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Construction and characterization of two BAC libraries representing a deep-coverage of the genome of chicory (Cichorium intybus L., Asteraceae).

ABSTRACT: 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.

SUBMITTER: Gonthier L

PROVIDER: S-EPMC2933585 | biostudies-literature | 2010

REPOSITORIES: biostudies-literature

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Construction and characterization of two BAC libraries representing a deep-coverage of the genome of chicory (Cichorium intybus L., Asteraceae).

Gonthier Lucy L Bellec Arnaud A Blassiau Christelle C Prat Elisa E Helmstetter Nicolas N Rambaud Caroline C Huss Brigitte B Hendriks Theo T Bergès Hélène H Quillet Marie-Christine MC

BMC research notes 20100811

<h4>Background</h4>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 c ...[more]

PMID: 20701751

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Project description:The Cichorium genus offers a unique opportunity to study the sporophytic self-incompatibility (SSI) system, being composed of species characterized by highly efficient self-incompatibility (e.g., C. intybus) and complete self-compatibility (e.g., C. endivia). To this end, the chicory genome was used to map seven previously identified SSI locus-associated markers. The region containing the S-locus was therefore restricted to an ~4 M bp window on chromosome 5. Among the genes predicted in this region, MDIS1 INTERACTING RECEPTOR LIKE KINASE 2 (ciMIK2) was particularly promising as a candidate for SSI. Its ortholog in Arabidopsis (atMIK2) is involved in pollen-stigma recognition reactions, and its protein structure is similar to that of S-receptor kinase (SRK), a key component of the SSI system in the Brassica genus. The amplification and sequencing of MIK2 in chicory and endive accessions revealed two contrasting scenarios. In C. endivia, MIK2 was fully conserved even when comparing different botanical varieties (i.e., smooth and curly endive). In C. intybus, 387 polymorphic positions and 3 INDELs were identified when comparing accessions of different biotypes all belonging to the same botanical variety (i.e., radicchio). The polymorphism distribution throughout the gene was uneven, with hypervariable domains preferentially localized in the LRR-rich extracellular region, putatively identified as the receptor domain. The gene was hypothesized to be under positive selection, as the nonsynonymous mutations were more than double the synonymous ones (dN/dS = 2.17). An analogous situation was observed when analyzing the first 500 bp of the MIK2 promoter: no SNPs were observed among the endive samples, whereas 44 SNPs and 6 INDELs were detected among the chicory samples. Further analyses are needed to confirm the role of MIK2 in SSI and to demonstrate whether the 23 species-specific nonsynonymous SNPs in the CDS and/or the species-specific 10 bp-INDEL found in a CCAAT box region of the promoter are responsible for the contrasting sexual behaviors of chicory and endive.

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: In our laboratory we use cultured chicory (Cichorium intybus) explants as a model to investigate cell reactivation and somatic embryogenesis and have produced 2 chicory genotypes (K59, C15) sharing a similar genetic background. K59 is a responsive genotype (embryogenic) capable of undergoing complete cell reactivation i.e. cell de- and re-differentiation leading to somatic embryogenesis (SE), whereas C15 is a non-responsive genotype (non-embryogenic) and is unable to undergo SE. Previous studies 1 showed that the use of the beta-D-glucosyl Yariv reagent (beta-GlcY) that specifically binds arabinogalactan-proteins (AGPs) blocked somatic embryo production in chicory root explants. This observation indicates that beta-GlcY is a useful tool for investigating somatic embryogenesis (SE) in chicory. In addition, a putative AGP (DT212818) encoding gene was previously found to be significantly up-regulated in the embryogenic K59 chicory genotype as compared to the non-embryogenic C15 genotype suggesting that this AGP could be involved in chicory re-differentiation 2. In order to improve our understanding of the molecular and cellular regulation underlying SE in chicory, we undertook a detailed cytological study of cell reactivation events in K59 and C15 genotypes, and used microarray profiling to compare gene expression in these 2 genotypes. In addition we also used beta-GlcY to block SE in order to identify genes potentially involved in this process. RESULTS: Microscopy confirmed that only the K59, but not the C15 genotype underwent complete cell reactivation leading to SE formation. beta-GlcY-treatment of explants blocked in vitro SE induction, but not cell reactivation, and induced cell wall modifications. Microarray analyses revealed that 78 genes were differentially expressed between induced K59 and C15 genotypes. The expression profiles of 19 genes were modified by beta-GlcY-treatment. Eight genes were both differentially expressed between K59 and C15 genotypes during SE induction and transcriptionally affected by beta-GlcY-treatment: AGP (DT212818), 26 S proteasome AAA ATPase subunit 6 (RPT6), remorin (REM), metallothionein-1 (MT1), two non-specific lipid transfer proteins genes (SDI-9 and DEA1), 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase), and snakin 2 (SN2). These results suggest that the 8 genes, including the previously-identified AGP gene (DT212818), could be involved in cell fate determination events leading to SE commitment in chicory. CONCLUSION: The use of two different chicory genotypes differing in their responsiveness to SE induction, together with beta-GlcY-treatment represented an efficient tool to discriminate cell reactivation from the SE morphogenetic pathway. Such an approach, together with microarray analyses, permitted us to identify several putative key genes related to the SE morphogenetic pathway in chicory.

Dataset Information

Construction and characterization of two BAC libraries representing a deep-coverage of the genome of chicory (Cichorium intybus L., Asteraceae).

Publications

Construction and characterization of two BAC libraries representing a deep-coverage of the genome of chicory (Cichorium intybus L., Asteraceae).

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