = 2x = 14, CC) and Ae. cylindrica Host. (2n = 4x = 28, DcDcCcCc), another tetraploid species containing the C-genome. Ae. cylindrica was further analyzed by genomic in situ hybridization (GISH) using C genome probe in order to identify any possible translocation.ConclusionsIn general, FISH patterns of the Ut- and Ct-genome chromosomes of Ae. triuncialis were similar to those of U- and C-genome chromosomes of the diploid progenitor species Ae. umbellulata and Ae. markgrafii respectively, although some differences were observed. Two major 45S rDNA loci were revealed in the short arm of chromosomes A and C, of the Ct genome which correspond to homoeologous groups 1 and 5 respectively. Minor 45S rDNA loci were mapped on the short arm of chromosomes 1Ut and 5Ut. GISH analysis revealed three different non-reciprocal homologous or heterologous translocations between Cc and Dc chromosomes in all studied accessions of Ae. cylindrica.Electronic supplementary materialThe online version of this article (doi:10.1186/s13039-014-0091-6) contains supplementary material, which is available to authorized users.

Cytological and molecular characterization of wheat-Hordeum chilense chromosome 7Hch introgression lines

Chromosome 7Hch of Hordeum chilense carries the Phytoene synthase 1 (Psy1) gene encoding the first step in the carotenoid biosynthetic pathway. As such it can be used in the improvement of seed carotenoid content in wheat. Four introgressions of chromosome 7Hch into wheat have been characterized by in situ hybridization of labeled DNA probes and by several sets of DNA markers. Chromosome-specific SSR were used for the identification of wheat chromosomes. Besides 113 conserved orthologous set (COS) markers were tested for homoeologous group 7, of which 97 amplified in H. chilense and 32 were polymorphic between H. chilense and wheat, and 28 expressed sequence tag (EST) barley markers previously allocated to chromosome 7. A total of 60 markers (32 COS and 28 EST) were allocated to chromosome 7Hch with 28 assigned to 7HchS and 22 to 7HchL. A combination of in situ probing and marker genotyping have shown that among the four introgressions there was a substitution line 7Hch (7D), a ditelosomic addition line for the long arm of 7Hch and two homozygous centric translocations 7HchS·2DS and 7HchS·5AL. The Psy1 gene was localized on the short arm of 7Hch. The positions of markers from the international barley consortium map (IBSC2012) were determined and the comparative arm location between H. chilense and H. vulgare is discussed. The genetic stocks characterized here include new wheat-H. chilense recombinations useful for genetic studies and with a potential for breeding.

Chromosome constitution and origin analysis in three derivatives of Triticum aestivum--Leymus mollis by molecular cytogenetic identification.

Leymus mollis (2n = 4x = 28, NsNsXmXm) is an important tetraploid species in Leymus (Poaceae: Triticeae) and a useful genetic resource for wheat breeding because of the stress tolerance and disease resistance of this species. The development of Triticum aestivum (common wheat) - L. mollis derivatives with desirable genes will provide valuable bridge materials for wheat improvement, especially regarding powdery mildew resistance genes, which are rarely documented in L. mollis. In the present study, three derivatives of common wheat cultivar 7182 and L. mollis, namely M47, M51, and M42, were subjected to chromosomal characterization via cytogenetic identification, the analysis of molecular markers, and genomic in situ hybridization. These derivatives were all morphologically and cytogenetically stable. M47 was highly resistant to powdery mildew and nearly immune to stripe rust at the adult stage, and the chromosome constitution of this derivative can be expressed as 2n = 56 = 42T.a + 14L.m (where T.a = T. aestivum chromosomes; L.m = L. mollis chromosomes). Compared to M47, M42 was also resistant to stripe rust but was susceptible to powdery mildew; the chromosome constitution of M42 was 2n = 54 = 42T.a + 12L.m, in which a pair of homoeologous group 7 L.m chromosomes was eliminated. Finally, M51 was susceptible to powdery mildew and stripe rust and had a chromosome constitution of 2n = 48 = 42T.a + 6L.m, in which four pairs of L.m chromosomes from homoeologous groups 2, 4, 5, and 7 were eliminated. The differing disease resistances of the three derivatives are discussed in this report in the context of their chromosomal variations; this information can thus contribute to breeding disease resistant wheat with the potential for applying these derivatives as useful bridge materials.

Evolutionary history of Methyltransferase 1 genes in hexaploid wheat.

BackgroundPlant and animal methyltransferases are key enzymes involved in DNA methylation at cytosine residues, required for gene expression control and genome stability. Taking advantage of the new sequence surveys of the wheat genome recently released by the International Wheat Genome Sequencing Consortium, we identified and characterized MET1 genes in the hexaploid wheat Triticum aestivum (TaMET1).ResultsNine TaMET1 genes were identified and mapped on homoeologous chromosome groups 2A/2B/2D, 5A/5B/5D and 7A/7B/7D. Synteny analysis and evolution rates suggest that the genome organization of TaMET1 genes results from a whole genome duplication shared within the grass family, and a second gene duplication, which occurred specifically in the Triticeae tribe prior to the speciation of diploid wheat. Higher expression levels were observed for TaMET1 homoeologous group 2 genes compared to group 5 and 7, indicating that group 2 homoeologous genes are predominant at the transcriptional level, while group 5 evolved into pseudogenes. We show the connection between low expression levels, elevated evolution rates and unexpected enrichment in CG-dinucleotides (CG-rich isochores) at putative promoter regions of homoeologous group 5 and 7, but not of group 2 TaMET1 genes. Bisulfite sequencing reveals that these CG-rich isochores are highly methylated in a CG context, which is the expected target of TaMET1.ConclusionsWe retraced the evolutionary history of MET1 genes in wheat, explaining the predominance of group 2 homoeologous genes and suggest CG-DNA methylation as one of the mechanisms involved in wheat genome dynamics.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-922) contains supplementary material, which is available to authorized users.

A novel wheat bZIP transcription factor, TabZIP60, confers multiple abiotic stress tolerances in transgenic Arabidopsis.

The basic region/leucine zipper (bZIP) transcription factors (TFs) play vital roles in the response to abiotic stress. However, little is known about the function of bZIP genes in wheat abiotic stress. In this study, we report the isolation and functional characterization of the TabZIP60 gene. Three homologous genome sequences of TabZIP60 were isolated from hexaploid wheat and mapped to the wheat homoeologous group 6. A subcellular localization analysis indicated that TabZIP60 is a nuclear-localized protein that activates transcription. Furthermore, TabZIP60 gene transcripts were strongly induced by polyethylene glycol, salt, cold and exogenous abscisic acid (ABA) treatments. Further analysis showed that the overexpression of TabZIP60 in Arabidopsis resulted in significantly improved tolerances to drought, salt, freezing stresses and increased plant sensitivity to ABA in seedling growth. Meanwhile, the TabZIP60 was capable of binding ABA-responsive cis-elements that are present in promoters of many known ABA-responsive genes. A subsequent analysis showed that the overexpression of TabZIP60 led to enhanced expression levels of some stress-responsive genes and changes in several physiological parameters. Taken together, these results suggest that TabZIP60 enhances multiple abiotic stresses through the ABA signaling pathway and that modifications of its expression may improve multiple stress tolerances in crop plants.

A Novel Wheat-Dasypyrum breviaristatum Substitution Line with Stripe Rust Resistance

The introduction of genetic variation from wild and cultivated Triticeae species has been a long-standing approach for wheat improvement. Dasypyrumbreviaristatum species harbor novel and agronomically important genes for resistance against multi-fungal diseases. The development of new wheat-D. breviaristatum introgression lines offers chances for the identification of stripe rust resistance gene(s). A wheat line, D11-5, was selected from a cross between wheat line MY11 and wheat-D. breviaristatum partial amphiploid TDH-2. It was characterized by FISH and PCR-based molecular markers. Chromosome counting revealed that the D11-5 line shows a hexaploid set of 2n = 6x = 42 chromosomes. FISH analysis using the Dasypyrum repetitive sequence pDb12H as a probe demonstrated that D11-5 contained a pair of D. breviaristatum chromosomes, while FISH with wheat D-genomic repetitive sequences revealed that the chromosome 2D was absent in D11-5. The functional molecular markers confirmed that the introduced D. breviaristatum chromosomes belong to the homoeologous group 2, indicating that D11-5 was a 2V^b (2D) disomic substitution line. Field resistance showed that the introduced D. breviaristatum chromosomes 2V^b were responsible for the stripe rust resistance at the adult plant stage. FISH, C-banding, and PCR-based molecular marker analysis indicated that the chromosome 2V^b of D. breviaristatum was completely different from the chromosome 2V of D. villosum. The identified wheat-D. breviaristatum chromosome substitution line D11-5 may be applied to produce agronomically desirable stripe rust resistance germplasm.

Mapping QTLs of yield-related traits using RIL population derived from common wheat and Tibetan semi-wild wheat.

QTLs controlling yield-related traits were mapped using a population derived from common wheat and Tibetan semi-wild wheat and they provided valuable information for using Tibetan semi-wild wheat in future wheat molecular breeding. Tibetan semi-wild wheat (Triticum aestivum ssp tibetanum Shao) is a kind of primitive hexaploid wheat and harbors several beneficial traits, such as tolerance to biotic and abiotic stresses. And as a wild relative of common wheat, heterosis of yield of the progeny between them was significant. This study focused on mapping QTLs controlling yield-related traits using a recombined inbred lines (RILs) population derived from a hybrid between a common wheat line NongDa3331 (ND3331) and the Tibetan semi-wild wheat accession Zang 1817. In nine location-year environments, a total of 148 putative QTLs controlling nine traits were detected, distributed on 19 chromosomes except for 1A and 2D. Single QTL explained the phenotypic variation ranging from 3.12 to 49.95%. Of these QTLs, 56 were contributed by Zang 1817. Some stable QTLs contributed by Zang 1817 were also detected in more than four environments, such as QPh-3A1, QPh-4B1 and QPh-4D for plant height, QSl-7A1 for spike length, QEp-4B2 for ears per plant, QGws-4D for grain weight per spike, and QTgw-4D for thousand grain weight. Several QTL-rich Regions were also identified, especially on the homoeologous group 4. The TaANT gene involved in floral organ development was mapped on chromosome 4A between Xksm71 and Xcfd6 with 0.8 cM interval, and co-segregated with the QTLs controlling floret number per spikelet, explaining 4.96-11.84% of the phenotypic variation. The current study broadens our understanding of the genetic characterization of Tibetan semi-wild wheat, which will enlarge the genetic diversity of yield-related traits in modern wheat breeding program.

Assessing genetic diversity of Egyptian hexaploid wheat (Triticum aestivum L.) using microsatellite markers

Genetic diversity was investigated in a set of thirty-three hexaploid wheat genotypes originated from Egypt, using 17 wheat microsatellites, representatives of fifteen wheat chromosomes. In total, ninety-five alleles were detected among Egyptian wheats. For 17 polymorphic microsatellite markers, the number of alleles per locus varied from 3 for Xgwm261-2DS, Xgwm3-3DL and Xgwm631-7AS to 11 for Xgwm437-7DL, with a mean of 5.59 alleles per locus. The highest average number of alleles per locus was detected in the B genome with 6.00, compared to 5.67 and 5.00 for genomes D and A, respectively. The highest and the lowest average number of alleles per locus among the wheat homoeologous groups were observed in group 7 and 3 with 7.00 and 4.00, respectively. Gene diversity for 17 microsatellites loci varied from 0.339 for Xgwm631-7AS to 0.845 for Xtaglgap-1BS with an average of 0.653. However, the gene diversity for three genomes A, B and D was 0.549, 0.718 and 0.674, respectively. A significant correlation coefficient between gene diversity and the number of alleles per locus, genomes and homoeologous groups was high, r = 0.649, 0.988 and 0.272 (P < 0.01), respectively. The gene diversity increased as the number of alleles increased. Cluster analysis was conducted based on microsatellites data. Five groups can be distinguished by truncating the dendrogram at genetic similarity (gs) value of 0.48. The present study indicated the presence of high diversity in Egyptian wheat genotypes.

Development of T. aestivum L.–H. californicum Alien Chromosome Lines and Assignment of Homoeologous Groups of Hordeum californicum Chromosomes

Hordeum californicum (2n = 2x = 14, HH) is resistant to several wheat diseases and tolerant to lower nitrogen. In this study, a molecular karyotype of H. californicum chromosomes in the Triticum aestivum L. cv. Chinese Spring (CS)–H. californicum amphidiploid (2n = 6x = 56, AABBDDHH) was established. By genomic in situ hybridization (GISH) and multicolor fluorescent in situ hybridization (FISH) using repetitive DNA clones (pTa71, pTa794 and pSc119.2) as probes, the H. californicum chromosomes could be differentiated from each other and from the wheat chromosomes unequivocally. Based on molecular karyotype and marker analyses, 12 wheat–alien chromosome lines, including four disomic addition lines (DAH1, DAH3, DAH5 and DAH6), five telosomic addition lines (MtH7L, MtH1S, MtH1L, DtH6S and DtH6L), one multiple addition line involving H. californicum chromosome H2, one disomic substitution line (DSH4) and one translocation line (TH7S/1BL), were identified from the progenies derived from the crosses of CS–H. californicum amphidiploid with common wheat varieties. A total of 482 EST (expressed sequence tag) or SSR (simple sequence repeat) markers specific for individual H. californicum chromosomes were identified, and 47, 50, 45, 49, 21, 51 and 40 markers were assigned to chromosomes H1, H2, H3, H4, H5, H6 and H7, respectively. According to the chromosome allocation of these markers, chromosomes H2, H3, H4, H5, and H7 of H. californicum have relationship with wheat homoeologous groups 5, 2, 6, 3, and 1, and hence could be designated as 5Hc, 2Hc, 6Hc, 3Hc and 1Hc, respectively. The chromosomes H1 and H6 were designated as 7Hc and 4Hc, respectively, by referring to SSR markers located on rye chromosomes.

Development and application of PCR markers specific to the 1Ns chromosome of Psathyrostachys huashanica Keng with leaf rust resistance

Wheat–Psathyrostachys huashanica Keng disomic addition line 12-3 was developed and characterized using genomic in situ hybridization (GISH), expressed sequence tag–sequence tagged site (EST–STS), and sequence characterized amplified region (SCAR) markers. Mitotic and meiotic GISH analyses indicated that it contained 42 wheat chromosomes and a pair of P. huashanica chromosomes. Eight EST–STS multiple-loci markers located on the homoeologous group 1 chromosomes of wheat amplified polymorphic bands in the 1Ns disomic addition line 12-3, which were unique to P. huashanica. These markers suggested that the introduced Ns chromosomes belonged to homoeologous group 1. Furthermore, diagnostic fragments of random amplified polymorphic DNA marker OPAG10986 and simple sequence repeat marker Xgwm601 135 were cloned, sequenced, and converted into SCAR markers, i.e., RHS153 and SHS10, respectively, which were validated using a range of distinct plant species and a complete set of wheat–P. huashanica disomic addition lines (1Ns–7Ns, 2n = 44 = 22 II). The results demonstrated that the SCAR markers targeted the Ns genome of P. huashanica and they were linked to the 1Ns chromosome. In addition, 12-3 was evaluated to test its leaf rust resistance in the adult stages and its agronomic traits. These newly developed EST–STS and SCAR markers will be powerful tools for wheat breeders who want to screen for genotypes containing the 1Ns chromosome, with low costs and high throughput.

Genome-wide association for grain morphology in synthetic hexaploid wheats using digital imaging analysis.

BackgroundGrain size and shape greatly influence grain weight which ultimately enhances grain yield in wheat. Digital imaging (DI) based phenomic characterization can capture the three dimensional variation in grain size and shape than has hitherto been possible. In this study, we report the results from using digital imaging of grain size and shape to understand the relationship among different components of this trait, their contribution to enhance grain weight, and to identify genomic regions (QTLs) controlling grain morphology using genome wide association mapping with high density diversity array technology (DArT) and allele-specific markers.ResultsSignificant positive correlations were observed between grain weight and grain size measurements such as grain length (r = 0.43), width, thickness (r = 0.64) and factor from density (FFD) (r = 0.69). A total of 231 synthetic hexaploid wheats (SHWs) were grouped into five different sub-clusters by Bayesian structure analysis using unlinked DArT markers. Linkage disequilibrium (LD) decay was observed among DArT loci > 10 cM distance and approximately 28% marker pairs were in significant LD. In total, 197 loci over 60 chromosomal regions and 79 loci over 31 chromosomal regions were associated with grain morphology by genome wide analysis using general linear model (GLM) and mixed linear model (MLM) approaches, respectively. They were mainly distributed on homoeologous group 2, 3, 6 and 7 chromosomes. Twenty eight marker-trait associations (MTAs) on the D genome chromosomes 2D, 3D and 6D may carry novel alleles with potential to enhance grain weight due to the use of untapped wild accessions of Aegilops tauschii. Statistical simulations showed that favorable alleles for thousand kernel weight (TKW), grain length, width and thickness have additive genetic effects. Allelic variations for known genes controlling grain size and weight, viz. TaCwi-2A, TaSus-2B, TaCKX6-3D and TaGw2-6A, were also associated with TKW, grain width and thickness. In silico functional analysis predicted a range of biological functions for 32 DArT loci and receptor like kinase, known to affect plant development, appeared to be common protein family encoded by several loci responsible for grain size and shape.ConclusionConclusively, we demonstrated the application and integration of multiple approaches including high throughput phenotyping using DI, genome wide association studies (GWAS) and in silico functional analysis of candidate loci to analyze target traits, and identify candidate genomic regions underlying these traits. These approaches provided great opportunity to understand the breeding value of SHWs for improving grain weight and enhanced our deep understanding on molecular genetics of grain weight in wheat.

Molecular characterization of a wheat–Psathyrostachys huashanica Keng 2Ns disomic addition line with resistance to stripe rust

We characterized a wheat-Psathyrostachys huashanica derived line 3-6-4-1 based on genomic in situ hybridization (GISH), molecular marker analysis, and agronomic trait evaluations. The GISH investigations showed that the 3-6-4-1 contained 42 wheat chromosomes and a pair of P. huashanica chromosomes. The homoeologous relationships of the introduced P. huashanica chromosomes were determined using EST-STS multiple loci markers from seven wheat homoeologous groups in the parents and the addition line. Twelve EST-STS markers located on the homoeologous group 2 chromosomes of wheat amplified polymorphic bands in 3-6-4-1, which were unique to P. huashanica. An introduced Ns chromosome pair that belonged to homoeologous group 2 was identified using chromosome-specific markers. Inoculation with isolates of the stripe rust pathotypes, CYR31, CYR32, and SY11-14, and mixed races (CYR31, CYR32, and SY11-14) in the seeding and adult stage, respectively, showed that 3-6-4-1 was generally resistant to stripe rust, which was probably attributable to its P. huashanica parent. We also compared a complete set of wheat-P. huashanica disomic addition lines (1Ns-7Ns, 2n=44=22II) to assess their agronomic traits and morphological characteristics, which showed that 3-6-4-1 had improved spike traits compared with its parents. The P. huashanica 2Ns chromosome-specific molecular markers in 3-6-4-1 could be useful for marker-assisted selection in breeding programs to combat stripe rust. This line can be used as a donor source to introduce novel excellent genes from P. huashanica into wheat to widen its genetic diversity, thereby providing new germplasms for wheat breeding.