Dataset Information

Integration of Solexa sequences on an ultradense genetic map in Brassica rapa L.

ABSTRACT: BACKGROUND: Sequence related amplified polymorphism (SRAP) is commonly used to construct high density genetic maps, map genes and QTL of important agronomic traits in crops and perform genetic diversity analysis without knowing sequence information. To combine next generation sequencing technology with SRAP, Illumina's Solexa sequencing was used to sequence tagged SRAP PCR products. RESULTS: Three sets of SRAP primers and three sets of tagging primers were used in 77,568 SRAP PCR reactions and the same number of tagging PCR reactions respectively to produce a pooled sample for Illumina's Solexa sequencing. After sequencing, 1.28 GB of sequence with over 13 million paired-end sequences was obtained and used to match Solexa sequences with their corresponding SRAP markers and to integrate Solexa sequences on an ultradense genetic map. The ultradense genetic bin map with 465 bins was constructed using a recombinant inbred (RI) line mapping population in B. rapa. For this ultradense genetic bin map, 9,177 SRAP markers, 1,737 integrated unique Solexa paired-end sequences and 46 SSR markers representing 10,960 independent genetic loci were assembled and 141 unique Solexa paired-end sequences were matched with their corresponding SRAP markers. The genetic map in B. rapa was aligned with the previous ultradense genetic map in B. napus through common SRAP markers in these two species. Additionally, SSR markers were used to perform alignment of the current genetic map with other five genetic maps in B. rapa and B. napus. CONCLUSION: We used SRAP to construct an ultradense genetic map with 10,960 independent genetic loci in B. rapa that is the most saturated genetic map ever constructed in this species. Using next generation sequencing, we integrated 1,878 Solexa sequences on the genetic map. These integrated sequences will be used to assemble the scaffolds in the B. rapa genome. Additionally, this genetic map may be used for gene cloning and marker development in B. rapa and B. napus.

SUBMITTER: Li W

PROVIDER: S-EPMC3125265 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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Integration of Solexa sequences on an ultradense genetic map in Brassica rapa L.

Li Wei W Zhang Jiefu J Mou Yanglong Y Geng Jianfeng J McVetty Peter B E PB Hu Shengwu S Li Genyi G

BMC genomics 20110519

<h4>Background</h4>Sequence related amplified polymorphism (SRAP) is commonly used to construct high density genetic maps, map genes and QTL of important agronomic traits in crops and perform genetic diversity analysis without knowing sequence information. To combine next generation sequencing technology with SRAP, Illumina's Solexa sequencing was used to sequence tagged SRAP PCR products.<h4>Results</h4>Three sets of SRAP primers and three sets of tagging primers were used in 77,568 SRAP PCR re ...[more]

PMID: 21595929

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Project description:BackgroundBrassica rapa is an economically important crop and a model plant for studies concerning polyploidization and the evolution of extreme morphology. The multinational B. rapa Genome Sequencing Project (BrGSP) was launched in 2003. In 2008, next generation sequencing technology was used to sequence the B. rapa genome. Several maps concerning B. rapa pseudochromosome assembly have been published but their coverage of the genome is incomplete, anchoring approximately 73.6% of the scaffolds on to chromosomes. Therefore, a new genetic map to aid pseudochromosome assembly is required.ResultsThis study concerns the construction of a reference genetic linkage map for Brassica rapa, forming the backbone for anchoring sequence scaffolds of the B. rapa genome resulting from recent sequencing efforts. One hundred and nineteen doubled haploid (DH) lines derived from microspore cultures of an F1 cross between a Chinese cabbage (B. rapa ssp. pekinensis) DH line (Z16) and a rapid cycling inbred line (L144) were used to construct the linkage map. PCR-based insertion/deletion (InDel) markers were developed by re-sequencing the two parental lines. The map comprises a total of 507 markers including 415 InDels and 92 SSRs. Alignment and orientation using SSR markers in common with existing B. rapa linkage maps allowed ten linkage groups to be identified, designated A01-A10. The total length of the linkage map was 1234.2 cM, with an average distance of 2.43 cM between adjacent marker loci. The lengths of linkage groups ranged from 71.5 cM to 188.5 cM for A08 and A09, respectively. Using the developed linkage map, 152 scaffolds were anchored on to the chromosomes, encompassing more than 82.9% of the B. rapa genome. Taken together with the previously available linkage maps, 183 scaffolds were anchored on to the chromosomes and the total coverage of the genome was 88.9%.ConclusionsThe development of this linkage map is vital for the integration of genome sequences and genetic information, and provides a useful resource for the international Brassica research community.

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.

Dataset Information

Integration of Solexa sequences on an ultradense genetic map in Brassica rapa L.

Publications

Integration of Solexa sequences on an ultradense genetic map in Brassica rapa L.

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