Molecular characterization of a diagnostic DNA marker for domesticated tetraploid wheat provides evidence for gene flow from wild tetraploid wheat to hexaploid wheat

All forms of domesticated tetraploid wheat (Triticum turgidum, genomes AABB) are nearly monomorphic for restriction fragment length polymorphism (RFLP) haplotype a at the Xpsr920 locus on chromosome 4A (Xpsr920-A1a), and wild tetraploid wheat is monomorphic for haplotype b. The Xpsr920-A1a/b dimorphism provides a molecular marker for domesticated and wild tetraploid wheat, respectively. Hexaploid wheat (Triticum aestivum, genomes AABBDD) is polymorphic for the 2 haplotypes. Bacterial artificial chromosome (BAC) clones hybridizing with PSR920 were isolated from Triticum urartu (genomes AA), Triticum monococcum (genomes AmAm), and T. turgidum ssp. durum (genomes AABB) and sequenced. PSR920 is a fragment of a putative ATP binding cassette (ABC) transporter gene (designated ABCT-1). The wheat ABCT-1 gene is more similar to the T. urartu gene than to the T. monococcum gene and diverged from the T. urartu gene about 0.7 MYA. The comparison of the sequence of the wheat A genome BAC clone with that of the T. urartu BAC clone provides the first insight into the microsynteny of the wheat A genome with that of T. urartu. Within 103 kb of orthologous intergenic space, 37 kb of new DNA has been inserted and 36 kb deleted leaving 49.7% of the region syntenic between the clones. The nucleotide substitution rate in the syntenic intergenic space has been 1.6 x 10(-8) nt(-1) year(-1), which is, respectively, 4 and 3 times as great as nucleotide substitution rates in the introns and the third codon positions of the juxtaposed gene. The RFLP is caused by a miniature inverted transposable element (MITE) insertion into intron 18 of the ABCT-A1 gene. Polymerase chain reaction primers were developed for the amplification of the MITE insertion site and its sequencing. The T. aestivum ABCT-A1a haplotype is identical to the haplotype of domesticated tetraploid wheat, and the ABCT-A1b haplotype is identical to that of wild tetraploid wheat. This finding shows for the first time that wild tetraploid wheat participated in the evolution of hexaploid wheat. A cline of the 2 haplotype frequencies exists across Euro-Asia in T. aestivum. It is suggested that T. aestivum in eastern Asia conserved the gene pool of the original T. aestivum more than wheat elsewhere.     

Genetic analysis of photosynthetic variation in hexaploid and tetraploid wheat and their interspecific hybrids

Intra- and inter-specific variation in CO₂ assimilation rate (A) in Triticum spp. is well documented for reproductive growth stages. Research was conducted to characterize early vegetative photosynthetic variation in a diverse set of cultivated hexaploid wheat (T. aestivum L.) germplasm and in wild tetraploid (T. dicoccoides Korn) and hexaploid x tetraploid populations. Choice of hexaploid genotypes was based on maximum genetic distance between cultivars within the HRW and SRW wheat classes of the USA. The tetraploid material was produced by hybridizing two accessions of T. dicoccoides previously shown to differ widely in A and A/Chl but with similar leaf morphology. Genetic variability in the HRW and SRW gene pools was attributed to more recently developed descendent lines and unrelated lines rather than parental lines. Phenotypic distributions for A, stomatal conductance (gs), and internal CO₂ concentration (Ci) in the F₂ tetraploid population were continuous and showed transgressive segregation, reflecting quantitative inheritance with intermediate heritability. Variability in A was not associated with chlorophyll content or CO₂ supply to the mesophyll measured as Ci. Genetic variability in A was also observed in the interspecific backcross population, 2*TAM W-101/PI 428109, thereby providing a germplasm pool to select for high A while restoring the D genome of hexaploid wheat. These results suggest that genetic improvement of vegetative assimilation rate is feasible in hexaploid wheat via homologous transfer from an alien source.Abbreviations HRW hard red winter - LA leaf area - r_G genotypic correlation - r_P phenotypic correlation - SRW soft red winter     

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.     

Genetic mapping of the gene affecting polyphenol oxidase activity in tetraploid durum wheat

The quality of durum wheat (Triticum turgidum ssp. durum) is influenced by polyphenol oxidase(PPO) activity and its corresponding substrates. A saturated molecular-marker linkage map was constructed previously by using a set of recombinant inbred (RI) lines, derived from a cross between durum wheat cultivars Jennah Khetifa and Cham 1. Quantitative trait loci (QTL) for PPO activity in seeds were mapped in this population. PPO activity in seeds of the parents and 110 RI lines was measured spectrophotometrically. The PPO activity of Cham 1 was significantly lower than that of Jennah Khetifa. QTL analysis of these data indicated that most of PPO activity was associated with major loci on the long arm of chromosome 2A. The trait was found to be strongly associated with the SSR marker Xgwm312@2A. With this knowledge, marker-assisted selection can be used to select genotypes with lower PPO activity in durum wheat populations.     

A new reduced height gene found in the tetraploid semi-dwarf wheat landrace Aiganfanmai

Aiganfanmai is a dwarf tetraploid wheat landrace (Triticum turgidum var. turgidum) that stably produces the semi-dwarf trait. Plant height varies from 80-105 cm under cultivation. Compared with tall durum wheat (T. turgidum var. durum) variety Langdon, we found it to have short spikes and seeds, besides a semi-dwarf character. We crossed Aiganfanmai with Langdon to analyze the genetic basis of the semi-dwarf trait. The F(2) population segregated at a 1:3 ratio for the short trait to the normal, which demonstrates that Aiganfanmai carries a recessive reduced height (Rht) gene. This gene was found to be located between the molecular markers Xgwm471 and Xgwm350 on chromosome arm 7AS by microsatellite analysis. No Rht gene had been reported from this chromosome; we designated it as Rht22. Rht 22, unlike other previously reported Rht genes, does not reduce internodal cell length. Reduced cell numbers might explain the short stem trait.     

Transfer and mapping of a gene conferring later-growth-stage powdery mildew resistance in a tetraploid wheat accession

Wheat powdery mildew is a severe foliar disease and causes significant yield losses in epidemic years. Breeding and using resistant cultivars is the most widely employed strategy to curb this disease. To identify and transfer powdery mildew resistance genes in wild emmer wheat accession TA1410 into common wheat, a resistant F3 line derived from the cross of TA1410 × durum wheat line Zhongyin1320 was crossed with common wheat cultivar Yangmai158. The homozygous resistant BC5F2 lines derived from the backcross with Yangmai158 exhibited susceptibility at seedling stage and conferred increasing resistance when the plants were closer to heading stage. In two segregating BC5F3 families investigated at heading stage, the segregation of the resistance fit a 3:1 ratio, suggesting that a single dominant gene controls the resistance. This resistance gene, designated HSM1, was mapped to the 0.6-cM Xmag5825.1–Xgwm344 interval on chromosome 7AL and co-segregated with Xrga-C3 and Xrga-C6. A mapping position comparison with other powdery mildew resistance genes on this chromosome suggested that HSM1 belongs to the Pm1 resistance gene cluster. HSM1 is a useful candidate gene for resistance breeding, particularly in winter-wheat growing areas.     

Genome inheritance in populations derived from hexaploid/tetraploid and tetraploid/hexaploid wheat crosses

Hexaploid/tetraploid and tetraploid/hexaploid wheat hybrids were established using the hexaploid (Triticum aestivum L.) bread wheat LRC2010-150 and the tetraploid durum wheat (T. turgidum spp. durum) WID802. Thirty F₂ progeny from each cross were characterised using Diversity Arrays Technology (DArTseq?) markers to determine whether there are differences between the crosses in the proportion of A, B and D genomic material inherited from each parent. Inheritance of the A and B genome from the tetraploid durum parent varied from 32 to 63% among the 60 lines assessed, and results indicated significant differences between the two F₂ populations in the mean overall proportion of chromosomes A and B inherited from each parent. Significant differences were also observed between the crosses in the proportion of chromosomal segments on 2B, 3A, 3B and 4A inherited from the tetraploid parent. The F₂ populations also showed significant differences in the average retention of D chromosomes per line with the tetraploid/hexaploid cross retaining a mean of 2.83 chromosomes while the reciprocal cross retained a mean of 1.8 chromosomes per line. A strong negative correlation was observed in individual lines from both populations between the proportion of the A and B genome inherited from the tetraploid durum parent and the retention of the D genome. The implication of these results for the design of efficient crossing strategies between hexaploid and tetraploid wheats is discussed.     

Inheritance of awn absence in tetraploid wheat species

Awn absence was shown to be inherited as a dominant character in the tetraploid wheat species Triticum dicoccum (Schrank) Schuebl. and T. durum Desf. but as a recessive one in T. aethiopicum Jakubz. The monogenic control of the character was demonstrated for all studied species. In accessions of emmer and durum wheat, the character is controlled by the dominant gene B1, located on chromosome 5A, and in Ethiopian wheat, by a recessive gene, which we designated as awn. The recessive awn gene was localized on chromosome 3B of T. aethiopicum with the use of D-genome disomic substitution lines of cultivar Langdon.     

The inheritance of tetraploid wheat seed peroxidases

Summary Embryo and endosperm peroxidases from dry mature seeds of three subspecies of tetraploid wheat (Triticum turgidum L.) were subjected to genetic analysis. The inheritance of eight isozymes (embryo isozymes a₂, d₁, d₂, e and f; and endosperm isozymes b, d and 4) were studied in F₂'s obtained from different wheat accessions. Simple monogenic inheritance producing three banded: one null segregation and two epistatic segregations (97 and 151) were found. In the case of isozymes b, d and 4, monogenic or epistatic segregation depended on the F₂ analyzed. Segregation data indicated that at least 9 different loci would determine the peroxidase isozymes of tetraploid wheat seed, all the loci studied containing null alleles. Furthermore, several loci determining embryo peroxidases were noticed to be mutually linked. All these data are discussed in context of the inheritance of seed peroxidases in hexaploid wheat and rye.     

Mapping of FHB resistance QTLs in tetraploid wheat.

Triticum turgidum L var. durum is known to be particularly susceptible to infection by Fusarium graminearum, the causal agent for Fusarium head blight (FHB), which results in severe yield losses and grain contaminated with mycotoxins. This research was aimed at identifying FHB resistance in tetraploid wheat and mapping the location of FHB resistance genes. A tetraploid cross of durum wheat ('Strongfield') x Triticum carthlicum ('Blackbird') was used to generate a doubled-haploid (DH) population. This population was evaluated for type II resistance to F. graminearum in replicated greenhouse trials, in which heads were innoculated and the percent of infected spikelets was determined 21 days later. The population was also genotyped with microsatellite markers to construct a map of 424 loci, covering 2 052 cM. The FHB reaction and genotypic data were used to identify FHB resistance quantitative trait loci (QTLs). It was determined that 2 intervals on chromosomes 2BL and 6BS controlled FHB resistance in this tetraploid cross. The FHB resistance allele on chromosome 2BL (r2=0.26, logarithm of odds (LOD)=8.5) was derived from 'Strongfield', and the FHB resistance allele on chromosome 6BS (r2=0.23, LOD=6.6) was derived from 'Blackbird'. Two other loci, on chromosomes 5AS and 2AL, were shown to regulate FHB infection and to have an epistatic effect on the FHB resistance QTL on chromosome 6BS. Further, the FHB resistance QTL peak on chromosome 6BS was clearly coincident with the known FHB resistance gene Fhb2, derived from Sumai 3. The results show that FHB resistance can be expressed in durum wheat, and that T. carthlicum and Triticum aestivum likely share a common FHB resistance gene on chromosome 6BS.     

Characterization of the wheat mitochondrial orf25 gene

The wheat mitochondrial orf25 nucleotide sequence of 576 pb has been determined. Its derived protein sequence shares 88% and 75% amino acid identity with those of maize and tobacco mitochondria, respectively. The wheat and tobacco orf25 sequences lack four inserts, of 6 bp to 36 bp, that are present in the maize homologue. The wheat orf25 gene is actively transcribed and is preceded by a regulatory sequence block very similar to those located upstream of the wheat coxII and atp6 genes. Our observations support the view that orf25 sequences encode a functional polypeptide in plant mitochondria.     

Chromosome pairing in tetraploid rye with monosomic-substitution wheat chromosomes

In tetraploid rye with single-substitution wheat chromosomes - 1A, 2A, 5A, 6A, 7A, 3B, 5B, 7B - chromosome pairing was analysed at metaphase I in PMCs with the C-banding method. The frequency of univalents of chromosome 1A was considerably higher than that of the other four wheat chromosomes of genome A (6A, 5A, 7A and 2A). Among chromosomes of genome B, the lowest mean frequency of univalents was observed for chromosome 5B. In monosomic lines, wheat chromosomes 1A, 2A, 5A, 6A, 7A and 5B paired with rye homoeologues most often in rod bivalents and in chain quadrivalents (also including 3B). The 47% pairing of 5B-5R chromosomes indicate that the rye genomes block the suppressor Ph1 gene activity. In monosomic plants with chromosomes 5A, 2A, 6A, 7A and 5B, a low frequency of rye univalents was observed. It was also found that the wheat chromosomes influenced the pairing of rye genome chromosomes, as well as the frequency of ring and rod bivalents and tri- and quadrivalents. However, the highest number of terminal chiasmata per chromosome occurred in the presence of chromosomes 5A and 2A, and the lowest - in the presence of chromosomes 3B and 7B. In the presence of chromosome 5B, the highest frequency of bivalents was observed. The results of the present study show that the rye genome is closer related to the wheat genome A of than to genome B. The high pairing of wheat-rye chromosomes, which occurs in tetraploid rye with substitution wheat chromosomes, indicates that there is a high probability of incorporating wheat chromosome segments into rye chromosomes.     

Physical distribution of recombination in B-genome chromosomes of tetraploid wheat

Summary Several studies have indicated a noncorrespondence between genetic and physical distances in wheat chromosomes. To study the physical distribution of recombination, polymorphism for C-banding patterns was used to monitor recombination in 67 segments in 11 B-genome chromosome arms of Triticum turgidum. Recombination was absent in proximal regions of all chromosome arms; its frequency increased exponentially with distance from the centromere. A significant difference was observed between the distribution of recombination in physically short and physically long arms. In physically short arms, recombination was almost exclusively concentrated in distal segments and only those regions were represented in their genetic maps. In physically long arms, while a majority of the genetic distance was again based upon recombination in distal chromosome segments, some interstitial recombination was observed. Consequently, these regions also contributed to the genetic maps. Such a pattern of recombination, skewed toward terminal segments of chromosomes, is probably a result of telomeric pairing initiation and strong positive chiasma interference. Interference averaged 0.81 in 35 pairs of adjacent segments and 0.57 across the entire recombining portions of chromosome arms. The total genetic map lengths of the arms corresponded closely to those expected on the basis of their metaphase-I chiasma frequencies. As a consequence of this uneven distribution of recombination there can be a 153-fold difference (or more) in the number of DNA base pairs per unit (centiMorgan) of genetic length.     

Synaptic behaviour of the tetraploid wheat Triticum timopheevii

Triticum timopheevii (2n=4x=A^tA^tGG) is an allotetraploid wheat which shows a diploid-like behaviour at metaphase-I. The synaptic process was analyzed in fully traced spread nuclei at mid-zygotene, late-zygotene and pachytene. The length and type of synaptonemal complexes, as well as the number of bivalent and multivalent associations, were determined in each nucleus. A high number of bivalents per nucleus was detected at all three stages. Nuclei at pachytene showed a lower frequency of multivalents than did zygotene nuclei, which suggests the existence of a pairing correction mechanism. At metaphase-I only homologous bivalents and, rarely, one pair of univalents were observed. Similarities between the diploidization mechanism of T. timopheevii and that of allohexaploid wheat, controlled by chromosome 5B, are discussed.     

Photosynthetic response of tetraploid and hexaploid wheat to water stress

Photosynthetic characteristics of ear and flag leaves of wheat species, tetraploid Triticum dicoccoides Kom and hexaploid Bima1, were studied in plants grown under well-watered (WW) and water-stressed (WS) conditions. Compared to ears, flag leaves exhibited higher photosynthetic rate (P _N) at the filling stage, but more severe decrease under WS. P _N in the tetraploid wheat ear remained higher than that in the hexaploid wheat during the grain-filling stage. Water stress decreased PN in both the organs; this decline was caused by a reduction in Rubisco activity, not by drought-induced stomatal limitation. Tetraploid wheat ears exhibited higher relative water content and water-use efficiency than that of hexaploid wheat, under WS. The change in phosphoenolpyruvate carboxylase activity and carbon isotope composition indicated the absence of C₄ metabolism in the ears of both species under both conditions. The improved performance of the tetraploid wheat ears under WS was associated with better water relations.     

Isozyme gene duplication in diploid and tetraploid potatoes

Summary Isozyme techniques allow the study of gene redundancy in different ploidy levels of potato (Solanum tuberosum). In tetraploid potatoes all isozyme loci are duplicated. No sign of structural or regulatory divergence was found, as is expected due to their tetrasomic inheritance patterns. In addition to this genetic redundancy, produced by a relatively recent polyploidization event, some additional redundancy was found for at least three enzymes even in diploid groups and species. These older duplicate genes show structural and regulatory divergence, indicating they appeared by a separate polyploidization event far in the past. Their common origin is still recognizable by both their expression in the same subcellular compartment and by the dimerizing ability of the isozymes they encode. To account for the present chromosome number x = 12 of the Solanaceae family, the most frequently found among the species, a hypothetical polyploidization event is proposed.     

Chromosome sorting in tetraploid wheat and its potential for genome analysis

This study evaluates the potential of flow cytometry for chromosome sorting in durum wheat (Triticum turgidum Desf. var. durum, 2n = 4x = 28). Histograms of fluorescence intensity (flow karyotypes) obtained after the analysis of DAPI-stained chromosomes consisted of three peaks. Of these, one represented chromosome 3B, a small peak corresponded to chromosomes 1A and 6A, and a large peak represented the remaining 11 chromosomes. Chromosomes sorted onto microscope slides were identified after fluorescence in situ hybridization (FISH) with probes for GAA microsatellite, pSc119.2, and Afa repeats. Genomic distribution of these sequences was determined for the first time in durum wheat and a molecular karyotype has been developed for this crop. Flow karyotyping in double-ditelosomic lines of durum wheat revealed that the lines facilitated sorting of any arm of the wheat A- and B-genome chromosomes. Compared to hexaploid wheat, flow karyotype of durum wheat is less complex. This property results in better discrimination of telosomes and high purities in sorted fractions, ranging from 90 to 98%. We have demonstrated that large insert libraries can be created from DNA purified using flow cytometry. This study considerably expands the potential of flow cytogenetics for use in wheat genomics and opens the possibility of sequencing the genome of this important crop one chromosome arm at a time.     

Independent evolution of functional Pm3 resistance genes in wild tetraploid wheat and domesticated bread wheat

The Pm3 alleles of cultivated bread wheat confer gene for gene resistance to the powdery mildew fungus. They represent a particular case of plant disease resistance gene evolution, because of their recent origin and possible evolution after the formation of hexaploid wheat. The Pm3 locus is conserved in tetraploid wheat, thereby allowing the comparative evolutionary study of the same resistance locus in a domesticated species and in one of its wild ancestors. We have identified 61 Pm3 allelic sequences from wild and domesticated tetraploid wheat subspecies. The Pm3 sequences corresponded to 24 different haplotypes. They showed low sequence diversity, differing by only a few polymorphic sequence blocks that were further reshuffled between alleles by gene conversion and recombination. Polymorphic sequence blocks are different from the blocks found in functional Pm3 alleles of hexaploid wheat, indicating an independent evolution of the Pm3 loci in the two species. A new functional gene was identified in a wild wheat accession from Syria. This gene, Pm3k , conferred intermediate race-specific resistance to powdery mildew, and consists of a mosaic of gene segments derived from non-functional alleles. This demonstrates that Pm3 -based resistance is not very frequent in wild tetraploid wheat, and that the evolution of functional resistance genes occurred independently in wild tetraploid and bread wheat. The Pm3 sequence variability and geographic distribution indicated that diversity was higher in wild emmer wheat from the Levant area, compared with the accessions from Turkey. Further screens for Pm3 functional genes in wild wheat should therefore focus on accessions from the Levant region.     

Characterization of the S7 ribosomal protein gene in wheat mitochondria

Summary By screening a wheat mitoplast cDNA bank, we have identified an open reading frame of 444 by that has a derived amino acid sequence homologous to bacterial-type S7 ribosomal proteins. This gene, designated rps7, is located upstream of one of two 26S rRNA gene copies in the wheat mitochondrial genome and is expressed as an abundant mRNA of approximately 0.7 kb. Its 5 terminus maps to the end of an 80 by element that is closely related to sequences preceding the wheat coxII, orf25 and atp6 genes. Southern hybridization analysis indicates that rps7-homologous sequences are present in the mitochondria of rice and pea, but not soybean.