Dataset Information

Development of genomic resources for Citrus clementina: characterization of three deep-coverage BAC libraries and analysis of 46,000 BAC end sequences.

ABSTRACT: BACKGROUND: Citrus species constitute one of the major tree fruit crops of the subtropical regions with great economic importance. However, their peculiar reproductive characteristics, low genetic diversity and the long-term nature of tree breeding mostly impair citrus variety improvement. In woody plants, genomic science holds promise of improvements and in the Citrus genera the development of genomic tools may be crucial for further crop improvements. In this work we report the characterization of three BAC libraries from Clementine (Citrus clementina), one of the most relevant citrus fresh fruit market cultivars, and the analyses of 46.000 BAC end sequences. Clementine is a diploid plant with an estimated haploid genome size of 367 Mb and 2n = 18 chromosomes, which makes feasible the use of genomics tools to boost genetic improvement. RESULTS: Three genomic BAC libraries of Citrus clementina were constructed through EcoRI, MboI and HindIII digestions and 56,000 clones, representing an estimated genomic coverage of 19.5 haploid genome-equivalents, were picked. BAC end sequencing (BES) of 28,000 clones produced 28.1 Mb of genomic sequence that allowed the identification of the repetitive fraction (12.5% of the genome) and estimation of gene content (31,000 genes) of this species. BES analyses identified 3,800 SSRs and 6,617 putative SNPs. Comparative genomic studies showed that citrus gene homology and microsyntheny with Populus trichocarpa was rather higher than with Arabidopsis thaliana, a species phylogenetically closer to citrus. CONCLUSION: In this work, we report the characterization of three BAC libraries from C. clementina, and a new set of genomic resources that may be useful for isolation of genes underlying economically important traits, physical mapping and eventually crop improvement in Citrus species. In addition, BAC end sequencing has provided a first insight on the basic structure and organization of the citrus genome and has yielded valuable molecular markers for genetic mapping and cloning of genes of agricultural interest. Paired end sequences also may be very helpful for whole-genome sequencing programs.

SUBMITTER: Terol J

PROVIDER: S-EPMC2561056 | biostudies-literature | 2008

REPOSITORIES: biostudies-literature

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Development of genomic resources for Citrus clementina: characterization of three deep-coverage BAC libraries and analysis of 46,000 BAC end sequences.

Terol Javier J Naranjo M Angel MA Ollitrault Patrick P Talon Manuel M

BMC genomics 20080918

<h4>Background</h4>Citrus species constitute one of the major tree fruit crops of the subtropical regions with great economic importance. However, their peculiar reproductive characteristics, low genetic diversity and the long-term nature of tree breeding mostly impair citrus variety improvement. In woody plants, genomic science holds promise of improvements and in the Citrus genera the development of genomic tools may be crucial for further crop improvements. In this work we report the characte ...[more]

PMID: 18801166

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Project description:BackgroundWith the increasing availability of EST databases and whole genome sequences, SNPs have become the most abundant and powerful polymorphic markers. However, SNP chip data generally suffers from ascertainment biases caused by the SNP discovery and selection process in which a small number of individuals are used as discovery panels. The ongoing International Citrus Genome Consortium sequencing project of the highly heterozygous Clementine and sweet orange genomes will soon result in the release of several hundred thousand SNPs. The primary goals of this study were: (i) to estimate the transferability within the genus Citrus of SNPs discovered from Clementine BACend sequencing (BES), (ii) to estimate bias associated with the very narrow discovery panel, and (iii) to evaluate the usefulness of the Clementine-derived SNP markers for diversity analysis and comparative mapping studies between the different cultivated Citrus species.ResultsFifty-four accessions covering the main Citrus species and 52 interspecific hybrids between pummelo and Clementine were genotyped on a GoldenGate array platform using 1,457 SNPs mined from Clementine BES and 37 SNPs identified between and within C. maxima, C. medica, C. reticulata and C. micrantha. Consistent results were obtained from 622 SNP loci. Of these markers, 116 displayed incomplete transferability primarily in C. medica, C. maxima and wild Citrus species. The two primary biases associated with the SNP mining in Clementine were an overestimation of the C. reticulata diversity and an underestimation of the interspecific differentiation. However, the genetic stratification of the gene pool was high, with very frequent significant linkage disequilibrium. Furthermore, the shared intraspecific polymorphism and accession heterozygosity were generally enough to perform interspecific comparative genetic mapping.ConclusionsA set of 622 SNP markers providing consistent results was selected. Of the markers mined from Clementine, 80.5% were successfully transferred to the whole Citrus gene pool. Despite the ascertainment biases in relation to the Clementine origin, the SNP data confirm the important stratification of the gene pools around C. maxima, C. medica and C. reticulata as well as previous hypothesis on the origin of secondary species. The implemented SNP marker set will be very useful for comparative genetic mapping in Citrus and genetic association in C. reticulata.

Project description:BackgroundManduca sexta, Heliothis virescens, and Heliconius erato represent three widely-used insect model species for genomic and fundamental studies in Lepidoptera. Large-insert BAC libraries of these insects are critical resources for many molecular studies, including physical mapping and genome sequencing, but not available to date.ResultsWe report the construction and characterization of six large-insert BAC libraries for the three species and sampling sequence analysis of the genomes. The six BAC libraries were constructed with two restriction enzymes, two libraries for each species, and each has an average clone insert size ranging from 152-175 kb. We estimated that the genome coverage of each library ranged from 6-9 x, with the two combined libraries of each species being equivalent to 13.0-16.3 x haploid genomes. The genome coverage, quality and utility of the libraries were further confirmed by library screening using 6 approximately 8 putative single-copy probes. To provide a first glimpse into these genomes, we sequenced and analyzed the BAC ends of approximately 200 clones randomly selected from the libraries of each species. The data revealed that the genomes are AT-rich, contain relatively small fractions of repeat elements with a majority belonging to the category of low complexity repeats, and are more abundant in retro-elements than DNA transposons. Among the species, the H. erato genome is somewhat more abundant in repeat elements and simple repeats than those of M. sexta and H. virescens. The BLAST analysis of the BAC end sequences suggested that the evolution of the three genomes is widely varied, with the genome of H. virescens being the most conserved as a typical lepidopteran, whereas both genomes of H. erato and M. sexta appear to have evolved significantly, resulting in a higher level of species- or evolutionary lineage-specific sequences.ConclusionThe high-quality and large-insert BAC libraries of the insects, together with the identified BACs containing genes of interest, provide valuable information, resources and tools for comprehensive understanding and studies of the insect genomes and for addressing many fundamental questions in Lepidoptera. The sample of the genomic sequences provides the first insight into the constitution and evolution of the insect genomes.

Project description:BACKGROUND: Eucalyptus species are among the most planted hardwoods in the world because of their rapid growth, adaptability and valuable wood properties. The development and integration of genomic resources into breeding practice will be increasingly important in the decades to come. Bacterial artificial chromosome (BAC) libraries are key genomic tools that enable positional cloning of important traits, synteny evaluation, and the development of genome framework physical maps for genetic linkage and genome sequencing. RESULTS: We describe the construction and characterization of two deep-coverage BAC libraries EG_Ba and EG_Bb obtained from nuclear DNA fragments of E. grandis (clone BRASUZ1) digested with HindIII and BstYI, respectively. Genome coverages of 17 and 15 haploid genome equivalents were estimated for EG_Ba and EG_Bb, respectively. Both libraries contained large inserts, with average sizes ranging from 135 Kb (Eg_Bb) to 157 Kb (Eg_Ba), very low extra-nuclear genome contamination providing a probability of finding a single copy gene ? 99.99%. Libraries were screened for the presence of several genes of interest via hybridizations to high-density BAC filters followed by PCR validation. Five selected BAC clones were sequenced and assembled using the Roche GS FLX technology providing the whole sequence of the E. grandis chloroplast genome, and complete genomic sequences of important lignin biosynthesis genes. CONCLUSIONS: The two E. grandis BAC libraries described in this study represent an important milestone for the advancement of Eucalyptus genomics and forest tree research. These BAC resources have a highly redundant genome coverage (> 15×), contain large average inserts and have a very low percentage of clones with organellar DNA or empty vectors. These publicly available BAC libraries are thus suitable for a broad range of applications in genetic and genomic research in Eucalyptus and possibly in related species of Myrtaceae, including genome sequencing, gene isolation, functional and comparative genomics. Because they have been constructed using the same tree (E. grandis BRASUZ1) whose full genome is being sequenced, they should prove instrumental for assembly and gap filling of the upcoming Eucalyptus reference genome sequence.

Project description:BACKGROUND: The Asteraceae represents an important plant family with respect to the numbers of species present in the wild and used by man. Nonetheless, genomic resources for Asteraceae species are relatively underdeveloped, hampering within species genetic studies as well as comparative genomics studies at the family level. So far, six BAC libraries have been described for the main crops of the family, i.e. lettuce and sunflower. Here we present the characterization of BAC libraries of chicory (Cichorium intybus L.) constructed from two genotypes differing in traits related to sexual and vegetative reproduction. Resolving the molecular mechanisms underlying traits controlling the reproductive system of chicory is a key determinant for hybrid development, and more generally will provide new insights into these traits, which are poorly investigated so far at the molecular level in Asteraceae. FINDINGS: Two bacterial artificial chromosome (BAC) libraries, CinS2S2 and CinS1S4, were constructed from HindIII-digested high molecular weight DNA of the contrasting genotypes C15 and C30.01, respectively. C15 was hermaphrodite, non-embryogenic, and S2S2 for the S-locus implicated in self-incompatibility, whereas C30.01 was male sterile, embryogenic, and S1S4. The CinS2S2 and CinS1S4 libraries contain 89,088 and 81,408 clones. Mean insert sizes of the CinS2S2 and CinS1S4 clones are 90 and 120 kb, respectively, and provide together a coverage of 12.3 haploid genome equivalents. Contamination with mitochondrial and chloroplast DNA sequences was evaluated with four mitochondrial and four chloroplast specific probes, and was estimated to be 0.024% and 1.00% for the CinS2S2 library, and 0.028% and 2.35% for the CinS1S4 library. Using two single copy genes putatively implicated in somatic embryogenesis, screening of both libraries resulted in detection of 12 and 13 positive clones for each gene, in accordance with expected numbers. CONCLUSIONS: This indicated that both BAC libraries are valuable tools for molecular studies in chicory, one goal being the positional cloning of the S-locus in this Asteraceae species.

Dataset Information

Development of genomic resources for Citrus clementina: characterization of three deep-coverage BAC libraries and analysis of 46,000 BAC end sequences.

Publications

Development of genomic resources for Citrus clementina: characterization of three deep-coverage BAC libraries and analysis of 46,000 BAC end sequences.

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