Construction of methylation-sensitive partial restriction bacterial artificial chromosome libraries in maize

Potato (Solanum tuberosum L.) is an important crop worldwide that is consumed fresh and processed in many different ways. The tuber shape is a critical trait in potato breeding, because it directly affects processing quality. Previous studies have demonstrated that the Ro locus on chromosome X is the major locus controlling tuber shape, and the round shape allele, Ro, is dominant over the long shape allele, ro. However, no reports on the cloning or physical mapping of this locus have been published. In this study, whole genome sequencing and bacterial artificial chromosome (BAC) library construction were applied to develop molecular markers and construct a physical map of Ro. Eight round-tuber genotypes were used to construct the BAC library, which contains about 40,000 clones with an average insert size of 85 kb and represents approximately four haploid potato genomes. A total of 66 individuals with extreme round or long tubers were selected to construct a round-tuber shape DNA bulk and a long-tuber shape DNA bulk for WGS. Single-nucleotide polymorphisms (SNPs) identified from WGS data were used to develop nine polymorphic markers linked to Ro. These markers were used to screen the BAC library, and eight positive BAC clones were ultimately identified and then analyzed by restriction enzyme digestion and BAC end sequencing and used to develop markers and identify new positive BAC clones. Finally, a primary physical map of Ro was constructed using a chromosome walking strategy. This map provides an important reference for cloning the potato tuber shape gene and studying tuber shape variation.

The first bacterial artificial chromosome (BAC) library of Robusta coffee (Coffea canephora) was constructed, with the aim of developing molecular resources to study the genome structure and evolution of this perennial crop. Clone 126, which is highly productive and confers good technological and organoleptic qualities of beverage, was chosen for development of this library. The BAC library contains 55,296 clones, with an average insert size of 135 Kb per plasmid, therefore representing theoretically nine haploid genome equivalents of C. canephora. Its validation was achieved with a set of 13 genetically anchored single-copy and 4 duplicated RFLP probes and yielded on average 9 BAC clones per probe. Screening of this BAC library was also carried out with partial cDNA probes coding for enzymes of sugar metabolism like invertases and sucrose synthase, with the aim of characterizing the organization and promoter structure of this important class of genes. It was shown that genes for both cell wall and vacuolar forms of invertases were probably unique in the Robusta genome whereas sucrose synthase was encoded by at least two genes. One of them (CcSUS1) was cloned and sequenced, showing that our BAC library is a valuable tool to rapidly identify genes of agronomic interest or linked to cup quality in C. canephora.

BackgroundThe lycophytes are an ancient lineage of vascular plants that diverged from the seed plant lineage about 400 Myr ago. Although the lycophytes occupy an important phylogenetic position for understanding the evolution of plants and their genomes, no genomic resources exist for this group of plants.ResultsHere we describe the construction of a large-insert bacterial artificial chromosome (BAC) library from the lycophyte Selaginella moellendorffii. Based on cell flow cytometry, this species has the smallest genome size among the different lycophytes tested, including Huperzia lucidula, Diphaiastrum digita, Isoetes engelmanii and S. kraussiana. The arrayed BAC library consists of 9126 clones; the average insert size is estimated to be 122 kb. Inserts of chloroplast origin account for 2.3% of the clones. The BAC library contains an estimated ten genome-equivalents based on DNA hybridizations using five single-copy and two duplicated S. moellendorffii genes as probes.ConclusionThe S. moellenforffii BAC library, the first to be constructed from a lycophyte, will be useful to the scientific community as a resource for comparative plant genomics and evolution.

Chinese pangolins as a representative species in the order Pholidota have highly specified morphological characters and occupy an important place in the mammalian phylogenetic tree. To obtain genomic data for this species, we have constructed a bacterial artificial chromosome (BAC) library of Chinese pangolin. The library contains 208,272 clones with an average insert size of 122.1 kb and represents approximately eight times the Chinese pangolin haploid genome (if we assume that the Chinese pangolins have a genome size similar to human). One hundred and twenty randomly-selected BAC clones were mapped onto Chinese pangolin chromosomes by fluorescence in situ hybridization (FISH), showing a largely unbiased chromosomal distribution. Several clones containing repetitive DNA and ribosomal DNA genes were also found. The BAC library and FISH mapped BAC clones are useful resources for comparative genomics and cytogenetics of mammals and in particular, the ongoing genome sequencing project of Chinese pangolins.

A bacterial artificial chromosome (BAC) library consisting of 235,000 clones with an average insert size of 130 kb was constructed from Capsicum annuum, 'CM334'. Based on a pepper haploid genome size of 2,702 Mbp/C, the BAC library is estimated to contain approximately 12 genome equivalents and represents at least 99% of the pepper genome. Screening of the library with mitochondrial DNA probes (coxII, coxIII, atp6 and atp9) and chloroplast DNA probes (atpB, rbcL) indicated that contamination with cytoplasmic DNA was less than 0.5%. To estimate the possibility of isolating a specific clone, the library was screened with single or low-copy gene-specific probes and RFLP probes. Screening of high density BAC filters with RFLP markers linked to L (TMV resistance), y (fruit color), C2 (fruit color) and C (pungency) loci under high stringency conditions revealed that at least three positive BAC clones were found per each probe. This fact indicates that the library is highly reliable and represents a resource for map-based cloning, physical mapping, and characterization of upstream and downstream regulations of the chili pepper genes.

Papaya is a major fruit crop in the tropics and has recently evolved sex chromosomes. Towards sequencing the papaya sex chromosomes, two bacterial artificial chromosome (BAC) libraries were constructed from papaya male and female genomic DNA. The female BAC library was constructed using restriction enzyme BstY I and consists of 36,864 clones with an average insert size of 104 kb, providing 10.3x genome equivalents. The male BAC library was constructed using restriction enzyme EcoR I and consists of 55,296 clones with an average insert size of 101 kb, providing 15.0x genome equivalents. The male BAC library was used in constructing the physical map of the male-specific region of the male Y chromosome (MSY) and in filling gaps and extending the physical map of the hermaphrodite-specific region of the Yh chromosome (HSY) and the X chromosome physical map. The female BAC library was used to extend the X physical map gap. The MSY, HSY, and X physical maps offer a unique opportunity to study chromosomal rearrangements, Y chromosome degeneration, and dosage compensation of the papaya nascent sex chromosomes.

In order to promote genome research on coffee trees, one of the most important tropical crops, a bacterial artificial chromosome (BAC) library of the coffee allotetraploid species, Coffea arabica, was constructed. The variety IAPAR 59, which is widely distributed in Latin America and exhibits a fair level of resistance to several pathogens, was chosen. High-efficiency BAC cloning of the high molecular weight genomic DNA partially digested by HindIII was achieved. In total, the library contains 88,813 clones with an average insert size of 130 kb, and represents approximately eight C. arabica dihaploid genome equivalents. One original feature of this library is that it can be divided into four sublibraries with mean insert sizes of 96, 130, 183 and 210 kb. Characterisation of the library showed that less than 4.5% of the clones contained organelle DNA. Furthermore, this library is representative and shows good genome coverage, as established by hybridisation screening of high-density filters using a number of nuclear probes distributed across the allotetraploid genome. This Arabica BAC library, the first large-insert DNA library so far constructed for the genus Coffea, is well-suited for many applications in genome research, including physical mapping, map-based cloning, functional and comparative genomics as well as polyploid genome analyses.

Cloned DNA libraries in bacterial artificial chromosome (BAC) are the most widely used form of large-insert DNA libraries. BAC libraries are typically represented by ordered clones derived from genomic DNA of a particular organism. In the case of large eukaryotic genomes, whole-genome libraries consist of a hundred thousand to a million clones, which make their handling and screening a daunting task. The labor and cost of working with whole-genome libraries can be greatly reduced by constructing a library derived from a smaller part of the genome. Here we describe construction of BAC libraries from mitotic chromosomes purified by flow cytometric sorting. Chromosome-specific BAC libraries facilitate positional gene cloning, physical mapping, and sequencing in complex plant genomes.

Utilizing improved methods, two bacterial artificial chromosome (BAC) libraries were constructed for the multidisease-resistant line of melon MR-1. The HindIII library consists of 177 microtiter plates in a 384-well format, while the EcoRI library consists of 222 microtiter plates. Approximately 95.6% of the HindIII library clones contain nuclear DNA inserts with an average size of 118 kb, providing a coverage of 15.4 genome equivalents. Similarly, 96% of the EcoRI library clones contain nuclear DNA inserts with an average size of 114 kb, providing a coverage of 18.7 genome equivalents. Both libraries were evaluated for contamination with high-copy vector, empty pIndigoBac536 vector, and organellar DNA sequences. High-density filters were screened with two genetic markers FM and AM that co-segregate with Fom-2, a gene conferring resistance to races 0 and 1 of Fusarium wilt. Fourteen and 18 candidate BAC clones were identified for the FM and AM probes, respectively, from the HindIII library, while 34 were identified for the AM probe from filters A, B, and C of the EcoRI library.Key words: bacterial artificial chromosome (BAC) library, Fusarium wilt, melon, pCUGIBAC1, resistant gene.

A bacterial artificial chromosome (BAC) library was constructed from DNA of a commercially grown winter wheat cultivar (Triticum aestivum L. cv. Norstar) that is highly tolerant to low temperatures (LT50 = −24°C). The library was produced from nuclear leaf DNA that was partially digested with HindIII and inserted into pCC1BAC vector. In excess of 1.2 × 106 clones propagated in E. coli were obtained, archived in 384‐well microtitre plates and stored at −80°C. More than 97.1% of the recombinant plasmids contained inserts and were free from organelle DNA contamination. Analysis of 119 randomly isolated clones revealed insert sizes ranging from 17 to 262 kb with 26% of the inserts exceeding 100 kb. The average insert size was 75 kb and the genome coverage of the library was theoretically 5.5 times the haploid genome equivalent. This corresponds to a 99.6% probability of recovering any specific Norstar DNA sequence from the library. Screening of the library with nine simple sequence repeat (SSR) locus‐specific markers supported a >4.4‐fold genome coverage. The BAC library was gridded onto high‐density filters and will be used for isolation of genetic loci associated with cold‐tolerance and grain quality traits.

We constructed a bacterial artificial chromosome (BAC) library for the indica rice variety Kasalath using a new BAC vector, pCHR8, suited for plasmid rescue, containing nine unique restriction sites in a multicloning site. This Kasalath BAC Iibrary consists of about 65, 000 clones with an average insert size of 106 kb using HindIII partially digested DNA fragments. It represents approximately sixteen rice haploid genome equivalents which are sufficient for the full coverage of the rice genome with BAC clone contigs. The library was constructed from only a single ligation mixture with high transformation efficiency. The quality of this BAC Iibrary was ascertained by successfully retrieving the BAC clones for seven rice RFLP markers. We could isolate eleven individual BAC clones using RFLP markers that linked to the lethal factor locus for segregation distortion on rice chromosome 3. The content of the chloroplast and mitochondrial DNA in this library was estimated to be 2.8% and 0.4%, respectively. These results demonstrate that this Kasalath BAC Iibrary should be suitable for map-based cloning and physical mapping of the rice genome.

Maize inbred line 77Ht2 contains agriculturally important genes and has been widely used in corn breeding in China. A bacterial artificial chromosome (BAC) library of 77Ht2 has been constructed in order to identify useful genes and to facilitate the study of the maize genome. The library contains 175104 clones with an average insert size of 57 kb and represents about 4 maize haploid genome equivalents. Characterization of the library showed less than 0.5% of clones to not contain large inserts. Significant contamination of chloroplast and mitochondria DNA was not detected. BAC clones (152 arrays) were stored in 96 microtiter plates, with each well containing 12 clones. This is the first maize BAC library constructed in China. It is well suited for map-based cloning of maize genes and genome physical mapping.

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.

A bacterial artificial chromosome (BAC) library of common carp Cyprinus carpio L. was constructed as a part of ongoing common carp genome project, which is aiming assembly of common carp genome. The library, containing a total of 92,160 BAC clones with an average insert size of 141 kb, was constructed into the restriction site of Hind III on BAC vector CopyControl pCC1BAC, covering 7.7 X haploid genome equivalents. Three dimension pools and superpools of the BAC library were established and 23 positive clones of 14 targets were identified from one-fifth of the BAC library. Pilot project of BAC end sequencing was conducted on 2,688 BAC ends from 1,344 clones and harvested 2,522 high-quality Q20 sequences with average length of 677 bp. The sequencing success rate was 93.8% and pair-end success rate was 92.3%. A total of 212 microsyntenies had been established between common carp and zebrafish genomes as a trial for genome-wide comparative genomics in these two closely related species.

A porcine bacterial artificial chromosome (BAC) library consisting of 103,488 clones has been constructed. The average insert size in the BAC vector was calculated to be 133 kb based on the examination of 189 randomly selected clones, indicating that the library contained 4.4 genome equivalents. The library can be screened by two-step PCR. The first screening step is performed on 22 superpools, each containing 4704 clones (49 x 96 well plates). In the second screening step, 49 plates comprising a superpool are arrayed in a 7 x 7 matrix and 4D-PCR is performed. Screening of the library superpools by PCR for 125 marker sequences selected from different regions of swine genome revealed 123 sequences, indicating that the library is not biased. Subsequent screenings (4D-PCR) were successfully applied for identification of clones containing each marker sequence. This porcine BAC library and the PCR screening system are useful for isolation of genomic DNA fragments containing desired sequences.