Phylogenetic analysis of tetraploid wheat based on nuclear DMC1 gene

Tetraploid wheat (AABB or AAGG, 2n = 4x = 28) holds an important place in Triticum. It includes two allopolyploid species, Triticum turgidum and Triticum timopheevii. Many problems concerning the phylogenetic relationships among tetraploid wheat species remain unresolved. In this study, sequences data for the nuclear DMC1 gene from 61 accessions of Triticum and Aegilops species, representing diploid and tetraploid species, were used to examine the phylogenetic relationships among tetraploid wheat. Phylogenetic trees were constructed using maximum-likelihood and neighbor-joining approaches, and gene flow and genetic differentiation values were computed. The results indicated that the A genome of tetraploid wheat originated from T. urartu rather than T. monococcum, and Aegilops speltoides was the donor of the B and G genomes. Hulled tetraploid wheat accessions formed a subclade, and naked tetraploid wheat got other subclade, indicating that at least two intermediary subspecies were involved in the evolution of T. turgidum. Triticum turgidum and T. timopheevii might have simultaneously originated from a hybridization events. These results indicated that the DMC1 gene sequences are useful for resolution of the molecular phylogenetic relationships of tetraploid wheat.     

Map-based analysis of the tenacious glume gene Tg-B1 of wild emmer and its role in wheat domestication

The domestication of wheat was instrumental in spawning the civilization of humankind, and it occurred through genetic mutations that gave rise to types with non-fragile rachises, soft glumes, and free-threshing seed. Wild emmer (Triticum turgidum ssp. dicoccoides), the tetraploid AB-genome progenitor of domesticated wheat has genes that confer tenacious glumes (Tg) that underwent genetic mutations to give rise to free-threshing wheat. Here, we evaluated disomic substitution lines involving chromosomes 2A and 2B of wild emmer accessions substituted for homologous chromosomes in tetraploid and hexaploid backgrounds. The results suggested that both chromosomes 2A and 2B of wild emmer possess genes that inhibit threshability. A population of recombinant inbred lines derived from the tetraploid durum wheat variety Langdon crossed with a Langdon — T. turgidum ssp. dicoccoides accession PI 481521 chromosome 2B disomic substitution line was used to develop a genetic linkage map of 2B, evaluate the genetics of threshability, and map the gene derived from PI 481521 that inhibited threshability. A 2BS linkage map comprised of 58 markers was developed, and markers delineated the gene to a 2.3 cM interval. Comparative analysis with maps containing the tenacious glume gene Tg-D1 on chromosome arm 2DS from Aegilops tauschii, the D genome progenitor of hexaploid wheat, revealed that the gene inhibiting threshability in wild emmer was homoeologous to Tg-D1 and therefore designated Tg-B1. Comparative analysis with rice and Brachypodium distachyon indicated a high level of divergence and poorly conserved colinearity, particularly near the Tg-B1 locus. These results provide a foundation for further studies involving Tg-B1, which, together with Tg-D1, had profound influences on wheat domestication.     

Genetic diversity of Triticum turgidum L. based on microsatellite markers

Using microsatellite (SSR) markers, the genetic diversity and genetic relationships among 48 Triticum turgidum L. accessions, including 30 Triticum turgidum L. ssp. turgidum, 7 Triticum turgidum L. ssp. durum, 4 Triticum turgidum L. ssp. carthlicum, 3 Triticum turgidum L. ssp. paleocolchicum, 2 Triticum turgidum L. ssp. turanicum, and 2 Triticum turgidum L. ssp. polonicum accessions, were investigated. A total of 97 alleles were detected at 16 SSR loci. At each locus, the number of alleles ranged from two to fourteen, with an average of 6.1. The genetic similarity (GS) value ranged from 0.20 to 0.92, with the mean of 0.59. In cluster analysis, it was found the 48 Triticum turgidum L. accessions could be distinguished easily by SSR markers, whereas the six subspecies taxonomic entities of T. turgidum L. could not differentiate with each other, indicating that the morphological differences present among the six subspecies could not be reflected by the SSR markers. These results suggested that SSR markers had superiority in detecting the genetic diversity of T. turgidum L., while they were not good for studies of the phylogenic relationships among the subspecies of T. turgidum L. The text was submitted by the authors in English.     

Genome-Wide Association of Stem Water Soluble Carbohydrates in Bread Wheat

Water soluble carbohydrates (WSC) in stems play an important role in buffering grain yield in wheat against biotic and abiotic stresses; however, knowledge of genes controlling WSC is very limited. We conducted a genome-wide association study (GWAS) using a high-density 90K SNP array to better understand the genetic basis underlying WSC, and to explore marker-based breeding approaches. WSC was evaluated in an association panel comprising 166 Chinese bread wheat cultivars planted in four environments. Fifty two marker-trait associations (MTAs) distributed across 23 loci were identified for phenotypic best linear unbiased estimates (BLUEs), and 11 MTAs were identified in two or more environments. Liner regression showed a clear dependence of WSC BLUE scores on numbers of favorable (increasing WSC content) and unfavorable alleles (decreasing WSC), indicating that genotypes with higher numbers of favorable or lower numbers of unfavorable alleles had higher WSC content. In silico analysis of flanking sequences of trait-associated SNPs revealed eight candidate genes related to WSC content grouped into two categories based on the type of encoding proteins, namely, defense response proteins and proteins triggered by environmental stresses. The identified SNPs and candidate genes related to WSC provide opportunities for breeding higher WSC wheat cultivars.     

Molecular Characterization of the pina Gene in Einkorn Wheat

Fifty-six sequences encoding the pina protein were characterized from three species or subspecies of einkorn wheat. These sequences contained 1,595 nucleotides, including 1,270 conserved sites, 21 single nucleotide polymorphisms (SNPs), and 16 indels. The average frequency of SNPs and indels was one out of 76.1 and 99.9 bases, respectively. Five SNPs and no indels were found in the translated sequences. Fourteen haplotypes were defined, and the accessions in each haplotype ranged from 1 to 18. There were nine haplotypes in Triticum monococcum ssp. aegilopoides, eight in T. monococcum ssp. monococcum, and two in T. urartu. Phylogenetic analysis showed that pina genes from different species or subspecies could be clearly differentiated based on the open reading frame. Genes from T. urartu grouped together, whereas genes from T. monococcum ssp. aegilopoides and T. monococcum ssp. monococcum were shared by three and two clusters, respectively. Both the haplotype and phylogenetic analyses indicated that T. monococcum ssp. aegilopoides was more diverse. These results would contribute to the understanding of functional aspects and efficient utilization of pina genes.     

In-depth genetic analysis reveals conditioning of polyphenol oxidase activity in wheat grains by cis regulation of TaPPO2A-1 expression level

Time-dependent darkening and discoloration of wheat product caused by high polyphenol oxidase enzymes (PPO) activity is the most undesirable character in wheat processing industry. We performed GWAS of PPO activity in wheat grains utilizing an association panel and identified 22 significant SNPs. The most significant GWAS peak on chromosome 2A was verified by QTL analysis of PPO activity. The candidate gene for this GWAS peak was identified as TaPPO2A-1, which was the highest expressed PPO gene in wheat grains. The expression level of TaPPO2A-1 was significantly correlated with PPO activity. The most significant association signal for GWAS of the expression values of TaPPO2A-1 pinpointed to the genomic region containing TaPPO2A-1. The results suggested that cis regulation of TaPPO2A-1 expression is the key factor in regulation of PPO activity in wheat grains. The conclusion was further enhanced by haplotype analysis of seven SNPs in the promoter of TaPPO2A-1.     

Reproductive and genetic roles of the maternal progenitor in the origin of common wheat (Triticum aestivum L.)

Common wheat (Triticum aestivum L., AABBDD genome) is thought to have emerged through natural hybridization between Triticum turgidum L. (AABB genome) and Aegilops tauschii Coss. (DD genome). Hybridization barriers and doubling of the trihaploid F₁ hybrids’ genome (ABD) via unreduced gamete fusion had key roles in the process. However, how T. turgidum, the maternal progenitor, was involved in these mechanisms remains unknown. An artificial cross‐experiment using 46 cultivated and 31 wild T. turgidum accessions and a single Ae. tauschii tester with a very short genetic distance to the common wheat D genome was conducted. Cytological and quantitative trait locus analyses of F₁ hybrid genome doubling were performed. The crossability and ability to cause hybrid inviability did not greatly differ between the cultivars and wild accessions. The ability to cause hybrid genome doubling was higher in the cultivars. Three novel T. turgidum loci for hybrid genome doubling, which influenced unreduced gamete production in F₁ hybrids, were identified. Cultivated T. turgidum might have increased the probability of the emergence of common wheat through its enhanced ability to cause genome doubling in F₁ hybrids with Ae. tauschii. The ability enhancement might have involved alterations at a relatively small number of loci.     

Genetic diversity for morphological traits and seed storage proteins in Spanish rivet wheat

The objective of the current work was to analyse the variability of high and low molecular mass (HMM and LMM) glutenin subunits, along with some morphological characteristics in sixty Spanish accessions of rivet wheat (Triticum turgidum L. ssp. turgidum). The lines were grouped in sixteen botanical varieties and five additional types, according the morphological criteria. Up to 13 allelic variants (four alleles for the Glu-A1 locus and nine alleles for the Glu-B1 locus) and 34 B-LMMGs patterns were found in the evaluated lines. The current data indicated a clear reduction of morphological variability, along with an asymmetric distribution of the alleles and patterns for seed storage proteins. This polymorphism could be useful for enlarging the genetic background of modern durum wheat.     

Genetic diversity in wild wheats and goat grass

The genetic structure of 35 populations of wild relatives of cultivated wheats, all collected in Syria and Lebanon, was assessed using ten isozymes. The populations consisted of diploid goat grass, Aegilops speltoides, diploid wild wheats, Triticum monococcum spp. aegilopoides and T. urartu, and tetraploid wild wheat, T. turgidum ssp. dicoccoides. The majority of the populations were polymorphic (P=0–70%) having low within-population mean genetic diversity (H_ep=0.05–0.10) and relatively high within-species genetic diversity (H_es=0.14–0.31). The linkage between loci did not seem to be one of the causes for the observed polymorphism. All four species showed significant inbreeding at both the population (0.31–0.64) and species (0.77–0.96) levels, and the extent of inbreeding did not correlate with mating systems. Despite their apparent common ecological and evolutionary history, between-population or between-species level genetic identity was low (I=0.43–0.86). Among the diploid species, populations of Ae. speltoides clustered distinctly from those overlapping clusters of T. monococcum ssp. aegilopoides and T. urartu. The tetraploid species T. turgidum ssp. dicoccoides had relatively less genetic diversity (H_es=0.14) and was highly homozygous (F=0.96). The results suggest that these wild progenitors of cultivated wheats have undergone extensive local differentiation and inbreeding. We discuss the implications of our results on the management of wild wheat and goat grass populations. Received: 12 September 1999 / Accepted: 10 November 1999     

Integration of dinucleotide microsatellites from hexaploid bread wheat into a genetic linkage map of durum wheat

 Seventy nine microsatellite markers from hexaploid bread wheat (T. aestivum L.) were integrated into a genetic linkage map of durum wheat (T. turgidum ssp. durum (Desf.) Huns.) created by RFLP segregation data from a population of 65 recombinant inbred lines. The results indicate a relatively even distribution of microsatellite loci and demonstrate that microsatellite markers from hexaploid wheat provide an excellent source of molecular markers for use in the genetics and breeding of durum wheat. Received: 16 July 1998 / Accepted: 13 October 1998     

Genetic Dissection of Drought and Heat Tolerance in Chickpea through Genome-Wide and Candidate Gene-Based Association Mapping Approaches

To understand the genetic basis of tolerance to drought and heat stresses in chickpea, a comprehensive association mapping approach has been undertaken. Phenotypic data were generated on the reference set (300 accessions, including 211 mini-core collection accessions) for drought tolerance related root traits, heat tolerance, yield and yield component traits from 1–7 seasons and 1–3 locations in India (Patancheru, Kanpur, Bangalore) and three locations in Africa (Nairobi, Egerton in Kenya and Debre Zeit in Ethiopia). Diversity Array Technology (DArT) markers equally distributed across chickpea genome were used to determine population structure and three sub-populations were identified using admixture model in STRUCTURE. The pairwise linkage disequilibrium (LD) estimated using the squared-allele frequency correlations (r²; when r²<0.20) was found to decay rapidly with the genetic distance of 5 cM. For establishing marker-trait associations (MTAs), both genome-wide and candidate gene-sequencing based association mapping approaches were conducted using 1,872 markers (1,072 DArTs, 651 single nucleotide polymorphisms [SNPs], 113 gene-based SNPs and 36 simple sequence repeats [SSRs]) and phenotyping data mentioned above employing mixed linear model (MLM) analysis with optimum compression with P3D method and kinship matrix. As a result, 312 significant MTAs were identified and a maximum number of MTAs (70) was identified for 100-seed weight. A total of 18 SNPs from 5 genes (ERECTA, 11 SNPs; ASR, 4 SNPs; DREB, 1 SNP; CAP2 promoter, 1 SNP and AMDH, 1SNP) were significantly associated with different traits. This study provides significant MTAs for drought and heat tolerance in chickpea that can be used, after validation, in molecular breeding for developing superior varieties with enhanced drought and heat tolerance.     

Heterochronic development of the floret meristem determines grain number per spikelet in diploid, tetraploid and hexaploid wheats

Background and Aims

The inflorescence of grass species such as wheat, rice and maize consists of a unique reproductive structure called the spikelet, which is comprised of one, a few, or several florets (individual flowers). When reproductive growth is initiated, the inflorescence meristem differentiates a spikelet meristem as a lateral branch; the spikelet meristem then produces a floret meristem as a lateral branch. Interestingly, in wheat, the number of fertile florets per spikelet is associated with ploidy level: one or two florets in diploid, two or three in tetraploid, and more than three in hexaploid wheats. The objective of this study was to identify the mechanisms that regulate the architecture of the inflorescence in wheat and its relationship to ploidy level.

Methods

The floral anatomy of diploid (Triticum monococcum), tetraploid (T. turgidum ssp. durum) and hexaploid (T. aestivum) wheat species were investigated by light and scanning electron microscopy to describe floret development and to clarify the timing of the initiation of the floret primordia. In situ hybridization analysis using Wknox1, a wheat knotted1 orthologue, was performed to determine the patterning of meristem formation in the inflorescence.

Key Results

The recessive natural mutation of tetraploid (T. turgidum ssp. turgidum) wheat, branching head (bh), which produces branched inflorescences, was used to demonstrate the utility of Wknox1 as a molecular marker for meristematic tissue. Then an analysis of Wknox1 expression was performed in diploid, tetraploid and hexaploid wheats and heterochronic development of the floret meristems was found among these wheat species.

Conclusions

It is shown that the difference in the number of floret primordia in diploid, tetraploid and hexaploid wheats is caused by the heterochronic initiation of floret meristem development from the spikelet meristem.Key words: Triticum, wheat, inflorescence, spikelet, floret, meristem, heterochrony, heterochronic development, knotted1, polyploidy     

Waxy genes from spelt wheat: new alleles for modern wheat breeding and new phylogenetic inferences about the origin of this species

Background and Aims

Waxy proteins are responsible for amylose synthesis in wheat seeds, being encoded by three waxy genes (Wx-A1, Wx-B1 and Wx-D1) in hexaploid wheat. In addition to their role in starch quality, waxy loci have been used to study the phylogeny of wheat. The origin of European spelt (Triticum aestivum ssp. spelta) is not clear. This study compared waxy gene sequences of a Spanish spelt collection with their homologous genes in emmer (T. turgidum ssp. dicoccum), durum (T. turgidum ssp. durum) and common wheat (T. aestivum ssp. aestivum), together with other Asian and European spelt that could be used to determine the origin of European spelt.

Methods

waxy genes were amplified and sequenced. Geneious Pro software, DNAsp and MEGA5 were used for sequence, nucleotide diversity and phylogenetic analysis, respectively.

Key Results

Three, four and three new alleles were described for the Wx-A1, Wx-B1 and Wx-D1 loci, respectively. Spelt accessions were classified into two groups based on the variation in Wx-B1, which suggests that there were two different origins for the emmer wheat that has been found to be part of the spelt genetic make-up. One of these groups was only detected in Iberian material. No differences were found between the rest of the European spelt and the Asiatic spelt, which suggested that the Iberian material had a different origin from the other spelt sources.

Conclusions

The results suggested that the waxy gene variability present in wheat is undervalued. The evaluation of this variability has permitted the detection of ten new waxy alleles that could affect starch quality and thus could be used in modern wheat breeding. In addition, two different classes of Wx-B1 were detected that could be used for evaluating the phylogenetic relationships and the origins of different types of wheat.     

Development and validation of a new Ppo-A1 marker useful for marker-assisted selection in tetraploid wheats

Polyphenol oxidase (PPO) activity is a major cause of undesirable brown color of semolina. In tetraploid wheat, the Ppo-A1 gene is significantly involved in the phenotypic expression of PPO activity. The main goal of this study was to develop and validate a more efficient marker for Ppo-A1 to facilitate marker-assisted selection for low PPO activity in tetraploid wheat breeding programs. A large tetraploid wheat collection, including durum cultivars, domesticated and wild accessions, was used to evaluate the PPO activity. The heritability values indicated that the phenotypic expression of PPO activity was mainly due to genotypic effect. PPO18, and a new marker named MG18, were used to study the Ppo-A1 allelic variation in a tetraploid wheat collection. PPO18 analysis detected four alleles (Ppo-A1b, Ppo-A1e, Ppo-A1f and Ppo-A1g). The high frequency of Ppo-A1g (no PCR product) detected in the tetraploid wheat collection, led to the development of a new genome-specific Ppo-A1 marker (MG18). MG18 analysis identified the same alleles as PPO18 which were associated with low or high PPO activity. The new MG18 marker was more efficient than PPO18 in detecting the four different alleles of Ppo-A1 in the tetraploid wheat collection. Indeed, the accessions assigned to the Ppo-A1g group, according to PPO18, when tested with MG18, were better classified in the four alleles of the Ppo-A1 gene. The MG18 analysis proved that the PPO18 marker overestimated the number of accessions with Ppo-A1g. Therefore, MG18 can be applied to large-scale marker-assisted selection for PPO activity in durum breeding programs.     

Molecular characterisation of the amino- and carboxyl-domains in different Glu-A1x alleles of Triticum urartu Thum. ex Gandil.

The wild diploid wheat (Triticum urartu Thum. ex Gandil.) is a potential gene source for wheat breeding, as this species has been identified as the A-genome donor in polyploid wheats. One important wheat breeding trait is bread-making quality, which is associated in bread wheat (T. aestivum ssp. aestivum L. em. Thell.) with the high-molecular-weight glutenin subunits. In T. urartu, these proteins are encoded by the Glu-A1x and Glu-A1Ay genes at the Glu-A ^u 1 locus. The Glu-A1x genes of 12 Glu-A ^u 1 allelic variants previously detected in this species were analysed using PCR amplification and sequencing. Data showed wide diversity for the Glu-A1x alleles in T. urartu, which also showed clear differences to the bread wheat alleles. This variation could enlarge the high-quality genetic pool of modern wheat and be used to diversify the bread-making quality in durum (T. turgidum ssp. durum Desf. em. Husn.) and common wheat.     

Association analysis for agronomic traits in wheat under terminal heat stress

Terminal heat stress causes irreversible damage to wheat crop productivity. It reduces the vegetative growth and flowering period that consequently declines the efficiency to capture available stem reserves (carbohydrates) in grains. Markers associated with thermotolerant traits ease in marker assisted selection (MAS) for crop improvement. It identifies the genomic regions associated with thermotolerant traits in wheat, but the scarcity of markers is the major hindrance in crop improvement. Therefore, 158 wheat genotypes were subjected to genotyping with 165 simple sequence repeat markers dispersed on three genomes (A, B and D). Allelic frequency and polymorphic information content values were highest on genome A (5.34 (14% greater than the lowest value at genome D) and 0.715 (3% greater than the lowest value at genome D)), chromosome 4 (5.40 (16% greater than the lowest value at chromosome 2) and 0.725 (5% greater than the lowest value at chromosome 6)) and marker xgwm44 (13.0 (84% greater than the lowest value at marker xbarc148) and 0.916 (46% greater than the lowest value at marker xbarc148)). Bayesian based population structure discriminated the wheat genotypes into seven groups based on genetic similarity indicating their ancestral origin and geographical ecotype. Linkage disequilibrium pattern had highest significant (P < 0.001) linked loci pairs 732 on genome A at r² > 0.1 whereas, 58 on genome B at r² > 0.5. Linkage disequilibrium decay (P < 0.01 and r² > 0.1) had larger LD block (5–10 cM) on genome A. Highly significant MTAs (P < 0.000061) under heat stress conditions were identified for flag leaf area (xwmc336), spikelet per spike (xwmc553), grains per spike (cxfa2147, xwmc418 and xwmc121), biomass (xbarc7) and grain yield (xcfa2147 and xwmc671). The identified markers in this study could facilitate in MAS and gene pyramiding against heat stress in wheat.     

Genetic Diversity in Old Portuguese Durum Wheat Cultivars Assessed by Retrotransposon-Based Markers

Retrotransposon-based markers, such as the inter-retrotransposon-amplified polymorphism (IRAP) and the retrotransposon microsatellite-amplified polymorphism (REMAP) are highly informative, multilocus, and reveal insertional polymorphisms among plant individuals. These markers have been used for evolutionary studies, genetic diversity assessment, DNA fingerprinting, genetic mapping linkage, and for the detection of genetic rearrangements induced by polyploidization. In this study, we used IRAP and REMAP markers to assess the genetic diversity among 51 old Portuguese durum wheat cultivars belonging to 27 botanical varieties and to define their genetic relationships. Five IRAP and four REMAP primer combinations were used. IRAP markers revealed 66.3% of polymorphism and an average of 18.4 bands per primer combination which ranged in size from 450 to 3,100?bp. The REMAP technique allowed the detection of 86.36% of inter-cultivar polymorphism and an average of 11 bands per primer combination. The molecular weight of the REMAP bands ranged from 250 to 2,750?bp. The durum wheat cultivars analyzed here belong to 27 botanical varieties of the subspecies Triticum turgidum subsp. turgidum L. [syn. T. turgidum] and Triticum turgidum L. subsp. durum [syn. T. durum] (Desf.) Husn.. Our results showed that the genetic variability assessed by both the IRAP and REMAP markers did not allow the clustering of the durum wheat cultivars according to their taxonomical criteria (subspecies or botanical variety) or homonymy. Nonetheless, these markers were useful for the assessment of genetic diversity at the individual level, for the definition of genetic relationships among cultivars, and for estimation of the genetic structure of the Old collection under analysis. The achieved data could be valuable for future experiments of DNA fingerprinting, genetic improvement, and germplasm conservation in wheat.     

Chromosomal structural changes and microsatellite variations in newly synthesized hexaploid wheat mediated by unreduced gametes

Allohexaploid wheat was derived from interspecific hybridization, followed by spontaneous chromosome doubling. Newly synthesized hexaploid wheat by crossing Triticum turgidum and Aegilops tauschii provides a classical model to understand the mechanisms of allohexaploidization in wheat. However, immediate chromosome level variation and microsatellite level variation of newly synthesized hexaploid wheat have been rarely reported. Here, unreduced gametes were applied to develop synthesized hexaploid wheat, NA0928, population by crossing T. turgidum ssp. dicoccum MY3478 and Ae. tauschii SY41, and further S0–S3 generations of NA0928 were assayed by sequential cytological and microsatellite techniques. We demonstrated that plentiful chromosomal structural changes and microsatellite variations emerged in the early generations of newly synthesized hexaploid wheat population NA0928, including aneuploidy with whole-chromosome loss or gain, aneuploidy with telosome formation, chromosome-specific repeated sequence elimination (indicated by fluorescence in situ hybridization) and microsatellite sequence elimination (indicated by sequencing), and many kinds of variations have not been previously reported. Additionally, we reported a new germplasm, T. turgidum accession MY3478 with excellent unreduced gametes trait, and then succeeded to transfer powdery mildew resistance from Ae. tauschii SY41 to synthesized allohexaploid wheat population NA0928, which would be valuable resistance resources for wheat improvement.