Dissecting large and complex genomes: flow sorting and BAC cloning of individual chromosomes from bread wheat.

The analysis of the complex genome of common wheat (Triticum aestivum, 2n = 6x = 42, genome formula AABBDD) is hampered by its large size ( approximately 17 000 Mbp) and allohexaploid nature. In order to simplify its analysis, we developed a generic strategy for dissecting such large and complex genomes into individual chromosomes. Chromosome 3B was successfully sorted by flow cytometry and cloned into a bacterial artificial chromosome (BAC), using only 1.8 million chromosomes and an adapted protocol developed for this purpose. The BAC library (designated as TA-3B) consists of 67 968 clones with an average insert size of 103 kb. It represents 6.2 equivalents of chromosome 3B with 100% coverage and 90% specificity as confirmed by genetic markers. This method was validated using other chromosomes and its broad application and usefulness in facilitating wheat genome analysis were demonstrated by target characterization of the chromosome 3B structure through cytogenetic mapping. This report on the successful cloning of flow-sorted chromosomes into BACs marks the integration of flow cytogenetics and genomics and represents a great leap forward in genetics and genomic analysis.

Safár J ，Bartos J ，Janda J ，Bellec A ，Kubaláková M ，Valárik M ，Pateyron S ，Weiserová J ，Tusková R ，Cíhalíková J ，Vrána J ，Simková H ，Faivre-Rampant P ，Sourdille P ，Caboche M ，Bernard M ，Dolezel J ，Chalhoub B ... -  《PLANT JOURNAL》

Advanced resources for plant genomics: a BAC library specific for the short arm of wheat chromosome 1B.

Common wheat (Triticum aestivum L., 2n = 6x = 42) is a polyploid species possessing one of the largest genomes among the cultivated crops (1C is approximately 17 000 Mb). The presence of three homoeologous genomes (A, B and D), and the prevalence of repetitive DNA make sequencing the wheat genome a daunting task. We have developed a novel 'chromosome arm-based' strategy for wheat genome sequencing to simplify this task; this relies on sub-genomic libraries of large DNA inserts. In this paper, we used a di-telosomic line of wheat to isolate six million copies of the short arm of chromosome 1B (1BS) by flow sorting. Chromosomal DNA was partially digested with HindIII and used to construct an arm-specific BAC library. The library consists of 65 280 clones with an average insert size of 82 kb. Almost half of the library (45%) has inserts larger than 100 kb, while 18% of the inserts range in size between 75 and 100 kb, and 37% are shorter than 75 kb. We estimated the chromosome arm coverage to be 14.5-fold, giving a 99.9% probability of identifying a clone corresponding to any sequence on the short arm of 1B. Each chromosome arm in wheat can be flow sorted from an appropriate cytogenetic stock, and we envisage that the availability of chromosome arm-specific BAC resources in wheat will greatly facilitate the development of ready-to-sequence physical maps and map-based gene cloning.

Janda J ，Safár J ，Kubaláková M ，Bartos J ，Kovárová P ，Suchánková P ，Pateyron S ，Cíhalíková J ，Sourdille P ，Simková H ，Faivre-Rampant P ，Hribová E ，Bernard M ，Lukaszewski A ，Dolezel J ，Chalhoub B ... -  《PLANT JOURNAL》

Development of chromosome-specific BAC resources for genomics of bread wheat.

The large bread wheat genome (1C approximately 17 Gbp) contains a preponderance of repetitive DNA and the species is polyploid. These characteristics together serve to hamper the molecular analysis of the wheat genome. Its complexity can, however, be reduced by using flow cytometry to isolate individual chromosomes, and these can be exploited to construct chromosome-specific BAC libraries. Such libraries simplify the task of physical map construction, positional cloning and the targeted development of genetic markers. Rapid improvements in the efficiency and cost of DNA sequencing provide an opportunity to contemplate sequencing the wheat genome by preparing sequence-ready physical maps for each chromosome or chromosome arm in turn. The quality of the chromosome-specific libraries depends on their chromosome coverage and the mean insert size. First-generation libraries suffered from a relatively low mean insert size, but improvements to the protocol have generated a second wave of libraries with a significantly increased mean insert size and better chromosome coverage. Each chromosome (arm)-specific library is composed of a manageable number of clones, and so represents a practical tool in the area of wheat genomics.

Safár J ，Simková H ，Kubaláková M ，Cíhalíková J ，Suchánková P ，Bartos J ，Dolezel J ... -  《-》

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 ... -  《PLANT JOURNAL》

A physical map of the 1-gigabase bread wheat chromosome 3B.

As the staple food for 35% of the world's population, wheat is one of the most important crop species. To date, sequence-based tools to accelerate wheat improvement are lacking. As part of the international effort to sequence the 17-billion-base-pair hexaploid bread wheat genome (2n = 6x = 42 chromosomes), we constructed a bacterial artificial chromosome (BAC)-based integrated physical map of the largest chromosome, 3B, that alone is 995 megabases. A chromosome-specific BAC library was used to assemble 82% of the chromosome into 1036 contigs that were anchored with 1443 molecular markers, providing a major resource for genetic and genomic studies. This physical map establishes a template for the remaining wheat chromosomes and demonstrates the feasibility of constructing physical maps in large, complex, polyploid genomes with a chromosome-based approach.

Paux E ，Sourdille P ，Salse J ，Saintenac C ，Choulet F ，Leroy P ，Korol A ，Michalak M ，Kianian S ，Spielmeyer W ，Lagudah E ，Somers D ，Kilian A ，Alaux M ，Vautrin S ，Bergès H ，Eversole K ，Appels R ，Safar J ，Simkova H ，Dolezel J ，Bernard M ，Feuillet C ... -  《-》