Localization of high level of sequence conservation and divergence regions in cotton

In a previous study, we observed that the variations in chromosome size are due to uneven expansion and contraction by comparing the structures and sizes of a pair of homoeologous high-resolution cytogenetic maps of chromosomes 12A and 12D in tetraploid cotton. To reveal the variation at the sequence level, in the present paper, we sequenced two pairs of homoeologous bacterial artificial chromosomes derived from high- to low-variable genomic regions. Comparisons of their sequence variations confirmed that the highly conserved and divergent sequences existed in the distal and pericentric regions, e.g., high- and low-variable genome size regions in these two pairs of cotton homoeologous chromosomes. Sequence analysis also confirmed that the differential accumulation of Gossypium retrotransposable gypsy-like element (Gorge3) accounted for the main contributions for the size difference between the pericentric regions. By fluorescence in situ hybridization analysis, we found that Gorge3 has a bias distribution in the A_T/A proximal regions and is associated with the heterochromatin along the chromosomes in the entire Gossypium genome. These results indicate that, between A_T/A and D_T/D genomes, the distal and pericentric regions usually possess high level of sequence conservation and divergence, respectively, in cotton.     

Phylogenetic diversity and relationship among Gossypium germplasm using SSRs markers

Gossypium species represent a vast resource of genetic multiplicity for the improvement of cultivated cotton. To determine genetic diversity and relationships within a diverse collection of Gossypium, we employed 120 SSR primers on 20 diploid species representing seven basic genome groups of the genus Gossypium, five AD allotetraploid cotton accessions while T. populnea served as an outgroup species. Out of 120 SSR primers, 49 pairs are polymorphic, which produced a total of 99 distinct alleles with an average of 2.0 alleles per primer pair. A total of 1139 major SSR bands were observed. Genetic similarities among all the diploid species ranged from 0.582 (between G. herbaceum and G. trilobum) up to 0.969 (between G. arboreum and G. herbaceum). Phylogenetic trees based on genetic similarities were consistent with known taxonomic relationships. The results also indicated that G. raimondii is the closest living relative of the ancestral D-genome donor of tetraploid species and the A-genome donor is much similar to the present-day G. herbaceum and G. arboreum. Ancient tetraploid cotton species were formed by hybridizing and chromosome doubling between them, then different tetraploid cotton species appeared by further geographical and genetic isolation and separating differentiation. The results showed that SSRs could be an ideal means for the identification of the genetic diversity and relationship of cotton resources at the genomic level.     

Distribution of 5S and 18S–28S rDNA loci in a tetraploid cotton (Gossypium hirsutum L.) and its putative diploid ancestors

The most widely cultivated species of cotton,Gossypium hirsutum, is a disomic tetraploid (2n=4x=52). It has been proposed previously that extant A- and D-genome species are most closely related to the diploid progenitors of the tetraploid. We used fluorescent in situ hybridization (FISH) to determine the distribution of 5S and 18S-28S rDNA loci in the A-genome speciesG. herbaceum andG. arboreum, the D-genome speciesG. raimondii andG. thurberi, and the AD tetraploidG. hirsutum. High signal-to-noise, single-label FISH was used to enumerate rDNA loci, and simultaneous, dual-label FISH was used to determine the syntenic relationships of 5S rDNA loci relative to 18S–28S rDNA loci. These techniques provided greater sensitivity than our previous methods and permitted detection of six newG. hirsutum 18S–28S rDNA loci, bringing the total number of observed loci to 11. Differences in the intensity of the hybrizization signal at these loci allowed us to designate them as major, intermediate, or minor 18–28S loci. Using genomic painting with labeled A-genome DNA, five 18S–28S loci were localized to theG. hirsutum A-subgenome and six to the D-subgenome. Four of the 11 18S–28S rDNA loci inG. hirsutum could not be accounted for in its presumed diploid progenitors, as both A-genome species has three loci and both D-genome species had four.G. hirsutum has two 5S rDNA loci, both of which are syntenic to major 18S–28S rDNA loci. All four of the diploid genomes wer examined contained a single 5S locus. InG. herbaceum (A₁) andG. thurberi (D₁), the 5S locus is syntenic to a major 18S–28S locus, but inG. arboreum (A₂) andG. raimondii (D₅), the proposed D-genome progenitor ofG. hirsutum, the 5S loci are syntenic tominor and intermediate 18S–28S loci, respecitively. The multiplicity, variation in size and site number, and lack of additivity between the tetraploid species and its putative diploid ancestors indicate that the behavior of rDNA loci in cotton is nondogmatic, and considerably more complex and dynamic than previously envisioned. The relative variability of 18S–28S rDNA loci versus 5S rDNA loci suggests that the behavior of tandem repearts can differ widely. Edited by: R. Appels     

Clustering, haplotype diversity and locations of MIC-3: a unique root-specific defense-related gene family in Upland cotton (Gossypium hirsutum L.)

MIC-3 is a recently identified gene family shown to exhibit increased root-specific expression following nematode infection of cotton plants that are resistant to root-knot nematode. Here, we cloned and sequenced MIC-3 genes from selected diploid and tetraploid cotton species to reveal sequence differences at the molecular level and identify chromosomal locations of MIC-3 genes in Gossypium species. Detailed sequence analysis and phylogenetic clustering of MIC-3 genes indicated the presence of multiple MIC-3 gene members in Gossypium species. Haplotypes of a MIC-3 gene family member were discovered by comparative analysis among consensus sequences across genotypes within an individual clade in the phylogram to overcome the problem of duplicated loci in the tetraploid cotton. Deficiency tests of the SNPs delimited six A_t-genome members of the MIC-3 family clustered to chromosome arm 4sh, and one D_t-genome member to chromosome 19. Clustering was confirmed by long-PCR amplification of the intergenic regions using A_t-genome-specific MIC-3 primer pairs. The clustered distribution may have been favored by selection for responsiveness to evolving disease and/or pest pressures, because large variants of the MIC-3 gene family may have been recovered from small physical areas by recombination. This could give a buffer against selection pressure from a broad range of pest and pathogens in the future. To our knowledge, these are the first results on the evolution of clustering and genome-specific haplotype members of a unique cotton gene family associated with resistant response against a major pathogen.     

Entire nucleotide sequences of Gossypium raimondii and G. arboreum mitochondrial genomes revealed A‐genome species as cytoplasmic donor of the allotetraploid species

• Cotton (Gossypium spp.) is commonly grouped into eight diploid genomic groups, designated A–G and K, and an allotetraploid genomic group, AD. Gossypium raimondii (D₅) and G. arboreum (A₂) are the putative contributors to the progenitor of G. hirsutum (AD₁), the economically important fibre‐producing cotton species.
• Mitochondrial DNA from week‐old etiolated seedlings was extracted from isolated organelles using discontinuous sucrose density gradient method. Mitochondrial genomes were sequenced, assembled, annotated and analysed in orderly.
• Gossypium raimondii (D₅) and G. arboreum (A₂) mitochondrial genomes were provided in this study. The mitochondrial genomes of two diploid species harboured circular genome of 643,914 bp (D₅) and 687,482 bp (A₂), respectively. They differ in size and number of repeat sequences, both contain illuminating triplicate sequences with 7317 and 10,246 bp, respectively, demonstrating dynamic difference and rearranged genome organisations. Comparing the D₅ and A₂ mitogenomes with mitogenomes of tetraploid Gossypium species (AD₁, G. hirsutum; AD₂, G. barbadense), a shared 11 kbp fragment loss was detected in allotetraploid species, three regions shared by G. arboreum (A₂), G. hirsutum (AD₁) and G. barbadense (AD₂), while eight regions were specific to G. raimondii (D₅). The presence/absence variations and gene‐based phylogeny supported that A‐genome is a cytoplasmic donor to the progenitor of allotetraploid species G. hirsutum and G. barbadense.
• The results present structure variations and phylogeny of Gossypium mitochondrial genome evolution.

     

Chromosomal Locations of 5S and 45S rDNA in Gossypium Genus and Its Phylogenetic Implications Revealed by FISH

We investigated the locations of 5S and 45S rDNA in Gossypium diploid A, B, D, E, F, G genomes and tetraploid genome (AD) using multi-probe fluorescent in situ hybridization (FISH) for evolution analysis in Gossypium genus. The rDNA numbers and sizes, and synteny relationships between 5S and 45S were revealed using 5S and 45S as double-probe for all species, and the rDNA-bearing chromosomes were identified for A, D and AD genomes with one more probe that is single-chromosome-specific BAC clone from G. hirsutum (A₁D₁). Two to four 45S and one 5S loci were found in diploid-species except two 5S loci in G . incanum (E₄), the same as that in tetraploid species. The 45S on the 7th and 9th chromosomes and the 5S on the 9th chromosomes seemed to be conserved in A, D and AD genomes. In the species of B, E, F and G genomes, the rDNA numbers, sizes, and synteny relationships were first reported in this paper. The rDNA pattern agrees with previously reported phylogenetic history with some disagreements. Combined with the whole-genome sequencing data from G . raimondii (D₅) and the conserved cotton karyotype, it is suggested that the expansion, decrease and transposition of rDNA other than chromosome rearrangements might occur during the Gossypium evolution.     

Exogenous hormone applications at pollination for in vitro and in vivo production of cotton interspecific hybrids

Interspecific hybridization of cotton (Gossypium) has been assisted by ovule and embryo culture. These culture methods were compared to exogenous hormone applications for efficient plant production from crosses between Upland cotton, G. hirsutum L., as the maternal parent, and various diploid and tetraploid wild species as the pollen donor. The best exogenous hormone treatment resulted in an average production of five seeds per boll and 4% boll abscission. Generally, exogenous hormones used with standard hybridization techniques were superior to in vitro methods, but for some crosses, embryo culture following hormone applications was warranted.Abbreviations GA gibberellic acid, >90% A₃ - NAA 1-naphthaleneacetic acid - NOA 2-naphthoxyacetic acid.     

Nuclear DNA,heterochromatin and phylogeny of Nicotiana amphidiploids

There are significant differences in nuclear DNA amount between both diploid and amphidiploid species of Nicotiana. Owing to the higher DNA density in the interphase nuclei of the amphidiploids DNA amounts tend to be underestimated by microdensitometry. After applying necessary corrections to amphidiploid readings it was found that: (1) The nuclear DNA amount in the tetraploid N. rustica is not significantly different from the sum of nuclear DNA amounts in reputed diploid parents, N. undulata and N. paniculata. (2) It is well established that N. sylvestris is one of the diploid progenitors of N. tabacum. The sum of the nuclear DNA amounts in N. sylvestris and N. tomentosiformis is not significantly different from that of the amphidiploid N. tabacum. In contrast the sum of the DNA amounts in N. sylvestris and N. otophora is significantly higher than that in N. tabacum. Observations and measurements of the amount and distribution of heterochromatin in interphase nuclei of the diploid and tetraploid species give further support to the conclusion that N. tomentosiformis rather than N. otophora is the second diploid progenitor of N. tabacum.     

Heterochromatin differentiation and phylogenetic relationship of the A genomes in diploid and polyploid wheats

Summary Heterochromatin differentiation, including band size, sites, and Giemsa staining intensity, was analyzed by the HKG (HCl-KOH-Giemsa) banding technique in the A genomes of 21 diploid (Triticum urartu, T. boeoticum and T. monococcum), 13 tetraploid (T. araraticum, T. timopheevi, T. dicoccoides and T. turgidum var. Dicoccon, Polonicum), and 7 cultivars of hexaploid (T. aestivum) wheats from different germplasm collections. Among wild and cultivated diploid taxa, heterochromatin was located mainly at centromeric regions, but the size and staining intensity were distinct and some accessions' genomes had interstitial and telomeric bands. Among wild and cultivated polyploid wheats, heterochromatin exhibited bifurcated differentiation. Heterochromatinization occurred in chromosomes 4A^t and 7A^t and in smaller amounts in 2A^t, 3A^t, 5A^t, and 6A^t within the genomes of the tetraploid Timopheevi group (T. araraticum, and T. timopheevi) and vice versa within those of the Emmer group (T. dicoccoides and T. turgidum). Similar divergence patterns occurred among chromosome 4A^a and 7A^a of cultivars of hexaploid wheat (T. aestivum). These dynamic processes could be related to geographic distribution and to natural and artifical selection. Comparison of the A genomes of diploid wheats with those of polyploid wheats shows that the A genomes in existing diploid wheats could not be the direct donors of those in polyploid wheats, but that the extant taxa of diploids and polyploids probably have a common origin and share a common A-genomelike ancestor.Contribution of the College of Agricultural Sciences, Texas Tech Univ. Journal No. T-4-233.     

Sequencing and Utilization of the Gossypium Genomes

Revealing the genetic underpinnings of cotton productivity will require understanding both the prehistoric evolution of spinnable fibers, and the results of independent domestication processes in both the Old and New Worlds. Progress toward a reference sequence for the smallest Gossypium genome is a logical stepping-stone toward revealing diversity in the remaining seven genomes (A, B, C, E, F, G, K) that permitted Gossypium species to adapt to a wide range of ecosystems in warmer arid regions of the world, and toward identifying the emergent properties that account for the superior productivity and quality of tetraploid cottons. The greatest challenge facing the cotton community is not genome sequencing per se but the conversion of sequence to knowledge.     

Comprehensive Analysis of the COBRA-Like (COBL) Gene Family in Gossypium Identifies Two COBLs Potentially Associated with Fiber Quality

COBRA-Like (COBL) genes, which encode a plant-specific glycosylphosphatidylinositol (GPI) anchored protein, have been proven to be key regulators in the orientation of cell expansion and cellulose crystallinity status. Genome-wide analysis has been performed in A. thaliana, O. sativa, Z. mays and S. lycopersicum, but little in Gossypium. Here we identified 19, 18 and 33 candidate COBL genes from three sequenced cotton species, diploid cotton G. raimondii, G. arboreum and tetraploid cotton G. hirsutum acc. TM-1, respectively. These COBL members were anchored onto 10 chromosomes in G. raimondii and could be divided into two subgroups. Expression patterns of COBL genes showed highly developmental and spatial regulation in G. hirsutum acc. TM-1. Of them, GhCOBL9 and GhCOBL13 were preferentially expressed at the secondary cell wall stage of fiber development and had significantly co-upregulated expression with cellulose synthase genes GhCESA4, GhCESA7 and GhCESA8. Besides, GhCOBL9 D_t and GhCOBL13 D_t were co-localized with previously reported cotton fiber quality quantitative trait loci (QTLs) and the favorable allele types of GhCOBL9 D_t had significantly positive correlations with fiber quality traits, indicating that these two genes might play an important role in fiber development.     

Development of chromosome-specific markers with high polymorphism for allotetraploid cotton based on genome-wide characterization of simple sequence repeats in diploid cottons (Gossypium arboreum L. and Gossypium raimondii Ulbrich)

Background

Tetraploid cotton contains two sets of homologous chromosomes, the At- and Dt-subgenomes. Consequently, many markers in cotton were mapped to multiple positions during linkage genetic map construction, posing a challenge to anchoring linkage groups and mapping economically-important genes to particular chromosomes. Chromosome-specific markers could solve this problem. Recently, the genomes of two diploid species were sequenced whose progenitors were putative contributors of the At- and Dt-subgenomes to tetraploid cotton. These sequences provide a powerful tool for developing chromosome-specific markers given the high level of synteny among tetraploid and diploid cotton genomes. In this study, simple sequence repeats (SSRs) on each chromosome in the two diploid genomes were characterized. Chromosome-specific SSRs were developed by comparative analysis and proved to distinguish chromosomes.

Results

A total of 200,744 and 142,409 SSRs were detected on the 13 chromosomes of Gossypium arboreum L. and Gossypium raimondii Ulbrich, respectively. Chromosome-specific SSRs were obtained by comparing SSR flanking sequences from each chromosome with those from the other 25 chromosomes. The average was 7,996 per chromosome. To confirm their chromosome specificity, these SSRs were used to distinguish two homologous chromosomes in tetraploid cotton through linkage group construction. The chromosome-specific SSRs and previously-reported chromosome markers were grouped together, and no marker mapped to another homologous chromosome, proving that the chromosome-specific SSRs were unique and could distinguish homologous chromosomes in tetraploid cotton. Because longer dinucleotide AT-rich repeats were the most polymorphic in previous reports, the SSRs on each chromosome were sorted by motif type and repeat length for convenient selection. The primer sequences of all chromosome-specific SSRs were also made publicly available.

Conclusion

Chromosome-specific SSRs are efficient tools for chromosome identification by anchoring linkage groups to particular chromosomes during genetic mapping and are especially useful in mapping of qualitative-trait genes or quantitative trait loci with just a few markers. The SSRs reported here will facilitate a number of genetic and genomic studies in cotton, including construction of high-density genetic maps, positional gene cloning, fingerprinting, and genetic diversity and comparative evolutionary analyses among Gossypium species.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1265-2) contains supplementary material, which is available to authorized users.     

Analysis of complete nucleotide sequences of 12 gossypium chloroplast genomes: origin and evolution of allotetraploids

Background

Cotton (Gossypium spp.) is a model system for the analysis of polyploidization. Although ascertaining the donor species of allotetraploid cotton has been intensively studied, sequence comparison of Gossypium chloroplast genomes is still of interest to understand the mechanisms underlining the evolution of Gossypium allotetraploids, while it is generally accepted that the parents were A- and D-genome containing species. Here we performed a comparative analysis of 13 Gossypium chloroplast genomes, twelve of which are presented here for the first time.

Methodology/Principal Findings

The size of 12 chloroplast genomes under study varied from 159,959 bp to 160,433 bp. The chromosomes were highly similar having >98% sequence identity. They encoded the same set of 112 unique genes which occurred in a uniform order with only slightly different boundary junctions. Divergence due to indels as well as substitutions was examined separately for genome, coding and noncoding sequences. The genome divergence was estimated as 0.374% to 0.583% between allotetraploid species and A-genome, and 0.159% to 0.454% within allotetraploids. Forty protein-coding genes were completely identical at the protein level, and 20 intergenic sequences were completely conserved. The 9 allotetraploids shared 5 insertions and 9 deletions in whole genome, and 7-bp substitutions in protein-coding genes. The phylogenetic tree confirmed a close relationship between allotetraploids and the ancestor of A-genome, and the allotetraploids were divided into four separate groups. Progenitor allotetraploid cotton originated 0.43–0.68 million years ago (MYA).

Conclusion

Despite high degree of conservation between the Gossypium chloroplast genomes, sequence variations among species could still be detected. Gossypium chloroplast genomes preferred for 5-bp indels and 1–3-bp indels are mainly attributed to the SSR polymorphisms. This study supports that the common ancestor of diploid A-genome species in Gossypium is the maternal source of extant allotetraploid species and allotetraploids have a monophyletic origin. G. hirsutum AD1 lineages have experienced more sequence variations than other allotetraploids in intergenic regions. The available complete nucleotide sequences of 12 Gossypium chloroplast genomes should facilitate studies to uncover the molecular mechanisms of compartmental co-evolution and speciation of Gossypium allotetraploids.     

Assessing the monophyly of polyploid Gossypium species

The origin and monophyly of the polyploid cotton (Gossypium) species has been largely accepted, despite the lack of explicit phylogenetic evidence. Recent studies in other polyploid systems have demonstrated that multiple origins for polyploid species are much more common than once thought, raising the possibility that Gossypium polyploids also had multiple origins, as postulated by some authors. To test the monophyly of polyploid cotton, we sequenced a 2.8-kb intergenic region from all diploid species belonging to the genome groups from which the polyploid originates. The resulting phylogenetic analyses strongly support a single origin of polyploid cotton involving a D-genome ancestor related to Gossypium raimondii and an A-genome ancestor that was sister to both extant A-genome species.     

Somatic embryo initiation and germination in diploid cotton (<Emphasis Type="Italic">Gossypium arboreum</Emphasis> L.)

Summary The diploid cotton species can constitute a valuable gene pool for the more agronomically desirable cultivated tetraploid cultivars and offer better opportunities to study gene structure and function through gene knockouts. In order to exploit these advantages, a regeneration system is required to achieve these transformation-based goals. Carbohydrate source and concentration were evaluated to improve somatic embryo (SE) production and desiccation treatments to improve the conversion efficiency of SEs to plants in a diploid Gossypium arboreum accession, A₂-9 (PI-529712). Improved SE numbers and their subsequent conversion into plantlets was achieved with a Murashige and Skoog (MS)/sucrose-based medium M₂ [0.04M sucrose, 0.3 μM α-naphthaleneacetic acid (NAA)] On this medium, 219 embryos per g initiated, and close to 11% of these embryos germinated into plantlets. Neither a 5-d desiccation treatment of embryogenic callus previously cultured in liquid medium nor filter paper insertion improved the numbers of SEs induced or their conversion to plantlets. A 3-d desiccation period resulted in improved plant regeneration. When immature G. arboreum SEs induced on M₁ (0.2M glucose, 2.6 μM NAA, and 0.2 μM kinetin) medium underwent a 3-d desiccation treatment, 49% of these immature SEs were converted to plantlets after a 4-wk period on M₂ medium. These improved results will help to pave the way for future genetic transformation and associated gene structure and function studies utilizing G. arboreum. These results, in particular the 3-d desiccation treatment, can also be incorporated into regeneration protocols to improve the regeneration efficiency of other Gossypium species.     

Analysis of [Gossypium capitis-viridis × (G.hirsutum × G.australe)2] Trispecific Hybrid and Selected Characteristics

Speciation is always a contentious and challenging issue following with the presence of gene flow. In Gossypium, there are many valuable resources and wild diploid cotton especially C and B genome species possess some excellent traits which cultivated cotton always lacks. In order to explore character transferring rule from wild cotton to upland tetraploid cotton, the [G. capitis-viridis × (G. hirsutum × G. australe)²] triple hybrid was synthesized by interspecies hybridization and chromosome doubling. Morphology comparisons were measured among this hybrid and its parents. It showed that trispecific hybrid F₁ had some intermediate morphological characters like leaf style between its parents and some different characters from its parents, like crawl growth characteristics and two kind flower color. It is highly resistant to insects comparing with other cotton species by four year field investigation. By cytogenetic analysis, triple hybrid was further confirmed by meiosis behavior of pollen mother cells. Comparing with regular meiosis of its three parents, it was distinguished by the occurrence of polyads with various numbers of unbalanced microspores and finally generating various abnormal pollen grains. All this phenomenon results in the sterility of this hybrid. This hybrid was further identified by SSR marker from DNA molecular level. It showed that 98 selected polymorphism primers amplified effective bands in this hybrids and its parents. The genetic proportion of three parents in this hybrid is 47.8% from G. hirsutum, 14.3% from G. australe, 7.0% from G. capitis-viridis, and 30.9% recombination bands respectively. It was testified that wild genetic material has been transferred into cultivated cotton and this new germplasm can be incorporated into cotton breeding program.     

Studies of multipolar mitoses in euploid tissue cultures

In tissue cultures of male Microtus agrestis, diploid mitoses with two X or two Y chromosomes were found. For identifiying the sex chromosomes in nonhypotonioally treated mitoses, the asynchrony of DNA replication of the sex chromosomes of both sexes was used. The constitutive heterochromatin of Y replicates later in the S period than X, and X₂ of the female replicates later than X₁. Autoradiographic studies of tetraploid tripolar mitoses showed that the diploid daughter nuclei contain either XX or YY in the male; in the female, X₁X₂ daughter nuclei were found less frequently than X₁X₁ and X₂X₂ cells.     

Incipient Genome Differentiation in Gossypium. III. Comparison of Chromosomes of G. HIRSUTUM and Asiatic Diploids Using Heterozygous Translocations

Hybrids between upland cotton (G. hirsutum, genome constitution 2A_hD_h) and either A-genome or D-genome diploid species exhibit 26 paired and 13 unpaired chromosomes at metaphase I. The A_h and D_h genomes are therefore considered homoeologous with those of the respective diploids. Previous studies, nevertheless, revealed a low level of ("incipient") differentiation between D_h and various diploid D genomes. The diploid A genomes have been regarded as more closely homologous to A_h on the basis of low preferential pairing and autotetraploid segregation ratios in allohexaploids.—The present study addressed the following questions: Are the diploid A genomes differentiated from A_h in meiotic homology? If so, is the differentiation manifested equally by all 13 chromosomes or is it localized in certain chromosomes?—Three diploid A-genome lines representing G. herbaceum and G. arboreum were hybridized by in ovulo culture of embryos (1) with a standard line of G. hirsutum, which differs from G. herbaceum by two and from G. arboreum by three naturally occurring reciprocal translocations involving chromosomes 1–5, and (2) with six lines homozygous for experimental translocations involving chromosomes 6, 7, 10, 11, 12 and 13. Chiasma frequencies in hybrids were compared with those in appropriate G. hirsutum controls. In every comparison overall chiasma frequencies were slightly lower in the hybrids. Therefore A_h appears to be differentiated from the diploid A genomes. No localized differentiation was detected in chromosomes marked by experimental translocations. The differentiation may be localized mainly in chromosomes 4 and 5.