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      Genome-wide assessment of population structure and genetic diversity of Chinese Lou onion using specific length amplified fragment (SLAF) sequencing

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          Abstract

          Lou onion ( Allium fistulosum L. var. viviparum) is an abundant source of flavonols which provides additional health benefits to diseases. Genome-wide specific length amplified fragment (SLAF) sequencing method is a rapidly developed deep sequencing technologies used for selection and identification of genetic loci or markers. This study aimed to elucidate the genetic diversity of 122 onion accessions in China using the SLAF-seq method. A set of 122 onion accessions including 107 A. fistulosum L. var. viviparum Makino, 3 A. fistulosum L. var. gigantum Makino, 3 A. mongolicum Regel and 9 A. cepa L. accessions (3 whites, 3 reds and 3 yellows) from different regions in China were enrolled. Genomic DNA was isolated from young leaves and prepared for the SLAF-seq, which generated a total of 1,387.55 M reads and 162,321 high quality SNPs (integrity >0.5 and MAF >0.05). These SNPs were used for the construction of neighbor-joining phylogenetic tree, in which 10 A. fistulosum L. var. viviparum Makino accessions from Yinchuan (Ningxia province) and Datong (Qinghai province) had close genetic relationship. The 3 A. cepa L. clusters (red, white and yellow) had close genetic relationship especially with the 97 A. fistulosum L. var. viviparum Makino accessions. Population structure analysis suggested entire population could be clustered into 3 groups, while principal component analysis (PCA) showed there were 4 genetic groups. We confirmed the SLAF-seq approach was effective in genetic diversity analysis in red onion accessions. The key findings would provide a reference to the Lou onion germplasm in China.

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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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            An improved allele-specific PCR primer design method for SNP marker analysis and its application

            Background Although Single Nucleotide Polymorphism (SNP) marker is an invaluable tool for positional cloning, association study and evolutionary analysis, low SNP detection efficiency by Allele-Specific PCR (AS-PCR) still restricts its application as molecular marker like other markers such as Simple Sequence Repeat (SSR). To overcome this problem, primers with a single nucleotide artificial mismatch introduced within the three bases closest to the 3’end (SNP site) have been used in AS-PCR. However, for one SNP site, nine possible mismatches can be generated among the three bases and how to select the right one to increase primer specificity is still a challenge. Results In this study, different from the previous reports which used a limited quantity of primers randomly (several or dozen pairs), we systematically investigated the effects of mismatch base pairs, mismatch sites and SNP types on primer specificity with 2071 primer pairs, which were designed based on SNPs from Brassica oleracea 01-88 and 02-12. According to the statistical results, we (1) found that the primers designed with SNP (A/T), in which the mismatch (CA) in the 3rd nucleotide from the 3’ end, had the highest allele-specificity (81.9%). This information could be used when designing primers from a large quantity of SNP sites; (2) performed the primer design principle which forms the one and only best primer for every SNP type. This is never reported in previous studies. Additionally, we further identified its availability in rapeseed (Brassica napus L.) and sesame (Sesamum indicum). High polymorphism percent (75%) of the designed primers indicated it is a general method and can be applied in other species. Conclusion The method provided in this study can generate primers more effectively for every SNP site compared to other AS-PCR primer design methods. The high allele-specific efficiency of the SNP primer allows the feasibility for low- to moderate- throughput SNP analyses and is much suitable for gene mapping, map-based cloning, and marker-assisted selection in crops.
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              An SNP-based saturated genetic map and QTL analysis of fruit-related traits in cucumber using specific-length amplified fragment (SLAF) sequencing

              Background Cucumber, Cucumis sativus L., is an economically important vegetable crop which is processed or consumed fresh worldwide. However, the narrow genetic base in cucumber makes it difficult for constructing high-density genetic maps. The development of massively parallel genotyping methods and next-generation sequencing (NGS) technologies provides an excellent opportunity for developing single nucleotide polymorphisms (SNPs) for linkage map construction and QTL analysis of horticultural traits. Specific-length amplified fragment sequencing (SLAF-seq) is a recent marker development technology that allows large-scale SNP discovery and genotyping at a reasonable cost. In this study, we constructed a high-density SNP map for cucumber using SLAF-seq and detected fruit-related QTLs. Results An F2 population of 148 individuals was developed from an intra-varietal cross between CC3 and NC76. Genomic DNAs extracted from two parents and 148 F2 individuals were subjected to high-throughput sequencing and SLAF library construction. A total of 10.76 Gb raw data and 75,024,043 pair-end reads were generated to develop 52,684 high-quality SLAFs, out of which 5,044 were polymorphic. 4,817 SLAFs were encoded and grouped into different segregation patterns. A high-resolution genetic map containing 1,800 SNPs was constructed for cucumber spanning 890.79 cM. The average distance between adjacent markers was 0.50 cM. 183 scaffolds were anchored to the SNP-based genetic map covering 46% (168.9 Mb) of the cucumber genome (367 Mb). Nine QTLs for fruit length and weight were detected, a QTL designated fl3.2 explained 44.60% of the phenotypic variance. Alignment of the SNP markers to draft genome scaffolds revealed two mis-assembled scaffolds that were validated by fluorescence in situ hybridization (FISH). Conclusions We report herein the development of evenly dispersed SNPs across cucumber genome, and for the first time an SNP-based saturated linkage map. This 1,800-locus map would likely facilitate genetic mapping of complex QTL loci controlling fruit yield, and the orientation of draft genome scaffolds. Electronic supplementary material The online version of this article (doi:10.1186/1471-2164-15-1158) contains supplementary material, which is available to authorized users.
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                Author and article information

                Contributors
                Role: ConceptualizationRole: Project administrationRole: ResourcesRole: SupervisionRole: Writing – original draft
                Role: Investigation
                Role: Data curation
                Role: Data curation
                Role: Investigation
                Role: InvestigationRole: Resources
                Role: Data curation
                Role: Formal analysis
                Role: ConceptualizationRole: InvestigationRole: Project administration
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                5 May 2020
                2020
                : 15
                : 5
                : e0231753
                Affiliations
                [1 ] School of Agriculture, Ningxia University, Yinchuan, China
                [2 ] Ningxia Key Laboratory for Food Microbial-Applications Technology and Safety Control, Ningxia University, Yinchuan, China
                [3 ] Technological Innovation Center of Protected Horticulture (Ningxia University) in Ningxia, Yinchuan, China
                [4 ] Technological Innovation center of Horticulture (Ningxia University), Ningxia Hui Autonomous Region, Yinchuan, China
                National Cheng Kung University, TAIWAN
                Author notes

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

                Author information
                http://orcid.org/0000-0003-3153-0648
                Article
                PONE-D-19-22783
                10.1371/journal.pone.0231753
                7199963
                32369481
                86354ed9-2efa-41d7-8073-763988fa717c
                © 2020 Fang 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
                : 12 August 2019
                : 30 March 2020
                Page count
                Figures: 5, Tables: 1, Pages: 14
                Funding
                Funded by: the Funds of Breeding project for Agricultural Science and Technology Park in Ningxia 
                Award ID: 201406
                Award Recipient :
                Funded by: Innovation Platform Funds of Ningxia Key Laboratory for Food Microbial-Applications Technology and Safety Control
                Award ID: 2018DPC05026
                Award Recipient :
                Funded by: Project of support local colleges and universities reform and development funds of China 
                Award ID: 2018
                Award Recipient :
                This work was financially supported by the Funds of Breeding project for Agricultural Science and Technology Park in Ningxia (201406), Innovation Platform Funds of Ningxia Key Laboratory for Food Microbial-Applications Technology and Safety Control (2018DPC05026), the Key R&D Program of Ningxia (Grant No. 2017BY071), and Funds of the Western First-Class Disciplines (Horticulture) in Ningxia University(2015-2018).
                Categories
                Research Article
                Biology and Life Sciences
                Organisms
                Eukaryota
                Plants
                Flowering Plants
                Onions
                Biology and Life Sciences
                Organisms
                Eukaryota
                Plants
                Vegetables
                Onions
                Biology and Life Sciences
                Evolutionary Biology
                Evolutionary Genetics
                Biology and Life Sciences
                Agriculture
                Agronomy
                Plant Breeding
                Biology and Life Sciences
                Evolutionary Biology
                Population Genetics
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                Genetics
                Population Genetics
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                Population Biology
                Population Genetics
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                Evolutionary Biology
                Evolutionary Systematics
                Phylogenetics
                Phylogenetic Analysis
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                Phylogenetic Analysis
                Computer and Information Sciences
                Data Management
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                Research and Analysis Methods
                Mathematical and Statistical Techniques
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                Multivariate Analysis
                Principal Component Analysis
                Physical Sciences
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                Multivariate Analysis
                Principal Component Analysis
                Biology and Life Sciences
                Evolutionary Biology
                Evolutionary Systematics
                Phylogenetics
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                Taxonomy
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                Phylogenetics
                Computer and Information Sciences
                Data Management
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                Molecular Biology
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                Molecular Biology Techniques
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                Custom metadata
                The original data has been supplied as Supporting Information. The raw sequence data reported in this paper have been deposited in the Genome Sequence Archive in BIG Data Center, Beijing Institute of Genomics (BIG), Chinese Academy of Sciences, under submit accession number CRA002156 that are publicly accessible at https://bigd.big.ac.cn/gsa

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