Using RNA-Seq for Genomic Scaffold Placement, Correcting Assemblies, and Genetic Map Creation in a Common Brassica rapa Mapping Population.
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ABSTRACT:
SUBMITTER: Markelz RJC
PROVIDER: S-EPMC5499133 | biostudies-literature | 2017 Jul
REPOSITORIES: biostudies-literature
Publications
Using RNA-Seq for Genomic Scaffold Placement, Correcting Assemblies, and Genetic Map Creation in a Common <i>Brassica rapa</i> Mapping Population.
Markelz R J Cody RJC Covington Michael F MF Brock Marcus T MT Devisetty Upendra K UK Kliebenstein Daniel J DJ Weinig Cynthia C Maloof Julin N JN
G3 (Bethesda, Md.) 20170705 7
<i>Brassica rapa</i> is a model species for agronomic, ecological, evolutionary, and translational studies. Here, we describe high-density SNP discovery and genetic map construction for a <i>B. rapa</i> recombinant inbred line (RIL) population derived from field collected RNA sequencing (RNA-Seq) data. This high-density genotype data enables the detection and correction of putative genome misassemblies and accurate assignment of scaffold sequences to their likely genomic locations. These assembl ...[more]
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Project description:BackgroundIn view of the immense value of Brassica rapa in the fields of agriculture and molecular biology, the multinational Brassica rapa Genome Sequencing Project (BrGSP) was launched in 2003 by five countries. The developing BrGSP has valuable resources for the community, including a reference genetic map and seed BAC sequences. Although the initial B. rapa linkage map served as a reference for the BrGSP, there was ambiguity in reconciling the linkage groups with the ten chromosomes of B. rapa. Consequently, the BrGSP assigned each of the linkage groups to the project members as chromosome substitutes for sequencing.ResultsWe identified simple sequence repeat (SSR) motifs in the B. rapa genome with the sequences of seed BACs used for the BrGSP. By testing 749 amplicons containing SSR motifs, we identified polymorphisms that enabled the anchoring of 188 BACs onto the B. rapa reference linkage map consisting of 719 loci in the 10 linkage groups with an average distance of 1.6 cM between adjacent loci. The anchored BAC sequences enabled the identification of 30 blocks of conserved synteny, totaling 534.9 cM in length, between the genomes of B. rapa and Arabidopsis thaliana. Most of these were consistent with previously reported duplication and rearrangement events that differentiate these genomes. However, we were able to identify the collinear regions for seven additional previously uncharacterized sections of the A genome. Integration of the linkage map with the B. rapa cytogenetic map was accomplished by FISH with probes representing 20 BAC clones, along with probes for rDNA and centromeric repeat sequences. This integration enabled unambiguous alignment and orientation of the maps representing the 10 B. rapa chromosomes.ConclusionWe developed a second generation reference linkage map for B. rapa, which was aligned unambiguously to the B. rapa cytogenetic map. Furthermore, using our data, we confirmed and extended the comparative genome analysis between B. rapa and A. thaliana. This work will serve as a basis for integrating the genetic, physical, and chromosome maps of the BrGSP, as well as for studies on polyploidization, speciation, and genome duplication in the genus Brassica.
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Project description:The purpose of this project is to investigate the molecular mechanism of CR gene Rcr1 by characterizing the proteomic regulation.
2018-10-27 | PXD004425 | Pride
Project description:Numerous regulatory genes participate in plant thermotolerance. In Arabidopsis, HEAT-INDUCED TAS1 TARGET2 (HTT2) is an important thermotolerance gene that is silenced by ta-siR255, a trans-acting siRNA. ta-siR255 is absent from heading Chinese cabbage (Brassica rapa ssp. pekinensis). Our previous attempt to overexpress the endogenous BrpHTT2 gene of heading Chinese cabbage (B. rapa ssp. pekinensis) failed because of cosuppression. In theory, heading Chinese cabbage can overexpress Arabidopsis HTT2 to improve thermotolerance in the absence of ta-siR255-mediated gene silencing and the weak potential of coexpression.To test the potential application of HTT2 in improving crop thermotolerance, we transferred p35S::HTT2 to heading Chinese cabbage. We tested the leaf electrical conductivity, hypocotyl elongation, and survival percentage of p35S::HTT2 plants subjected to high-temperature (38 °C) and heat-shock (46 °C) treatment. The leaf electrical conductivity of p35S::HTT2 seedlings under high temperature decreased but did negligibly change under heat shock. The hypocotyl length of p35S::HTT2 seedlings increased under high temperature and heat shock. The survival rate of p35S::HTT2 seedlings increased under heat shock. BrpHsfs, a subset of heat-shock factor genes, were upregulated in p35S::HTT2 plants under high-temperature and heat shock conditions. In the field, transgenic plants with HTT2 appeared greener and formed leafy heads earlier than wild-type plants.Exogenous HTT2 increased the survival rates of heat-shocked heading Chinese cabbage by promoting thermotolerance through decreasing electrical conductivity and extending hypocotyl length. Our work provides a new approach to the genetic manipulation of thermotolerance in crops through the introduction of exogenous thermotolerance genes.
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Project description:BACKGROUND:Plasmodiophora brassicae is a soil-borne plant pathogen that causes clubroot disease, which results in crop yield loss in cultivated Brassica species. Here, we investigated whether a quantitative trait locus (QTL) in B. rapa might confer resistance to a Korean P. brassicae pathotype isolate, Seosan. We crossed resistant and susceptible parental lines and analyzed the segregation pattern in a F2 population of 348 lines. We identified and mapped a novel clubroot resistance QTL using the same mapping population that included susceptible Chinese cabbage and resistant turnip lines. Forty-five resistant and 45 susceptible F2 lines along with their parental lines were used for double digest restriction site-associated DNA sequencing (ddRAD-seq). High resolution melting (HRM)-based validation of SNP positions was conducted to confirm the novel locus. RESULTS:A 3:1 ratio was observed for resistant: susceptible genotypes, which is in accordance with Mendelian segregation. ddRAD-seq identified a new locus, CRs, on chromosome A08 that was different from the clubroot resistance (CR) locus, Crr1. HRM analysis validated SNP positions and constricted CRs region. Four out of seventeen single nucleotide polymorphisms (SNPs) positions were within a 0.8-Mb region that included three NBS-LRR candidate genes but not Crr1. CONCLUSION:The newly identified CRs locus is a novel clubroot resistance locus, as the cultivar Akimeki bears the previously known Crr1 locus but remains susceptible to the Seosan isolate. These results could be exploited to develop molecular markers to detect Seosan-resistant genotypes and develop resistant Chinese cabbage cultivars.
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