Analysis of intraspecific divergence of hexaploid wheat Triticum spelta L. by chromosome C-banding

Intraspecific divergence of hexaploid wheat Triticum spelta was studied by chromosome C-banding in 41 accessions of different geographic origins. The spelt accessions did not differ in karyotype structure or heterochromatin distribution from common wheat, but showed greater intraspecific polymorphism for chromosome rearrangements (translocations, inversions) and banding patterns. On evidence of C-banding patterns, spelt was assumed to occupy an intermediate position between tetraploid and hexaploid wheat species. Accessions of the Asian spelt subspecies had more diverse banding patterns than European accessions. A relatively high frequency of chromosome rearrangements was observed in Iranian accessions. Visual analysis revealed high uniformity of chromosome banding patterns in T. spelta populations of Afghanistan, Spain, and Germany (Bavarian group), suggesting a significant role of the founder effect in their evolution.     

Analysis of Intraspecific Divergence of Hexaploid Wheat Triticum spelta L. by C-Banding of Chromosomes

Intraspecific divergence of hexaploid wheat Triticum spelta was studied by C-banding method in 41 accessions of different geographic origins. The spelt accessions did not differ in karyotype structure or heterochromatin distribution from common wheat, but showed greater intraspecific polymorphism by chromosome rearrangements (translocations, inversions) and banding patterns. On evidence of C-banding patterns, spelt was assumed to occupy an intermediate position between tetraploid and hexaploid wheat species. Accessions of the Asian spelt subspecies had more diverse banding patterns than European accessions. A relatively high frequency of chromosome rearrangements was observed in Iranian accessions. Visual analysis revealed high uniformity of chromosome banding patterns in T. spelta populations of Afghanistan, Spain, and Germany (Bavarian group), suggesting a significant role of the founder effect in their evolution.     

Pairing affinities of the B- and G-genome chromosomes of polyploid wheats with those of Aegilops speltoides.

Chromosome pairing at metaphase I was studied in different interspecific hybrids involving Aegilops speltoides (SS) and polyploid wheats Triticum timopheevii (AtAtGG), T. turgidum (AABB), and T. aestivum (AABBDD) to study the relationships between the S, G, and B genomes. Individual chromosomes and their arms were identified by means of C-banding. Pairing between chromosomes of the G and S genomes in T. timopheevii x Ae. speltoides (AtGS) hybrids reached a frequency much higher than pairing between chromosomes of the B and S genomes in T. turgidum x Ae. speltoides (ABS) hybrids and T. aestivum x Ae. speltoides (ABDS) hybrids, and pairing between B- and G-genome chromosomes in T. turgidum x T. timopheevii (AAtBG) hybrids or T. aestivum x T. timopheevii (AAtBGD) hybrids. These results support a higher degree of closeness of the G and S genomes to each other than to the B genome. Such relationships are consistent with independent origins of tetraploid wheats T. turgidum and T. timopheevii and with a more recent formation of the timopheevi lineage.     

Detection of intergenomic translocations with centromeric and noncentromeric breakpoints in Triticum araraticum: mechanism of origin and adaptive significance.

Triticum araraticum Jakubz. (2n = 4x = 28, AtAtGG), a wild progenitor of the polyploid cultivated wheat T. timopheevii, shows extensive chromosome translocation polymorphism in natural populations from the Middle East and Transcaucasia. From an extensive survey, eight intergenomic translocation types were observed and their breakpoints analyzed by genomic in situ hybridization. The previously reported species-specific 6At-1G-4G cyclic translocation was found in all accessions studied. In four translocation types, the breakpoints were in interstitial regions of chromosomes and the other four arose via centric-breakage-fusion. A model is presented on the mechanism of origin and the adaptive significance of translocations with centromeric and noncentromeric breakpoints.     

Natural or induced nucleocytoplasmic heterogeneity in Triticum longissimum.

Alien cytoplasms produce a variety of phenotypes in durum wheat (Triticum turgidum) and common wheat (Triticum aestivum) cultivars, which indicate the prevalence of cytoplasmic variability in the subtribe Triticinae. Intraspecific cytoplasmic differences have been demonstrated between the subspecies of Triticum speltoides, Triticum dichasians, and Triticum comosum. In this study, durum wheat lines with cytoplasm from two accessions, B and C, of Triticum longissimum were compared, and meiotic chromosome pairing between the group 4 homoeologues from the same two accessions was examined in common wheat. First, monosomic addition or monosomic substitution lines of common wheat with cytoplasm and one chromosome (designated B) from accession B were crossed with those having cytoplasm and a chromosome designated C-1 or C-2 from accession C. In each substitution line, an alien chromosome substituted for a group 4 homoeologue. Each alien chromosome had a "selfish" (Sf) gene, which remained fixed in the wheat nucleus. The F1s had greatly reduced meiotic pairing between chromosomes B and C-1 and B and C-2, which indicated greatly reduced homology between the group 4 homoeologues from the two accessions. Second, by using Triticum timopheevii as a bridging species, chromosome B in a common wheat line was eliminated and an euploid durum line with cytoplasm from accession B was obtained. This line was fertile. In contrast, a similarly produced durum line with cytoplasm from accession C was male sterile and retained a species cytoplasm specific (scs) nuclear gene from T. timopheevii. In conclusion, nuclear and cytoplasmic heterogeneity pre-existed between accessions B and C and they represent varieties or incipient subspecies in T. longissimum. Alternatively, the Sf genes produced chromosomal heterogeneity and mutated cytoplasmic genes from one or both accessions. Key words : meiotic drive, selfish gene (Sf), gametocidal gene (Gc), Triticum, Aegilops.     

Homoeoallelic gene Ncc-tmp of Triticum timopheevii conferring compatibility with the cytoplasm of Aegilops squarrosa in the tetraploid wheat nuclear background.

A nuclear gene, Ncc-tmp1A, of Triticum timopheevii is required for the nucleus-cytoplasm (NC) compatibility in tetraploid NC hybrids with the cytoplasm of Aegilops squarrosa. A euploid NC hybrid of T. durum was previously produced by introgressing the gene from chromosome 1A of T. timopheevii. To examine the possible presence of a functional homoeoallele in the G genome of T. timopheevii, segregation of seed viability was studied as a marker phenotype in BC1s involving the two types of NC hybrids, (Ae. squarrosa)-T. timopheevii and (Ae. squarrosa)-T. turgidum. The result of these test crosses suggested that the G genome possesses a functional homoeoallele Ncc-tmp1G. Segregation of two RAPD (random amplified polymorphic DNA) markers that were closely linked to Ncc-tmp1A was further studied among the viable BC1s obtained from a test cross of (Ae. squarrosa)-T. timopheevii x T. turgidum. Some viable BC1 segregants without the markers were obtained, suggesting a limited degree of transmission of chromosome 1G carrying Ncc-tmp1G. However, a similar RAPD analysis of BC1s obtained after backcrosses of reciprocal F1s of T. timopheevii/T. turgidum with T. turgidum showed random marker segregation. Thus, it was concluded that Ncc-tmp1A is not required for compatibility with its own cytoplasm. Southern blot analysis of the euploid NC hybrid using RFLP (restriction fragment length polymorphism) markers on the homologous group 1 chromosomes showed that Ncc-tmp1A locates in the centromeric region.     

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 on 16 SSR loci. At each locus, the number of alleles ranged from 2 to 14, 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 6 subspecies taxonomic entities of T. turgidum L. could not differentiate with each other, indicating that the morphological differences present among the 6 subspecies could not be reflected by the SSR markers. These results suggested that SSR markers had the superiority in detecting the genetic diversity of T. turgidum L., while it was not good for the studies of the phylogenic relationships among the subspecies of T. turgidum L.     

Intraspecific karyotype divergence inTriticum araraticum (Poaceae)

Karyotypes of 185 accessions ofTriticum araraticum Jakubz. (2n = 28 = 4x = A^tA^tGG) from Iraq, Iran, Turkey, and Transcaucasia were analyzed using C-banding technique. All accessions showed a certain degree of C-banding polymorphism and further karyotypic diversity was generated by structural rearrangements, mainly translocations. Eighty-one accessions had the normal karyotype similar to that ofT. timopheevii (cultivation), i.e., they showed C-banding polymorphism but no chromosomal rearrangements based on the resolving power of the C-banding technique. One-hundred four accessions showed 34 karyotypic variants, 31 had reciprocal translocations with the breakpoints in the centromeric regions of chromosomes. Three showed reciprocal translocations with the breakpoints in intercalary regions of chromosomes. A paracentric inversion for 7A^t chromosome was observed in some accessions. The rearranged karyotypes differed from the normal by one translocation in 21 variants, by two in 9 variants, by three in 1 variant, and by four in 2 variants of karyotypes. Translocations occurred more frequenty in the chromosomes of G-genome than of A^t-genome. Individual chromosomes differed in the frequencies of their involvement in translocations. Each geographical region contained a unique spectrum of translocations. Karyotypic diversity was the highest in Iraq followed by Transcaucasia and Turkey. Iran showed little karyotypic variation. Based on karyotypic analysis, Iraq should be considered as a centre of origin and primary centre of diversity ofT. araraticum.     

Structural chromosome differentiation between Triticum timopheevii and T. turgidum and T. aestivum

 Chromosome pairing at metaphase-I was analyzed in F₁ hybrids among T. turgidum (AABB), T. aestivum (AABBDD), and T. timopheevii (A^tA^tGG) to study the chromosome structure of T. timopheevii relative to durum (T. turgidum) and bread (T. aestivum) wheats. Individual chromosomes and their arms were identified by means of C-banding. Homologous pairing between the A-genome chromosomes was similar in the three hybrid types AA^tBG, AA^tBGD, and AABBD. However, associations of B-G were less frequent than B-B. Homoeologous associations were also observed, especially in the AA^tBGD hybrids. T. timopheevii chromosomes 1A^t, 2A^t, 5A^t, 7A^t, 2G, 3G, 5G, and 6G do not differ structurally from their counterpart in the A and B genomes. Thus, these three polyploid species inherited translocation 5AL/4AL from the diploid A-genome donor. Chromosome rearrangements that occurred at the tetraploid level were different in T. turgidum and T. timopheevii. Translocation 4AL/7BS and a pericentric inversion of chromosome 4A originated only in the T. turgidum lineage. The two lines of T. timophevii studied carry four different translocations, 6A^tS/1GS, 1GS/4GS, 4GS/4A^tL, and 4A^tL/3A^tL, which most likely arose in that sequence. These structural differences support a diphyletic origin of polyploid wheats. Received: 15 June 1998 / Accepted: 19 August 1998     

RAPD markers linked to the nuclear gene from Triticum timopheevii that confers compatibility with Aegilops squarrosa cytoplasm on alloplasmic durum wheat.

Alien cytoplasms cause a wide range of phenotypic alterations in the nucleus-cytoplasm (NC) hybrids in the Triticeae. Nuclear genomes of timopheevii wheat (Triticum timopheevii and Triticum araraticum) are fully compatible with the cytoplasm of Aegilops squarrosa, while those of a majority of emmer or durum wheat cultivars and more than half the wild emmer wheats are incompatible, and a maternal 1D chromosome is required to restore seed viability and male fertility in the NC hybrids. A euploid NC hybrid of Triticum durum cv. Langdon with Ae. squarrosa cytoplasm produced by introgressing the NC compatibility (Ncc) gene from T. timopheevii was used to identify random amplified polymorphic DNA (RAPD) markers linked to it. After a survey of 200 random decamer primers, four markers were selected, all of which were completely linked in 64 individuals of a SB8 mapping population. One marker was derived from a single locus, while three others were from interspersed repetitive sequences. Also, the hybrid chromosomes and those of the parental T. durum had identical C-banding patterns. RAPD-PCR analysis of 65 accessions from wild and cultivated tetraploid wheat species showed the exclusive presence of the markers in timopheevii wheat. In conclusion, the chromosomal region flanking Ncc of T. timopheevii is highly conserved in the genome of this group of tetraploid wheats.     

Genotypic variation in tetraploid wheat affecting homoeologous pairing in hybrids with Aegilops peregrina.

The Ph1 gene has long been considered the main factor responsible for the diploid-like meiotic behavior of polyploid wheat. This dominant gene, located on the long arm of chromosome 5B (5BL), suppresses pairing of homoeologous chromosomes in polyploid wheat and in their hybrids with related species. Here we report on the discovery of genotypic variation among tetraploid wheats in the control of homoeologous pairing. Compared with the level of homoeologous pairing in hybrids between Aegilops peregrina and the bread wheat cultivar Chinese Spring (CS), significantly higher levels of homoeologous pairing were obtained in hybrids between Ae. peregrina and CS substitution lines in which chromosome 5B of CS was replaced by either 5B of Triticum turgidum ssp. dicoccoides line 09 (TTD09) or 5G of Triticum timopheevii ssp. timopheevii line 01 (TIMO1). Similarly, a higher level of homoeologous pairing was found in the hybrid between Ae. peregrina and a substitution line of CS in which chromosome arm 5BL of line TTD140 substituted for 5BL of CS. It appears that the observed effect on the level of pairing is exerted by chromosome arm 5BL of T turgidum ssp. dicoccoides, most probably by an allele of Ph1. Searching for variation in the control of homoeologous pairing among lines of wild tetraploid wheat, either T turgidum ssp. dicoccoides or T timopheevii ssp. armeniacum, showed that hybrids between Ae. peregrina and lines of these two wild wheats exhibited three different levels of homoeologous pairing: low, low intermediate, and high intermediate. The low-intermediate and high-intermediate genotypes may possess weak alleles of Ph1. The three different T turgidum ssp. dicoccoides pairing genotypes were collected from different geographical regions in Israel, indicating that this trait may have an adaptive value. The availability of allelic variation at the Ph1 locus may facilitate the mapping, tagging, and eventually the isolation of this important gene.     

Characterization of waxy proteins and waxy genes of Triticum timopheevii and T. zhukovskyi and implications for evolution of wheat.

The granule-bound starch (GBSS I, waxy protein) in Triticum timopheevii (AtAtGG) and T. zhukovskyi (AtAtAzAzGG) and a diagnostic section of the genes encoding GBSS-I from the Wx-TtA and Wx-G loci of T. timopheevii and the Wx-TtA, Wx-G, and Wx-TzA loci of T. zhukovskyi were investigated in this study. The waxy proteins in these two polyploid wheats could not be separated into distinct bands, in contrast to those in the T. turgidum (AABB)-T. aestivum (AABBDD) lineage. Alignment of sequences of the section covering exon4-intron4-exon5 of the various waxy genes led to the identification of gene-specific sequences in intron 4. The sequences specific to the Wx-TtA and Wx-G genes of T. timopheevii were different from those of the Wx-A1 gene and Wx-B1 genes of T. turgidum and T. aestivum. A surprising observation was that the Wx-TzA of T. zhukovskyi did not match with the Wx-TmA of T. monococcum, a putative donor of the Az genome, but matched unexpectedly and perfectly with the Wx-B1 gene on chromosome 4A, which is proposed to have translocated from the chromosome 7B of T. aestivum. The possible genetic mechanism explaining these observations is discussed.     

Studies on the origin and evolution of tetraploid wheats based on the internal transcribed spacer (ITS) sequences of nuclear ribosomal DNA

In this study, the internal transcribed spacer (ITS) sequences of nuclear ribosomal DNA in the tetraploid wheats, Triticum turgidum (AABB) and Triticum timopheevii (AAGG), their possible diploid donors, i.e., Triticum monococcum (AA), Triticum urartu (AA), and five species in Aegilops sect. Sitopsis (SS genome), and a related species Aegilops tauschii were cloned and sequenced. ITS1 and ITS2 regions of 24 clones from the above species were compared. Phylogenetic analysis demonstrated that Aegilops speltoides was distinct from other species in Aegilops sect. Sitopsis and was the most-likely donor of the B and G genomes to tetraploid wheats. Two types of ITS repeats were cloned from Triticum turgidum ssp. dicoccoides, one markedly similar to that from T. monococcum ssp. boeoticum (AA), and the other to that from Ae. speltoides (SS). The former might have resulted from a recent integression event. The results also indicated that T. turgidum and T. timopheevii might have simultaneously originated from a common ancestral tetraploid species or be derived from two hybridization events but within a very short interval time. ITS paralogues in tetraploid wheats have not been uniformly homogenized by concerted evolution, and high heterogeneity has been found among repeats within individuals of tetraploid wheats. In some tetraploid wheats, the observed heterogeneity originated from the same genome (B or G). Three kinds of ITS repeats from the G genome of an individual of T. timopheevii ssp. araraticum were more divergent than that from inter-specific taxa. This study also demonstrated that hybridization and polyploidization might accelerate the evolution rate of ITS repeats in tetraploid wheats.     

Sequential chromosome banding and in situ hybridization analysis.

Different combinations of chromosome N- or C-banding with in situ hybridization (ISH) or genomic in situ hybridization (GISH) were sequentially performed on metaphase chromosomes of wheat. A modified N-banding-ISH/GISH sequential procedure gave best results. Similarly, a modified C-banding - ISH/GISH procedure also gave satisfactory results. The variation of the hot acid treatment in the standard chromosome N- or C-banding procedures was the major factor affecting the resolution of the subsequent ISH and GISH. By the sequential chromosome banding - ISH/GISH analysis, multicopy DNA sequences and the breakpoints of wheat-alien translocations were directly allocated to specific chromosomes of wheat. The sequential chromosome banding- ISH/GISH technique should be widely applicable in genome mapping, especially in cytogenetic and molecular mapping of heterochromatic and euchromatic regions of plant and animal chromosomes.     

The synaptic behaviour of the wild forms of Triticum turgidum and T. timopheevii.

Different wild allopolyploid species of Triticeae show extensive bivalent formation at zygotene while a considerable number of multivalents is present in cultivated polyploid wheats. To study the chromosome behaviour at early meiotic stages in wild forms of tetraploid wheats Triticum turgidum and T timopheevii (2n = 4x = 28) we have analysed the synaptic pattern in fully traced spread nuclei at mid- and late zygotene and at pachytene of wild accessions of these species. The mean number of synaptonemal complex (SC) bivalents at mid-zygotene ranged from 12.22 to 13.14 among the accessions studied indicating a strong restriction of synapsis initiation to homologous chromosomes. The mean of bivalents increased at pachytene because of the transformation of multivalents into bivalents. Ring bivalents observed at metaphase I support that SC bivalents were formed by homologous chromosomes. The average values of SC bivalents at mid-zygotene in the wild forms are much higher than the average values observed in the cultivated tetraploid wheats but similar to that of a mutant line of T turgidum with a duplication that includes Ph1, the major homoeologous pairing suppressor locus. These results suggest that the efficiency of the mechanism operating in the homologous recognition for synapsis is higher in wild wheat populations than in cultivated varieties. Apparently, a relatively detrimental modification of the pairing regulating genetic system accompanied the domestication of the wild wheat forms.     

The evolution of polyploid wheats: identification of the A genome donor species.

Cytogenetic work has shown that the tetraploid wheats, Triticum turgidum and T. timopheevii, and the hexaploid wheat T. aestivum have one pair of A genomes, whereas hexaploid T. zhukovskyi has two. Variation in 16 repeated nucleotide sequences was used to identify sources of the A genomes. The A genomes of T. turgidum, T. timopheevii, and T. aestivum were shown to be contributed by T. urartu. Little divergence in the repeated nucleotide sequences was detected in the A genomes of these species from the genome of T. urartu. In T. zhukovskyi one A genome was contributed by T. urartu and the other was contributed by T. monococcum. It is concluded that T. zhukovskyi originated from hybridization of T. timopheevii with T. monococcum. The repeated nucleotide sequence profiles in the A genomes of T. zhukovskyi showed reduced correspondence with those in the genomes of both ancestral species, T. urartu and T. monococcum. This differentiation is attributed to heterogenetic chromosome pairing and segregation among chromosomes of the two A genomes in T. zhukovskyi.     

Analysis of intraspecific diversity of cultivated emmer <Emphasis Type="Italic">Triticum dicoccum</Emphasis> (Schrank.) schuebl using C-banding technique

Ninety-four lines of Triticum dicoccum isolated from 86 wheat accessions from Vavilov All-Russia Research Institute of Plant Industry (VIR, Russia) and INRA (Clermont-Ferrand, France) germ-plasm collections were studied using C-banding technique. Visual comparison of karyotypes of different accessions was performed to establish genetic relationships and evaluate features inherent for ecological-geographical groups. The level of C-banding polymorphism in the whole sample of tetraploid emmer proved to be relatively low. The diversity within groups was higher than the differences between them. The material studied contained 39 lines carrying 16 different types of chromosomal rearrangements including single and multiple translocations and inversions. The level of translocation polymorphism was comparable with that detected earlier for polyploid wheat species. The frequencies of individual translocation types varied from 18 (T7A:5B) to 1 (nine types). Analysis of the distribution of the most frequent translocations 7A:5B suggested that it has significant adaptive value on the territory of Europe. Similarity of the C-banding patterns of European emmer and the accessions with the same translocation of the Asian origin points to their possible common origin. The occurrence of the same translocation in several T. dicoccoides accessions from Syria and Lebanon may indicate that such forms of wild emmer could have taken part in the origin of cultivate emmer from Western Europe. Similarity of the C-banding patterns of some chromosomes of European emmer and spelt could serve as an indirect evidence of their close genetic relationships.     

Intraspecific chromosomal polymorphism ofTriticum araraticum (Poaceae) detected by C-banding technique

DifferentTriticum araraticum lines were studied by C-banding method. The intraspecific divergence ofT. araraticum was shown to be caused mainly by large chromosomal rearrangements. Two main chromosomal types were distinguished among the studied lines: (1) a karyotype similar to that ofT. timopheevii and (2) different one. The first type includes some lines ofT. araraticum subspp.kurdistanicum andararaticum; the second comprises most lines ofT. araraticum subsp.araraticum. The lines of the first type can give fertile F₁ hybrids withT. timopheevii.     

Molecular cytogenetic analysis of Aegilops cylindrica host.

The genomic constitution of Aegilops cylindrica Host (2n = 4x = 28, DcDcCcCc) was analyzed by C-banding, genomic in situ hybridization (GISH), and fluorescence in situ hybridization (FISH) using the DNA clones pSc119, pAs1, pTa71, and pTA794. The C-banding patterns of the Dc- and Cc-genome chromosomes of Ae. cylindrica are similar to those of D-and C-genome chromosomes of the diploid progenitor species Ae. tauschii Coss. and Ae. caudata L., respectively. These similarities permitted the genome allocation and identification of the homoeologous relationships of the Ae. cylindrica chromosomes. FISH analysis detected one major 18S-5.8S-25S rDNA locus in the short arm of chromosome 1Cc. Minor 18S-5.8S-25S rDNA loci were mapped in the short arms of 5Dc and 5Cc. 5S rDNA loci were identified in the short arm of chromosomes 1Cc, 5Dc, 5Cc, and 1Dc. GISH analysis detected intergenomic translocation in three of the five Ae. cylindrica accessions. The breakpoints in all translocations were non-centromeric with similar-sized segment exchanges.     

Fixation of translocation 2A·4B infers the monophyletic origin of Ethiopian tetraploid wheat

Analysis of structural chromosomal polymorphism revealed the presence of a previously reported 2A·4B translocation common to all 15 strains of Ethiopian tetraploid wheat examined. Using the C-banding technique, we found two new translocations,T1B·6B and T5B·6B, and a pericentric inversion of chromosome 5A. The C-banding pattern indicated that in all three translocations the breakpoint was located in the centromeric region. Sequential N-banding and genomic in situ hybridization (GISH) confirmed the location of the breakpoint of translocation 2A·4B, and revealed that the breakpoint of another known translocation, 2A-2B, was in the proximal region of 2BL. The fixation of the 2A·4B translocation indicates the monophyletic origin of Ethiopian tetraploid wheat and the presence of a very severe bottleneck effect during its dispersal. Received: 29 June 1999 / Accepted: 18 February 2000