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      Rapid Identification of Candidate Genes for Seed Weight Using the SLAF-Seq Method in Brassica napus

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          Abstract

          Seed weight is a critical and direct trait for oilseed crop seed yield. Understanding its genetic mechanism is of great importance for yield improvement in Brassica napus breeding. Two hundred and fifty doubled haploid lines derived by microspore culture were developed from a cross between a large-seed line G-42 and a small-seed line 7–9. According to the 1000-seed weight (TSW) data, the individual DNA of the heaviest 46 lines and the lightest 47 lines were respectively selected to establish two bulked DNA pools. A new high-throughput sequencing technology, Specific Locus Amplified Fragment Sequencing (SLAF-seq), was used to identify candidate genes of TSW in association analysis combined with bulked segregant analysis (BSA). A total of 1,933 high quality polymorphic SLAF markers were developed and 4 associated markers of TSW were procured. A hot region of ~0.58 Mb at nucleotides 25,401,885–25,985,931 on ChrA09 containing 91 candidate genes was identified as tightly associated with the TSW trait. From annotation information, four genes (GSBRNA2T00037136001, GSBRNA2T00037157001, GSBRNA2T00037129001 and GSBRNA2T00069389001) might be interesting candidate genes that are highly related to seed weight.

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          Most cited references33

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          Gene Ontology: tool for the unification of biology

          Genomic sequencing has made it clear that a large fraction of the genes specifying the core biological functions are shared by all eukaryotes. Knowledge of the biological role of such shared proteins in one organism can often be transferred to other organisms. The goal of the Gene Ontology Consortium is to produce a dynamic, controlled vocabulary that can be applied to all eukaryotes even as knowledge of gene and protein roles in cells is accumulating and changing. To this end, three independent ontologies accessible on the World-Wide Web (http://www.geneontology.org) are being constructed: biological process, molecular function and cellular component.
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            High-throughput genotyping by whole-genome resequencing.

            The next-generation sequencing technology coupled with the growing number of genome sequences opens the opportunity to redesign genotyping strategies for more effective genetic mapping and genome analysis. We have developed a high-throughput method for genotyping recombinant populations utilizing whole-genome resequencing data generated by the Illumina Genome Analyzer. A sliding window approach is designed to collectively examine genome-wide single nucleotide polymorphisms for genotype calling and recombination breakpoint determination. Using this method, we constructed a genetic map for 150 rice recombinant inbred lines with an expected genotype calling accuracy of 99.94% and a resolution of recombination breakpoints within an average of 40 kb. In comparison to the genetic map constructed with 287 PCR-based markers for the rice population, the sequencing-based method was approximately 20x faster in data collection and 35x more precise in recombination breakpoint determination. Using the sequencing-based genetic map, we located a quantitative trait locus of large effect on plant height in a 100-kb region containing the rice "green revolution" gene. Through computer simulation, we demonstrate that the method is robust for different types of mapping populations derived from organisms with variable quality of genome sequences and is feasible for organisms with large genome sizes and low polymorphisms. With continuous advances in sequencing technologies, this genome-based method may replace the conventional marker-based genotyping approach to provide a powerful tool for large-scale gene discovery and for addressing a wide range of biological questions.
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              SLAF-seq: An Efficient Method of Large-Scale De Novo SNP Discovery and Genotyping Using High-Throughput Sequencing

              Large-scale genotyping plays an important role in genetic association studies. It has provided new opportunities for gene discovery, especially when combined with high-throughput sequencing technologies. Here, we report an efficient solution for large-scale genotyping. We call it specific-locus amplified fragment sequencing (SLAF-seq). SLAF-seq technology has several distinguishing characteristics: i) deep sequencing to ensure genotyping accuracy; ii) reduced representation strategy to reduce sequencing costs; iii) pre-designed reduced representation scheme to optimize marker efficiency; and iv) double barcode system for large populations. In this study, we tested the efficiency of SLAF-seq on rice and soybean data. Both sets of results showed strong consistency between predicted and practical SLAFs and considerable genotyping accuracy. We also report the highest density genetic map yet created for any organism without a reference genome sequence, common carp in this case, using SLAF-seq data. We detected 50,530 high-quality SLAFs with 13,291 SNPs genotyped in 211 individual carp. The genetic map contained 5,885 markers with 0.68 cM intervals on average. A comparative genomics study between common carp genetic map and zebrafish genome sequence map showed high-quality SLAF-seq genotyping results. SLAF-seq provides a high-resolution strategy for large-scale genotyping and can be generally applicable to various species and populations.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                29 January 2016
                2016
                : 11
                : 1
                : e0147580
                Affiliations
                [001]Oil Crops Research Institute of the Chinese Academy of Agricultural Sciences/Key Laboratory of Biology and Genetic Improvement of Oil Crops, Ministry of Agriculture, Wuhan, 430062, China
                Huazhong university of Science and Technology, CHINA
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Conceived and designed the experiments: XMW WHW. Performed the experiments: XXG CHJ. Analyzed the data: JY LJW. Contributed reagents/materials/analysis tools: XMW WHW. Wrote the paper: XXG.

                Article
                PONE-D-15-35936
                10.1371/journal.pone.0147580
                4732658
                26824525
                02c39f81-2da6-468d-9355-1f674bcaba5a
                © 2016 Geng et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 8 September 2015
                : 4 January 2016
                Page count
                Figures: 3, Tables: 7, Pages: 14
                Funding
                This work was supported by the National Natural Science Foundation of China (grant no. 31371664 and 31470088), the Scientific and Technological Project of Wuhan City (grant no. 2013020501010174) and the National Nonprofit Institute Research Grant (grant no. 1610172012001). WHW received all of the funding. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Genetics
                Genetic Loci
                Quantitative Trait Loci
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                Molecular Biology
                Molecular Biology Techniques
                Gene Mapping
                Research and Analysis Methods
                Molecular Biology Techniques
                Gene Mapping
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                Plant Science
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