Construction of a representative genomic library from a hexaploid wheat

The large genome size and the great amount of DNA repeats make it rather diffiult to obtain a representative hexaploid wheat genomic library. The protocol is given with modifications to phage isolation and to purification of vector and plant DNAs by electrophoresis in low-concentration SeaKem agarose gels. Representative genomic libraries of the soft wheat variety carrying a translocated rye chromosome are 1RS were constructed in arecD-minusE. coli strain to reduce recombinational loss. Erratum: This is a corrected version of an article that appeared in thePlant Molecular Biology Reporter 8(2):85–91. The online version of the original article can be found at     

Development of a D genome specific marker resource for diploid and hexaploid wheat

Background

Mapping and map-based cloning of genes that control agriculturally and economically important traits remain great challenges for plants with complex highly repetitive genomes such as those within the grass tribe, Triticeae. Mapping limitations in the Triticeae are primarily due to low frequencies of polymorphic gene markers and poor genetic recombination in certain genetic regions. Although the abundance of repetitive sequence may pose common problems in genome analysis and sequence assembly of large and complex genomes, they provide repeat junction markers with random and unbiased distribution throughout chromosomes. Hence, development of a high-throughput mapping technology that combine both gene-based and repeat junction-based markers is needed to generate maps that have better coverage of the entire genome.

Results

In this study, the available genomics resource of the diploid Aegilop tauschii, the D genome donor of bread wheat, were used to develop genome specific markers that can be applied for mapping in modern hexaploid wheat. A NimbleGen array containing both gene-based and repeat junction probe sequences derived from Ae. tauschii was developed and used to map the Chinese Spring nullisomic-tetrasomic lines and deletion bin lines of the D genome chromosomes. Based on these mapping data, we have now anchored 5,171 repeat junction probes and 10,892 gene probes, corresponding to 5,070 gene markers, to the delineated deletion bins of the D genome. The order of the gene-based markers within the deletion bins of the Chinese Spring can be inferred based on their positions on the Ae. tauschii genetic map. Analysis of the probe sequences against the Chinese Spring chromosome sequence assembly database facilitated mapping of the NimbleGen probes to the sequence contigs and allowed assignment or ordering of these sequence contigs within the deletion bins. The accumulated length of anchored sequence contigs is about 155 Mb, representing ~ 3.2 % of the D genome. A specific database was developed to allow user to search or BLAST against the probe sequence information and to directly download PCR primers for mapping specific genetic loci.

Conclusions

In bread wheat, aneuploid stocks have been extensively used to assign markers linked with genes/traits to chromosomes, chromosome arms, and their specific bins. Through this study, we added thousands of markers to the existing wheat chromosome bin map, representing a significant step forward in providing a resource to navigate the wheat genome. The database website (http://probes.pw.usda.gov/ATRJM/) provides easy access and efficient utilization of the data. The resources developed herein can aid map-based cloning of traits of interest and the sequencing of the D genome of hexaploid wheat.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1852-2) contains supplementary material, which is available to authorized users.Keyword: Wheat deletion bins, Molecular markers, Repeat junction markers, NimbleGen array, Recombination, Genetic map     

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.     

Dosage effects of chromosomes of homoeologous groups 1 and 6 upon bread-making quality in hexaploid wheat

Summary The endosperm storage proteins, glutenin and gliadin, are major determinants of bread-making quality in hexaploid wheat. Genes encoding them are located on chromosomes of homoeologous groups 1 and 6. Aneuploid lines of these groups in spring wheat cultivar Chinese Spring have been used to investigate the effect of varying the dosage of chromosomes and chromosome arms upon bread-making quality, where quality has been assessed using the SDS-sedimentation test. Differences between the group 1 chromosomes for quality were greater than those between the group 6 chromosomes. The chromosomes were ranked within homoeologous groups for their effect on quality as follows (>=better quality): 1D>1B>1A and 6A>6B=6D. The relationship of chromosome dosage with quality was principally linear for four of the chromosomes, but not for 6B and 6D. Increases in the dosage of 1B, 6A and, especially, 1D, were associated with significant improvements in quality, whereas increases in the dosage of 1A were associated with reductions in quality. The effects of 1A and 1D were such that the best genotype for quality was nullisomic 1A-tetrasomic 1D. For group 1, effects of the long arm appeared in general to be more important than effects of the short arm. For group 6, effects were found associated with the long arms as well as with the short arms, a surprising result in view of the absence of genes encoding storage proteins on the long arms. Significant interactions were found between chromosomes and genetic backgrounds, and between individual chromosomes. Analysis of trials grown over two years demonstrated that, although additive environmental differences over years and genotype x years interaction were present, they were relatively small in magnitude compared with purely genetic differences.     

Construction of a chromosome-enriched HpaII library from flow-sorted wheat chromosomes.

We report here the first successful generation of a chromosome-enriched library from flow sorted plant chromosomes. Chromosomes with a characteristic DNA content (a peak) were sorted from a synchronized cell culture (TaKB1, derived from Triticum aestivum). A HpaII library was constructed from the sorted chromosomes and half of the cloned DNA sequences analysed are unique or low copy. Approximately half of these sequences when used as probes detect sequences on wheat chromosome 4A. The generation and analysis of the chromosome library is described in detail and the prospects of using flow-sorted plant chromosomes discussed.     

Non-gridded library: a new approach for BAC (bacterial artificial chromosome) exploitation in hexaploid wheat (Triticum aestivum)

The feasibility of exploiting non-gridded bacterial artificial chromosome (BAC) libraries and some major factors affecting the efficiency of handling such libraries were studied in hexaploid wheat. Even for a bacterial culture containing only 55% recombinants, some 2000 BAC clones with inserts ranging from 45 to 245 kb could be pooled. The pooled BAC clones could be amplified by culturing for up to 6 h without losing any target clones. These results imply that even for hexaploid wheat, which has an extremely large genome, some 250 pools are sufficient for a BAC library that should satisfy many research objectives. This non-gridded strategy would dramatically reduce the cost and make robotic equipment non-essential in exploiting BAC technology. To construct a representative library and to minimise clone competition, thawing and re-freezing ligation mixtures and bacterial cultures should be avoided in BAC library construction and application.     

Construction of a representative genomic library from a hexaploid

The large genome size and the great amount of DNA repeats make it rather difficult to obtain a representative hexaploid wheat genomic library. The protocol is given with modifications to phage isolation and to purification of vector and plant DNAs by electrogenomic libraries of the soft wheat variety carrying a translocated rye chromosome arm 1RS were constructed in arecD E. coli strain to reduce recombinational loss.     

Construction and characterization of a peanut HindIII BAC library

Bacterial artificial chromosome (BAC) libraries have been an essential tool for physical analyses of genomes of many crops. We constructed and characterized the first large-insert DNA library for Arachis hypogaea L. The HindIII BAC library contains 182,784 clones; only 5,484 (3%) had no inserts; and the average insert size is 104.05 kb. Chloroplast DNA contamination was very low, only nine clones, and r-DNA content was 1,208, 0.66% of clones. The depth of coverage is estimated to be 6.5 genome-equivalents, allowing the isolation of virtually any single-copy locus. This rate of coverage was confirmed with the application of 20 overgos, which identified 305 positive clones from the library. The identification of multiple loci by most probes in polyploids complicates anchoring of physical and genetic maps. We explored the practicality of a hybridization-based approach for determination of map locations of BAC clones in peanut by analyzing 94 clones detected by seven different overgos. The banding patterns on Southern blots were good predictors of contig composition; that is, the clones that shared the same size bands and ascribed to the same overgos usually also located in the same contigs. This BAC library has great potential to advance future research about the peanut genome.Requests for the BAC library (or subsets) should be directed to Dr. A. Paterson (paterson@uga.edu).     

Construction and characterization of a gridded cattle BAC library

A bovine genomic large-insert bacterial artificial chromosome (BAC) library has been constructed from leukocytes of a Holstein-Friesian male. Size fractionated DpnII-digested genomic DNA was ligated to the dephosphorylated BamH1 ends of a pBACe3.6 vector. Approximately 8.3 x 10(4) individual BAC clones were picked into 384-well plates. Two-hundred and sixty-seven randomly chosen clones were characterized by pulsed-field gel electrophoresis (PFGE). The average insert size was 104 kb with a frequency of clones without inserts of 5.5%. Thirty-four BAC clones were mapped by fluorescence in situ hybridization (FISH) to cattle chromosomes. Three showed signals at more than one location, one of them on the centromeric regions of all autosomes, indicating that the clone contains centromeric repeats. A subset of these BAC clones was used for the development of sequence tagged sites. Both subcloning and direct sequencing of the BACs were used for generating sequence tagged site information. The clones from the library were gridded onto high-density membranes, and PCR superpools were produced from the same set of clones. Membranes and superpools are available through the Resource Centre of the German Human Genome Project in Berlin (http:// www.rzpd.de).     

Construction of a BAC library of pearl millet, Pennisetum glaucum

A bacterial artificial chromosome (BAC) library was constructed using nuclear DNA from pearl millet (Pennisetum glaucum), and used as a resource for the isolation of microsatellite sequences. The library contains a total of 159,100 clones with an average insert size of 90 kb, and corresponds to 5.8 haploid genome equivalents. The BAC library was pooled for screening by the polymerase chain reaction (PCR) as well as robotically gridded on high-density filters. PCR-based screening of a subset of the library (4.7 haploid genome equivalents) using five sequence-tagged site (STS) and six microsatellite markers identified between 2 and 11 positives superpools (5.4 on average). The frequency of BAC clones carrying inserts of chloroplast DNA was estimated to be less than 1% by hybridisation with a rice chloroplast probe. Received: 30 January 2000 / Accepted: 16 October 2000     

Differentiation between wheat chromosomes 4B and 4D.

A linkage map based on homoeologous recombination, induced by the absence of the Ph1 locus, between chromosome 4D of Triticum aestivum L. (genomes AABBDD) and chromosome 4B of T. turgidum L. (genomes AABB) was compared with a linkage map of chromosome 4Am of T. monococcum L. and a consensus map of chromosomes 4B and 4D of T. aestivum based on homologous recombination. The 4D/4B homoeologous map was only one-third the length of the homologous maps and all intervals were reduced relative to the 4B-4D consensus map. After the homoeologous map was corrected for this overall reduction in recombination, the distribution of recombination in the short arm was similar in both types of maps. In the long arm, homoeologous recombination declined disproportionally in the distal to proximal direction. This gradient was shown to be largely caused by severe segregation distortion reflecting selection against 4D genetic material. The segregation distortion had a maximum that coincided with the centromere and likely had a polygenic cause. Chromosomes 4D and 4B were colinear and recombination between them occurred in almost all intervals where homologous recombination occurred. These findings suggest that these chromosomes are not differentiated structurally and that the differentiation is not segmental. In the presence of Ph1, metaphase I chromosome pairing between chromosomes composed of homologous and differentiated regions correlated with the lengths of the homologous regions. No compensatory allocation of crossovers into the homologous regions was detected. In this respect, the present results are in dramatic contrast with the crossover allocation into the pseudoautosomal region in the mammalian male meiosis.     

Construction and characterization of a bacterial artificial chromosome (BAC) library for the A genome of wheat.

A genomic bacterial artificial chromosome (BAC) library of the A genome of wheat has been constructed. Triticum monococcum accession DV92 was selected for this purpose because it is a cultivated diploid wheat and one of the parental lines used in the construction of a saturated genetic map. Leaves from this accession were used to isolate high-molecular-weight DNA from nuclei. This DNA was partially digested with restriction enzyme Hind III, subjected to double size selection, electroeluted and cloned into the pINDIGO451 BAC vector. The library consists of 276,480 clones with an average insert size of 115 kb. Excluding the 1.33% of empty clones and 0.14% of clones with chloroplast DNA, the coverage of this library is 5.6 genome equivalents. With this genome coverage the probability of having any DNA sequence represented in this library is higher than 99.6%. Clones were sorted in 720,384-well plates and blotted onto 15 high-density filters. High-density filters were screened with several single or low-copy clones and five positive BAC clones were selected for further analysis. Since most of the T. monococcum BAC ends included repetitive sequences, a modification was introduced into the classical end-isolation procedure to select low copy sequences for chromosome walking.     

Construction and characterization of a bacterial artificial chromosome (BAC) library of hexaploid wheat (Triticum aestivum L.) and validation of genome coverage using locus-specific primers.

A BAC library of hexaploid wheat was constructed using the spring wheat cultivar Triticum aestivum L. 'Glenlea'. Fresh shoot tissue from 7- to 10-day-old seedlings was used to obtain HMW DNA. The library was constructed using the HindIII site of pIndigoBAC-5 and the BamHI site of pIndigoBAC-5 and pECBAC1. A total of 12 ligations were used to construct the entire library, which contains over 650 000 clones. Ninety-six percent of the clones had inserts. The insert size ranged from 5 to 189 kb with an average of 79 kb. The entire library was gridded onto 24 high-density filters using a 5 x 5 array. A subset of these membranes was hybridized with two intergenic chloroplast probes and the percentage of clones containing chloroplast DNA (cpDNA) was calculated to be 2.2%. The genome coverage was estimated to be 3.1 x haploid genome equivalents, giving a 95.3% probability of identifying a clone corresponding to any wheat DNA sequence. BAC pools were constructed and screened using markers targeting the Glu-B1 locus (1BL), the hardness loci (5AS, 5BS, 5DS), the leaf rust resistance locus Lr1 (5DL), and the major fusarium head blight QTL locus located on 3BS. These markers were either locus-specific amplicons or microsatellites. A total of 49 BAC clones were identified for 14 markers giving an average of 3.5 clones/marker, thereby corroborating the estimated 3.1x genome coverage. An example using the gene encoding the HMW glutenin Bx7 is illustrated.     

Construction of a BAC library for Haplochromis chilotes,a cichlid fish from Lake Victoria

Cichlid fishes in Lake Victoria are model organisms for studying rapid radiation and speciation. On the way to examine the molecular basis of how these cichlid fishes achieved such a remarkable morphological diversification, we constructed a bacterial artificial chromosome (BAC) library derived from a cichlid species, Haplochromis chilotes, from Lake Victoria. The library includes 157,056 clones with the average insert size of 128 kb, corresponding to a 10-fold coverage of the H. chilotes genome. Given that the cichlid fishes endemic to Lake Victoria are closely related to one another phylogenetically and their genomes are nearly identical, this BAC library can be utilized to isolate genes from the more than 200 Haplochromine cichlid species in Lake Victoria.     

Locus-specific primers for LMW glutenin genes on each of the group 1 chromosomes of hexaploid wheat

To reveal the chromosomal location of three known low-molecular-weight (LMW) glutenin genes in wheat, we designed and used three sets of sequence-specific primers in polymerase chain reactions (PCR) on Chinese Spring and its derived group 1 aneuploid nullisomic-tetrasomic stocks. Two sets proved to be chromosome specific and amplified sequences from the Glu-A3 and Glu-D3 loci, respectively. The third set was apparently composed of conserved sequences as it produced PCR products in each of the aneuploids. Two of these products were cloned, and their sequences differed from the known LMW glutenin genes at several positions. Again, primer sets specific for these sequences were designed. One set was directed to the Glu-A3 locus, the second set resulted in two PCR products differing in length, one of which was located on chromosome 1B and the other on 1D. Primer sets constructed for the latter two sequences were specific for the Glu-B3 and Glu-D3 loci, respectively. Hence, primer sets specific for each of the three homoeologous chromosomes of the group 1 (1A, 1B, 1D) are available. In addition, these locus-specific primers were assayed for their ability to distinguish among wheat cultivars. PCR products amplified with one of the Glu-A3-specific primer sets showed length polymorphisms in various wheat varieties. Varieties carrying the 1RS.1BL translocated chromosomes could be recognized by the absence of a PCR product when the Glu-B3 primer set was used. These results suggest that PCR with locus-specific primers can be useful in the molecular genetic analysis of hexaploid wheat.     

Construction of BAC library for the amphibian Xenopus tropicalis

Xenopus tropicalis has become an alternative model to the amphibian Xenopus laevis because it is better suited for genetic and genomic studies. We have constructed a genomic BAC library consisting of over 100,000 clones from sperm of Xenopus tropicalis. Analysis by pulsed field gel electrophoresis of representative BAC clones indicated the average size of insert DNA to be 100 kb, and we estimated the library covers 6 times the Xenopus tropicalis genome of 1.7 x 10(9) base pairs. To evaluate the BAC library, we attempted to isolate BAC clones which contain a protocadherin gamma (Pcdh gamma) gene and found that the isolated BAC clones are assembled as two separate contigs. This result suggests the presence of at least two clusters for the Pcdh gamma gene in the genome of X. tropicalis.     

Construction and characterization of a Lipotes vexillifer genomic DNA BAC library

We constructed a genomic DNA library for Lipotes vexillifer (L. vexillifer), the Baiji or Yangtze River dolphin, one of the most endangered mammals in the world. The library consists of 149,000 BAC clones, with an average insert size of 83 kb, representing approximately 3.4 haploid genome equivalents. PCR amplification of four known L. vexillifer genes yielded two to four positive clones each. To demonstrate the utility of this library, we isolated and sequenced the L. vexillifer alpha lactalbumin gene, which is a gene specific to mammals and one which has been widely used as molecular tool in phylogenetic analysis. We also end-sequenced 20 randomly selected clones, resulting in the identification of at least five new L. vexillifer genes, five SSR loci, and one SINE locus. These results suggest that this library is a valuable resource for candidate gene cloning, physical mapping, and genome sequencing of this important and threatened species.     

白氏文昌鱼单个体基因组BAC文库的构建

隶属于头索动物亚门的文昌鱼是现存生物中最近似于脊椎动物亚门直接祖先的一个类群, 具有重要的进化地位, 是研究脊椎动物原始祖先的重要材料和模式动物。随着文昌鱼实验室连续繁殖的成功, 全基因组测序成为中国文昌鱼模式化急需完成的工作之一。文章从单条雄性白氏文昌鱼精巢组织中提取高质量的基因组DNA, 经EcoRⅠ限制性内切酶和EcoRⅠ甲基化酶酶切, 脉冲场电泳选择合适酶切DNA片段, 连接线性磷酸化的载体pCC1BAC, 转化大肠杆菌EPI300 E. coli, 构建了含有44 706个克隆的全基因组BAC( Bacterial artificial chromosome)文库, 该文库平均插入片段80 kb,具有9倍的基因组覆盖率, 基本能够满足功能基因等研究需要, 为中国文昌鱼全基因组测序打下基础。