Chromosome arm-specific BAC end sequences permit comparative analysis of homoeologous chromosomes and genomes of polyploid wheat.

Bread wheat, one of the world's staple food crops, has the largest, highly repetitive and polyploid genome among the cereal crops. The wheat genome holds the key to crop genetic improvement against challenges such as climate change, environmental degradation, and water scarcity. To unravel the complex wheat genome, the International Wheat Genome Sequencing Consortium (IWGSC) is pursuing a chromosome- and chromosome arm-based approach to physical mapping and sequencing. Here we report on the use of a BAC library made from flow-sorted telosomic chromosome 3A short arm (t3AS) for marker development and analysis of sequence composition and comparative evolution of homoeologous genomes of hexaploid wheat. The end-sequencing of 9,984 random BACs from a chromosome arm 3AS-specific library (TaaCsp3AShA) generated 11,014,359 bp of high quality sequence from 17,591 BAC-ends with an average length of 626 bp. The sequence represents 3.2% of t3AS with an average DNA sequence read every 19 kb. Overall, 79% of the sequence consisted of repetitive elements, 1.38% as coding regions (estimated 2,850 genes) and another 19% of unknown origin. Comparative sequence analysis suggested that 70-77% of the genes present in both 3A and 3B were syntenic with model species. Among the transposable elements, gypsy/sabrina (12.4%) was the most abundant repeat and was significantly more frequent in 3A compared to homoeologous chromosome 3B. Twenty novel repetitive sequences were also identified using de novo repeat identification. BESs were screened to identify simple sequence repeats (SSR) and transposable element junctions. A total of 1,057 SSRs were identified with a density of one per 10.4 kb, and 7,928 junctions between transposable elements (TE) and other sequences were identified with a density of one per 1.39 kb. With the objective of enhancing the marker density of chromosome 3AS, oligonucleotide primers were successfully designed from 758 SSRs and 695 Insertion Site Based Polymorphisms (ISBPs). Of the 96 ISBP primer pairs tested, 28 (29%) were 3A-specific and compared to 17 (18%) for 96 SSRs. This work reports on the use of wheat chromosome arm 3AS-specific BAC library for the targeted generation of sequence data from a particular region of the huge genome of wheat. A large quantity of sequences were generated from the A genome of hexaploid wheat for comparative genome analysis with homoeologous B and D genomes and other model grass genomes. Hundreds of molecular markers were developed from the 3AS arm-specific sequences; these and other sequences will be useful in gene discovery and physical mapping.

Sehgal SK ，Li W ，Rabinowicz PD ，Chan A ，Simková H ，Doležel J ，Gill BS ... -  《BMC PLANT BIOLOGY》

Characterizing the composition and evolution of homoeologous genomes in hexaploid wheat through BAC-end sequencing on chromosome 3B.

Bread wheat (Triticum aestivum) is one of the most important crops worldwide. However, because of its large, hexaploid, highly repetitive genome it is a challenge to develop efficient means for molecular analysis and genetic improvement in wheat. To better understand the composition and molecular evolution of the hexaploid wheat homoeologous genomes and to evaluate the potential of BAC-end sequences (BES) for marker development, we have followed a chromosome-specific strategy and generated 11 Mb of random BES from chromosome 3B, the largest chromosome of bread wheat. The sequence consisted of about 86% of repetitive elements, 1.2% of coding regions, and 13% remained unknown. With 1.2% of the sequence length corresponding to coding sequences, 6000 genes were estimated for chromosome 3B. New repetitive sequences were identified, including a Triticineae-specific tandem repeat (Fat) that represents 0.6% of the B-genome and has been differentially amplified in the homoeologous genomes before polyploidization. About 10% of the BES contained junctions between nested transposable elements that were used to develop chromosome-specific markers for physical and genetic mapping. Finally, sequence comparison with 2.9 Mb of random sequences from the D-genome of Aegilops tauschii suggested that the larger size of the B-genome is due to a higher content in repetitive elements. It also indicated which families of transposable elements are mostly responsible for differential expansion of the homoeologous wheat genomes during evolution. Our data demonstrate that BAC-end sequencing from flow-sorted chromosomes is a powerful tool for analysing the structure and evolution of polyploid and highly repetitive genomes.

Paux E ，Roger D ，Badaeva E ，Gay G ，Bernard M ，Sourdille P ，Feuillet C ... -  《-》

A highly conserved gene island of three genes on chromosome 3B of hexaploid wheat: diverse gene function and genomic structure maintained in a tightly linked block.

The complexity of the wheat genome has resulted from waves of retrotransposable element insertions. Gene deletions and disruptions generated by the fast replacement of repetitive elements in wheat have resulted in disruption of colinearity at a micro (sub-megabase) level among the cereals. In view of genomic changes that are possible within a given time span, conservation of genes between species tends to imply an important functional or regional constraint that does not permit a change in genomic structure. The ctg1034 contig completed in this paper was initially studied because it was assigned to the Sr2 resistance locus region, but detailed mapping studies subsequently assigned it to the long arm of 3B and revealed its unusual features. BAC shotgun sequencing of the hexaploid wheat (Triticum aestivum cv. Chinese Spring) genome has been used to assemble a group of 15 wheat BACs from the chromosome 3B physical map FPC contig ctg1034 into a 783,553 bp genomic sequence. This ctg1034 sequence was annotated for biological features such as genes and transposable elements. A three-gene island was identified among >80% repetitive DNA sequence. Using bioinformatics analysis there were no observable similarity in their gene functions. The ctg1034 gene island also displayed complete conservation of gene order and orientation with syntenic gene islands found in publicly available genome sequences of Brachypodium distachyon, Oryza sativa, Sorghum bicolor and Zea mays, even though the intergenic space and introns were divergent. We propose that ctg1034 is located within the heterochromatic C-band region of deletion bin 3BL7 based on the identification of heterochromatic tandem repeats and presence of significant matches to chromodomain-containing gypsy LTR retrotransposable elements. We also speculate that this location, among other highly repetitive sequences, may account for the relative stability in gene order and orientation within the gene island.Sequence data from this article have been deposited with the GenBank Data Libraries under accession no. GQ422824.

Breen J ，Wicker T ，Kong X ，Zhang J ，Ma W ，Paux E ，Feuillet C ，Appels R ，Bellgard M ... -  《-》

A first survey of the rye (Secale cereale) genome composition through BAC end sequencing of the short arm of chromosome 1R.

Rye (Secale cereale L.) belongs to tribe Triticeae and is an important temperate cereal. It is one of the parents of man-made species Triticale and has been used as a source of agronomically important genes for wheat improvement. The short arm of rye chromosome 1 (1RS), in particular is rich in useful genes, and as it may increase yield, protein content and resistance to biotic and abiotic stress, it has been introgressed into wheat as the 1BL.1RS translocation. A better knowledge of the rye genome could facilitate rye improvement and increase the efficiency of utilizing rye genes in wheat breeding. Here, we report on BAC end sequencing of 1,536 clones from two 1RS-specific BAC libraries. We obtained 2,778 (90.4%) useful sequences with a cumulative length of 2,032,538 bp and an average read length of 732 bp. These sequences represent 0.5% of 1RS arm. The GC content of the sequenced fraction of 1RS is 45.9%, and at least 84% of the 1RS arm consists of repetitive DNA. We identified transposable element junctions in BESs and developed insertion site based polymorphism markers (ISBP). Out of the 64 primer pairs tested, 17 (26.6%) were specific for 1RS. We also identified BESs carrying microsatellites suitable for development of 1RS-specific SSR markers. This work demonstrates the utility of chromosome arm-specific BAC libraries for targeted analysis of large Triticeae genomes and provides new sequence data from the rye genome and molecular markers for the short arm of rye chromosome 1.

Bartos J ，Paux E ，Kofler R ，Havránková M ，Kopecký D ，Suchánková P ，Safár J ，Simková H ，Town CD ，Lelley T ，Feuillet C ，Dolezel J ... -  《-》

Analysis and mapping of randomly chosen bacterial artificial chromosome clones from hexaploid bread wheat.

The current view of wheat genome composition is that genes are compartmentalized into gene-rich and gene-poor regions. This model can be tested by analyzing randomly selected bacterial artificial chromosome (BAC) clones for gene content, followed by placement of these BACs onto physical and genetic maps. Map localization could be difficult for BACs that consist entirely of repeated elements. We therefore developed a technique where repeat junctions are used to generate unique markers. Four BAC clones from hexaploid wheat variety Chinese Spring were randomly selected and sequenced at 4- to 6-fold redundancy. About 50% of the BAC sequences corresponded to previously identified repeats, mainly LTR-retrotransposons, whereas most of the remaining DNA consisted of sequences with unknown origin or function. The average gene content was <1%, although each BAC contained one or two identified genes. Repeat boundaries were amplified and used to map each clone to a chromosome arm. Extrapolation from wheat-rice comparative knowledge suggests that three of the four BAC clones originate from "gene-rich" regions of the wheat genome. Nevertheless, because these BACs carry only a single gene (two BACs) or two genes (one BAC), the predicted gene density is approximately 1 gene per 75 kb, which is considerably lower than previously estimated gene densities (one gene per 5-20 kb) for gene-rich regions in wheat. This analysis of randomly selected wheat BAC clones suggests that genes are more evenly distributed in wheat than previously believed and substantiates the need for large-scale random BAC sequencing to determine wheat genome organization.

Devos KM ，Ma J ，Pontaroli AC ，Pratt LH ，Bennetzen JL ... -  《-》