N. Jouve

Next-generation sequencing and syntenic integration of flow-sorted arms of wheat chromosome 4A exposes the chromosome structure and gene content

Wheat is the third most important crop for human nutrition in the world. The availability of high-resolution genetic and physical maps and ultimately a complete genome sequence holds great promise for breeding improved varieties to cope with increasing food demand under the conditions of changing global climate. However, the large size of the bread wheat (Triticum aestivum) genome (approximately 17 Gb/1C) and the triplication of genic sequence resulting from its hexaploid status have impeded genome sequencing of this important crop species. Here we describe the use of mitotic chromosome flow sorting to separately purify and then shotgun-sequence a pair of telocentric chromosomes that together form chromosome 4A (856 Mb/1C) of wheat. The isolation of this much reduced template and the consequent avoidance of the problem of sequence duplication, in conjunction with synteny-based comparisons with other grass genomes, have facilitated construction of an ordered gene map of chromosome 4A, embracing ≥85% of its total gene content, and have enabled precise localization of the various translocation and inversion breakpoints on chromosome 4A that differentiate it from its progenitor chromosome in the A genome diploid donor. The gene map of chromosome 4A, together with the emerging sequences of homoeologous wheat chromosome groups 4, 5 and 7, represent unique resources that will allow us to obtain new insights into the evolutionary dynamics between homoeologous chromosomes and syntenic chromosomal regions.

Mapping and organization of highly-repeated DNA sequences by means of simultaneous and sequential FISH and C-banding in 6×-triticale

Three families of highly repeated sequences from rye and the rRNA multigenes (NOR and 5S) have been mapped by FISH and C-banding, in chromosomes of triticale. The pSc119.2 probe showed interstitial hybridization in chromosome arms 1RS, 1RL, 4RL, 5RL, 6RS, 6RL, 7RS and 7RL, and is very effective for chromosome identification of rye chromosomes in triticale. This sequence also hybridizes to the 4A, 5A and the seven B-genome wheat chromosomes. Simultaneous hybridization with the pSc119.2 and pTa794 (5S rRNA) is very useful to distinguish the metacentric chromosomes 2R and 3R. The pSc74 probe appears at interstitial sites in the long arm of the most heterobrachial chromosomes (5R and 6R). The three repetitive sequences of 120 bp, 480 bp, and 610 bp hybridize to telomeric regions in rye chromosomes. Different arrangements and complex organizations consisting of arrays of three or more family sequences were found. The results demonstrate a great variation in the relative arrangement of the repetitive sequences in the telomeres of the rye chromosomes. There were quantitative differences in each cytological marker between triticale lines in bothin situ labelling and C-banding, probably as the result of differences in the number and/or kind of repeat sequence.

Molecular characterisation of the inactive allele of the gene Glu-A1 and the development of a set of AS-PCR markers for HMW glutenins of wheat.

Abstract The present work reports new PCR markers that amplify the complete coding sequence of the specific alleles of the high molecular weight (HMW) glutenin genes. A set of AS-PCR molecular markers was designed which use primers from nucleotide sequences of the Glu-A1 and Glu-D1 genes, making use of the minor diffeences between the sequences of the x1, x2* of Glu-A1, and the x5 and y10 of Glu-D1. These primers were able to distinguish between x2* and the x1 or xNull of Glu-A1. Also x5 was distinguishable from x2, and y10 from y12. The primers amplified the complete coding regions and corresponded to the upstream and downstream flanking positions of Glu-A1 and Glu-D1. Primers designed to amplify the Glu-A1 gene amplified a single product when used with genomic DNA of common wheats and the xNull allele of this gene. This work also describes the cloning and characterisation of the nucleotide sequence of this allele. It possesses the same general structure as x2* and x1 (previously determined) and differs from these alleles in the extension of the coding sequence for a presumptive mature protein with only 384 residues. This is due to the presence of a stop codon (TAA) 1215-bp downstream from the start codon. A further stop codon (TAG), 2280-bp downstream from the starting codon is also found. The open reading frame of xNull and x1 alleles has the same size in bp. Both are larger than x2* which shows two small deletions. The reduced size of the presumptive mature protein encoded by xNull could explain the negative effect of this allele on grain quality.

Identification of different chromatin classes in wheat using in situ hybridization with simple sequence repeat oligonucleotides.

Abstract Clusters of four simple sequence repeats (SSRs), AAC, AAG, AG and CAT, have been mapped physically to hexaploid wheat chromosomes; 15—24-bp synthetic oligonucleotides were labelled by random-primer labelling and used as probes for fluorescent in situ hybridization with standard formamide and low-salt conditions. AAC hybridized strongly to the pericentromeric regions and several intercalary sites of all seven chromosomes of the B-genome corresponding to N bands and enabling their identification. Most of the AAC sites also co-localize with AAG, although the strength of the AAC and AAG signal was often different at the same location. Not all heterochromatic bands showed AAC signals and a few AAC sites were detected that are neither AAG nor N band positive, revealing the complex and heterogeneous genome organization of wheat and identifying the four most frequent classes of banded chromatin. Clusters characterised by a high concentration of AG repeats were detected on chromosome arms 3BS, 4BL, 5BS and 5BL, adjacent to AAG sites. The only detectable CAT cluster was found on chromosome arm 3BL, making this oligonucleotide valuable in identifying this particular chromosome. SSR in situ hybridization is useful as a diagnostic tool in cytogenetics and for understanding genome organization in wheat.

Physical mapping of repetitive DNA sequences and 5S and 18S-26S rDNA in five wild species of the genus Hordeum.

The genetic relationships between several wild species and subspecies of the genusHordeum were assessed using fluorescencein situ hybridization (FISH). Plant material included natural populations of wild barley growing in Spain of the annual species,H. marinum ssp.marinum (2n=14) andgussoneanum (2n=14), andH. murinum ssp.murinum (2n=28), andleporinum (2n=28) and the perennial speciesH. bulbosum (2n=14) andH. secalinum (2n=28), plus the South American perennial speciesH. chilense (2n=14). FISH was used to locate the chromosomal sites of two rDNA multigene families 5S and 18S–26S (pTa71 and pTa794) and three repetitive DNA sequences (pSc119.2, pAs1 and pHch950) isolated from different species and genera. The seven chromosomes of the diploid species were readily distinguished by their external morphology and hybridization patterns to pTa71, pTa794, pSc119.2 and pAs1. These DNA probes were also useful for the identification of homologous chromosomes and in differentiating these from unidentified chromosomes in the tetraploid taxa. The use of the probe pHch950 permitted intergenomic differentiation in tetraploids and supports the diphyletic origin ofH. murinum andH. secalinum. Thein situ experiments yielded the following conclusions: (1) differences between the subspeciesmarinum andgussoneanum; (2) close relationships between the subspeciesmurinum andLeporinum; and (3) major differences in physical mapping betweenH. bulbosum and the remaining taxa. The genomic and phylogenetic relationships between taxa, as inferred from the results, are discussed.

RAPD variation in wild populations of four species of the genus Hordeum (Poaceae)

Abstract The genetic variation of 102 natural populations of wild barley growing in Spain was assessed using RAPDs (random amplified polymorphic DNA). The plant material included the annual species H. marinum subsp. marinum (22 populations) and subsp. gussoneanum (14), H. murinum subsp. murinum (7) and subsp. leporinum (35), and the perennial species H. bulbosum (17) and H. secalinum (7). Ten of the tested 64 arbitrary 10-mer primers amplified polymorphic DNA in all taxonomic units. Analyses was performed within and between populations, species and subspecies. The primers gave a total of 250 RAPD products. The level of polymorphism varied between taxonomic units depending on the primers employed and the plant reproductive system. In general, the most variable were the allogamous species H. secalinum and H. bulbosum and the autogamous H. marinum subsp. marinum. Among the amplified bands, 69 (27%) were shared by at least two different taxonomic units. The remaining bands were specific. The results demonstrate differences in the degree of similarity between taxonomic units. Jaccard’s similarity coefficients for interval measure within and between populations were used to produce a cluster diagram using the unweighted pair-group method (UPGMA). The different populations of the species and subspecies of Hordeum fell into three groups. The first group contained the populations belonging to both subspecies of H. marinum, plus those of H. secalinum. The populations of H. marinum subsp. gussoneanum were very closely associated. Those of H. marinum subsp. marinum were grouped in a broad cluster. The second group, occupying the innermost position of the tree, was very closely associated with the populations of both subspecies of H. murinum. The third branch segregated H. bulbosum. A series of RAPD markers were investigated by cleaving the amplified products of the same size with restriction endonucleases that recognize targets of 4- or 6-bp. The production of equivalent fragments following cleavage by the same enzyme would seem to demonstrate their homology in samples from different individuals, populations or taxonomic units.

Physical organisation of simple sequence repeats (SSRs) in Triticeae: structural, functional and evolutionary implications

A significant fraction of the nuclear DNA of all eukaryotes is occupied by simple sequence repeats (SSRs) or microsatellites. This type of sequence has sparked great interest as a means of studying genetic variation, linkage mapping, gene tagging and evolution. Although SSRs at different positions in a gene help determine the regulation of expression and the function of the protein produced, little attention has been paid to the chromosomal organisation and distribution of these sequences, even in model species. This review discusses the main achievements in the characterisation of long-range SSR organisation in the chromosomes of Triticum aestivum L., Secale cereale L., and Hordeum vulgare L. (all members of Triticeae). We have detected SSRs using an improved FISH technique based on the random primer labelling of synthetic oligonucleotides (15–24 bases) in multi-colour experiments. Detailed information on the presence and distribution of AC, AG and all the possible classes of trinucleotide repeats has been acquired. These data have revealed the motif-dependent and non-random chromosome distributions of SSRs in the different genomes, and allowed the correlation of particular SSRs with chromosome areas characterised by specific features (e.g., heterochromatin, euchromatin and centromeres) in all three species. The present review provides a detailed comparative study of the distribution of these SSRs in each of the seven chromosomes of the genomes A, B and D of wheat, H of barley and R of rye. The importance of SSRs in plant breeding and their possible role in chromosome structure, function and evolution is discussed.

Characterisation and analysis of new HMW-glutenin alleles encoded by the Glu-R1 locus of Secale cereale

This work reports the molecular characterisation of new alleles of the previously reported Glu-R1 locus. Wheat lines carrying the chromosome substitution 1R(1D), rye cultivars and related wild species were analysed. Five new x-type and four y-type Glu-R1 glutenin subunits were isolated and characterised. The coding region of the sequences shows the typical structure of the HMW glutenin genes previously described in wheat, with the N and C-terminal domains flanking the central repetitive region. Tri-, hexa- and nona-peptides found in the central repetitive region of wheat glutenin genes were also present in the rye genes. Duplications and deletions of these motifs are responsible for allelic variation at the Glu-R1 locus. Orthologous genes (from different genomes) were more closely related than paralogous genes (x- and y-type), supporting the hypothesis of gene duplication before Triticeae speciation. Differences in the number and position of cysteine residues identified alleles which in wheat are associated with good dough quality. SDS proteins encoded by some characterised alleles were presumptively identified.

Phylogenetic relationships of the genus Secale based on the characterisation of rDNA ITS sequences

Abstract. Sequence analysis of the internal transcribed spacer of the 18S-5.8S-26S rDNA (ITS-1) region was performed in order to analyse the phylogenetic relationships of eleven taxa of cultivated and wild rye species. The ITS regions were amplified using designed primers. At least ten positive clones of each taxonomic unit were sequenced and compared. Two different ITS sequences were found in three taxa: Secale sylvestre Host, Secale strictum ssp. kuprijanovii Grossh. and Secale strictum ssp. africanum Stapf. Secale sylvestre Host was the species that showed the greatest number of comparative differences in the sequences, and was the most distant of all the taxonomic units analysed. A certain degree of variation was found among all four subspecies of S. strictum analysed. S. strictum Presl ssp. strictum was most closely related to S. strictum ssp. africanum Stapf and S. strictum ssp. kuprijanovii Grossh to S. strictum ssp. anatolicum (Boiss.) Hammer. S. vavilovii showed similarities with this group of subspecies and with the S. cereale group. No differences were found between the weed forms of S. cereale and cultivated rye.

Fluorescence in situ hybridization with multiple repeated DNA probes applied to the analysis of wheat-rye chromosome pairing

Abstract Fluorescence in situ hybridization (FISH) with multiple probes has been applied to meiotic chromosome spreads derived from ph1b common wheat x rye hybrid plants. The probes used included pSc74 and pSc 119.2 from rye (the latter also hybridizes on wheat, mainly B genome chromosomes), the Ae. squarrosa pAs1 probe, which hybridizes almost exclusively on D genome chromosomes, and wheat rDNA probes pTa71 and pTa794. Simultaneous and sequential FISH with a two-by-two combination of these probes allowed unequivocal identification of all of the rye (R) and most of the wheat (W) chromosomes, either unpaired or involved in pairing. Thus not only could wheat-wheat and wheat-rye associations be easily discriminated, which was already feasible by the sole use of the rye-specific pSc74 probe, but the individual pairing partners could also be identified. Of the wheat-rye pairing observed, which averaged from about 7% to 11% of the total pairing detected in six hybrid plants of the same cross combination, most involved B genome chromosomes (about 70%), and to a much lesser degree, those of the D (almost 17%) and A (14%) genomes. Rye arms 1RL and 5RL showed the highest pairing frequency (over 30%), followed by 2RL (11%) and 4RL (about 8%), with much lower values for all the other arms. 2RS and 5RS were never observed to pair in the sample analysed. Chromosome arms 1RL, 1RS, 2RL, 3RS, 4RS and 6RS were observed to be exclusively bound to wheat chromosomes of the same homoeologous group. The opposite was true for 4RL (paired with 6BS and 7BS) and 6RL (paired with 7BL). 5RL, on the other hand, paired with 4WL arms or segments of them in more than 80% of the cases and with 5WL in the remaining ones. Additional cases of pairing involving wheat chromosomes belonging to more than one homoeologous group occurred with 3RL, 7RS and 7RL. These results, while adding support to previous evidence about the existence of several translocations in the rye genome relative to that of wheat, show that FISH with multiple probes is an efficient method by which to study fundamental aspects of chromosome behaviour at meiosis, such as interspecific pairing. The type of knowledge attainable from this approach is expected to have a significant impact on both theoretical and applied research concerning wheat and related Triticeae.