Fast and Cost-Effective Genetic Mapping in Apple Using Next-Generation Sequencing

Next-generation DNA sequencing (NGS) produces vast amounts of DNA sequence data, but it is not specifically designed to generate data suitable for genetic mapping. Recently developed DNA library preparation methods for NGS have helped solve this problem, however, by combining the use of reduced representation libraries with DNA sample barcoding to generate genome-wide genotype data from a common set of genetic markers across a large number of samples. Here we use such a method, called genotyping-by-sequencing (GBS), to produce a data set for genetic mapping in an F1 population of apples (Malus × domestica) segregating for skin color. We show that GBS produces a relatively large, but extremely sparse, genotype matrix: over 270,000 SNPs were discovered but most SNPs have too much missing data across samples to be useful for genetic mapping. After filtering for genotype quality and missing data, only 6% of the 85 million DNA sequence reads contributed to useful genotype calls. Despite this limitation, using existing software and a set of simple heuristics, we generated a final genotype matrix containing 3967 SNPs from 89 DNA samples from a single lane of Illumina HiSeq and used it to create a saturated genetic linkage map and to identify a known QTL underlying apple skin color. We therefore demonstrate that GBS is a cost-effective method for generating genome-wide SNP data suitable for genetic mapping in a highly diverse and heterozygous agricultural species. We anticipate future improvements to the GBS analysis pipeline presented here that will enhance the utility of next-generation DNA sequence data for the purposes of genetic mapping across diverse species.

[1]  J. Keulemans,et al.  Genetic linkage maps of two apple cultivars (Malus × domestica Borkh.) based on AFLP and microsatellite markers , 2005, Molecular Breeding.

[2]  Marco C. A. M. Bink,et al.  Genomic Selection for Fruit Quality Traits in Apple (Malus×domestica Borkh.) , 2012, PloS one.

[3]  L. Cadle-Davidson,et al.  Grapevine powdery mildew resistance and susceptibility loci identified on a high-resolution SNP map , 2013, Theoretical and Applied Genetics.

[4]  Brian Boyle,et al.  An Improved Genotyping by Sequencing (GBS) Approach Offering Increased Versatility and Efficiency of SNP Discovery and Genotyping , 2013, PloS one.

[5]  José Crossa,et al.  Genomic Selection in Wheat Breeding using Genotyping‐by‐Sequencing , 2012 .

[6]  Trevor W. Rife,et al.  Genotyping‐by‐Sequencing for Plant Breeding and Genetics , 2012 .

[7]  Robert J. Elshire,et al.  A Robust, Simple Genotyping-by-Sequencing (GBS) Approach for High Diversity Species , 2011, PloS one.

[8]  S. Korban,et al.  Integration of physical and genetic maps in apple confirms whole-genome and segmental duplications in the apple genome , 2011, Journal of experimental botany.

[9]  J. Poland,et al.  Application of Genotyping-by-Sequencing on Semiconductor Sequencing Platforms: A Comparison of Genetic and Reference-Based Marker Ordering in Barley , 2013, PloS one.

[10]  A. Allan,et al.  QTL and candidate gene mapping for polyphenolic composition in apple fruit , 2012, BMC Plant Biology.

[11]  A. Zharkikh,et al.  Genetic diversity of the genus Malus and implications for linkage mapping with SNPs , 2011, Tree Genetics & Genomes.

[12]  Hao Wu,et al.  R/qtl: QTL Mapping in Experimental Crosses , 2003, Bioinform..

[13]  M. DePristo,et al.  The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. , 2010, Genome research.

[14]  Robert J. Elshire,et al.  Comprehensive genotyping of the USA national maize inbred seed bank , 2013, Genome Biology.

[15]  R. Doerge,et al.  Empirical threshold values for quantitative trait mapping. , 1994, Genetics.

[16]  C. Gessler,et al.  Mapping quantitative physiological traits in apple (Malus × domestica Borkh.) , 2003, Plant Molecular Biology.

[17]  A. R. Walker,et al.  Light-Induced Expression of a MYB Gene Regulates Anthocyanin Biosynthesis in Red Apples1 , 2006, Plant Physiology.

[18]  Richard Durbin,et al.  Sequence analysis Fast and accurate short read alignment with Burrows – Wheeler transform , 2009 .

[19]  D. Sargent,et al.  A genetic linkage map of an apple rootstock progeny anchored to the Malus genome sequence , 2012, Tree Genetics & Genomes.

[20]  F. Cattonaro,et al.  Application of genomics to grapevine improvement , 2010 .

[21]  Pere Arús,et al.  Comparative mapping and marker-assisted selection in Rosaceae fruit crops. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[22]  D. Sargent,et al.  Development of a dense SNP-based linkage map of an apple rootstock progeny using the Malus Infinium whole genome genotyping array , 2012, BMC Genomics.

[23]  Schuyler S. Korban,et al.  A Multi-Population Consensus Genetic Map Reveals Inconsistent Marker Order among Maps Likely Attributed to Structural Variations in the Apple Genome , 2012, PloS one.

[24]  M. Bink,et al.  Novel genomic approaches unravel genetic architecture of complex traits in apple , 2013, BMC Genomics.

[25]  Mihaela M. Martis,et al.  A physical, genetic and functional sequence assembly of the barley genome , 2012, Nature.

[26]  Gonçalo R. Abecasis,et al.  The Sequence Alignment/Map format and SAMtools , 2009, Bioinform..

[27]  Joseph L. Gage,et al.  Bridging the genotyping gap: using genotyping by sequencing (GBS) to add high-density SNP markers and new value to traditional bi-parental mapping and breeding populations , 2013, TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik.

[28]  R. Hellens,et al.  Red colouration in apple fruit is due to the activity of the MYB transcription factor, MdMYB10 , 2007, The Plant journal : for cell and molecular biology.

[29]  Robert J. Elshire,et al.  Switchgrass Genomic Diversity, Ploidy, and Evolution: Novel Insights from a Network-Based SNP Discovery Protocol , 2013, PLoS genetics.

[30]  V. Bus,et al.  Marker assisted selection for pest and disease resistance in the New Zealand apple breeding programme. , 2000 .

[31]  Riccardo Velasco,et al.  An Ancient Duplication of Apple MYB Transcription Factors Is Responsible for Novel Red Fruit-Flesh Phenotypes1[C][W] , 2012, Plant Physiology.

[32]  J. Celton,et al.  Construction of a dense genetic linkage map for apple rootstocks using SSRs developed from Malus ESTs and Pyrus genomic sequences , 2008, Tree Genetics & Genomes.

[33]  Susan McCouch,et al.  Bridging the genotyping gap: using genotyping by sequencing (GBS) to add high-density SNP markers and new value to traditional bi-parental mapping and breeding populations , 2013, Theoretical and Applied Genetics.

[34]  C. Peace,et al.  Utility testing of an apple skin color MdMYB1 marker in two progenies , 2011, Molecular Breeding.

[35]  Edward S. Buckler,et al.  TASSEL: software for association mapping of complex traits in diverse samples , 2007, Bioinform..

[36]  S. Myles.  Improving fruit and wine: what does genomics have to offer? , 2013, Trends in genetics : TIG.

[37]  R. Hellens,et al.  High temperature reduces apple fruit colour via modulation of the anthocyanin regulatory complex. , 2011, Plant, cell & environment.

[38]  Riccardo Velasco,et al.  Saturated linkage map construction in Rubus idaeus using genotyping by sequencing and genome-independent imputation , 2013, BMC Genomics.

[39]  Manuel A. R. Ferreira,et al.  PLINK: a tool set for whole-genome association and population-based linkage analyses. , 2007, American journal of human genetics.

[40]  Allison J. Miller,et al.  Vitis Phylogenomics: Hybridization Intensities from a SNP Array Outperform Genotype Calls , 2013, PloS one.

[41]  Gonçalo R. Abecasis,et al.  The variant call format and VCFtools , 2011, Bioinform..

[42]  Riccardo Velasco,et al.  Genome-Wide SNP Detection, Validation, and Development of an 8K SNP Array for Apple , 2012, PloS one.

[43]  P. Etter,et al.  Rapid SNP Discovery and Genetic Mapping Using Sequenced RAD Markers , 2008, PloS one.

[44]  J. Poland,et al.  Development of High-Density Genetic Maps for Barley and Wheat Using a Novel Two-Enzyme Genotyping-by-Sequencing Approach , 2012, PloS one.

[45]  J. Chapman,et al.  Anchoring and ordering NGS contig assemblies by population sequencing (POPSEQ) , 2013, The Plant journal : for cell and molecular biology.

[46]  P. Stam,et al.  Construction of integrated genetic linkage maps by means of a new computer package: JOINMAP. , 1993 .

[47]  Roger E Bumgarner,et al.  The genome of the domesticated apple (Malus × domestica Borkh.) , 2010, Nature Genetics.