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讲 座 提 纲

讲 座 提 纲. 1 什么是分子育种 2 历史回顾 3 全基因组策略 4 基因型鉴定 5 表现型鉴定 6 环境 型鉴定 ( etyping ) 7 标记 - 性状关联分析 8 标记 辅助 选择 9 决策支撑系统 10 展望. Evolution of Genotyping (1980-2010s). G. Systems From gels to chips and sequencing (GBS) Throughput From singles to millions

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讲 座 提 纲

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  1. 讲 座 提 纲 1 什么是分子育种 2 历史回顾 3 全基因组策略 4 基因型鉴定 5 表现型鉴定 6 环境型鉴定 (etyping) 7 标记-性状关联分析 8 标记辅助选择 9 决策支撑系统 10 展望

  2. Evolution of Genotyping (1980-2010s) G Systems From gels to chips and sequencing (GBS) Throughput From singles to millions Resolution 10-30 cM to many markers per gene Cost (per data point) Several US dollars to 1/1000 cent

  3. Marker type Morphological Cytological Protein DNA RFLP RAPD AFLP SSR SNP Xu 2010 Molecular Plant Breeding CABI Publisher

  4. Molecular basis of DNA markers A single-nucleotide polymorphism (SNP) is a DNA sequence variation occurring when a single nucleotide — A, T, C, or G — in the genome differs between members of a biological species. (Wiki) Revised from: Xu 2010 Molecular Plant Breeding CABI Publisher

  5. Copy-number variations (CNVs) — a form of structural variation—are alterations of the DNA of a genome that results in the cell having an abnormal number of copies of one or more sections of the DNA

  6. Presence/Absence Variation (PAV) Chromosomedistribution Sample Presence A B Absence Presence/Absence Variation (PAV)results in many genes that cannot be mapped based on regular linkage mapping with SNP markers

  7. 单倍型的概念及其发展 A haplotype is a group of genes within an organism that was inherited together from a single parent. A haplotype can describe a pair of genes inherited together from one parent on one chromosome, or it can describe all of the genes on a chromosome that were inherited together from a single parent. This group of genes was inherited together because of genetic linkage. The term "haplotype" can also refer to the inheritance of a cluster of single nucleotide polymorphisms (SNPs), which are variations at single positions in the DNA sequence among individuals. 功能区域SNP构成的单倍型 基因内SNP构成的单倍型 染色体内SNP构成的单倍型 全基因组范围 SNP构成的单倍型

  8. SNP2 SNP3 SNP1 SNPs Chromosome 1 AACACGCCA …. TTCGGGGTC….AGTCGACCG …. Chromosome 2 AACACGCCA …. TTCGAGGTC….AGTCAACCG …. Chromosome 3 AACATGCCA …. TTCGGGGTC….AGTCAACCG …. Chromosome 4 AACACGCCA …. TTCGGGGTC….AGTCGACCG …. 从SNP 到单倍型和标签SNP Haplotype Individual 01 CTCAAAGTACGGTTCAGGCA Haplotype 1 Individual 02 CTCAAAGTACGGTTCAGGCA Individual 03 CTCAAAGTACGGTTCAGGCA Individual 04 CTCAAAGCACGGTTGAGGCA Haplotype 2 Individual 05 CTCAAAGCACGGTTGAGGCA Individual 06 CTCAAAGCACGGTTGAGGCA Individual 07 CTCGAAGTACGGTTCAGGCA Haplotype 3 Individual 08 CTCGAAGTACGGTTCAGGCA Individual 09 CTCGAAGTACGGTTCAGGCA Individual 10 CTCAAAGCACGGTTCAGGCA Haplotype 4 Individual 11 CTCAAAGCACGGTTCAGGCA Individual 12 CTCAAAGCACGGTTCAGGCA A T C / / /G C G Tag SNPs

  9. SNP Genotyping Platforms • Winner? • # Markers • Throughput • Cost • Data deliverry • Service

  10. Genotyping by Arraying (Chips) • Three Illumina 1536-SNP chips: • Illumina-Cornell-CIMMYT collaboration Yan et al 2009; Yan et al 2010 • Illumina MaizeSNP50 Beadchip: • Up to 56,110 SNPs, 1 SNPs/40 kb • Covering 19,540 genes, 2 SNPs/gene • Functionally tested with over 30 diverse maize lines • Developed by Illumina in collaboration with TraitGenetics, INRA, and Syngenta

  11. SNP genotyping by Array Tape Douglas Scientific Array Tape 平台包括： Nexar Inline Liquid Handling System Soellex Thermal Cycler Araya Inline Fluorescence Scanner Centrifuge Kraken SNPline XL System 高通量数据： 每天处理400 张384孔反应数据（15万个） 低运行成本：极微量反应体系， 节省80-90% 的反应试剂 模块化程序设计： NEXAR微量液体转移系统 SOELLEX高通量PCR反应系统 ARAYA扫描系统 特别适合于大量样本少量标记的分析

  12. Genotyping By Sequencing (GBS) • GBS technology enables the detection of a wider range of polymorphisms: SNPs plus small indels • No pre-discovery or validation • Applicable to any species or population • GBS approaches • Simply sequence the entire genomes of individuals: expensive • Several extant methods. Each enriches for a portion of the genome which is then sequenced. Enrichment is most often achieved via restriction enzyme (RE) digestion. The existence of only 4, 6 or 8bp recognition sites limits the “tunability” of extant methods. Huang et al., 2010 Nature Genetics; Andolfatto et al., 2011 Genome Research; Elshire et al., 2011, PLoS ONE; Davey et al., 2011 Nature Reviews Genetics

  13. Genotyping-By-Sequencing GBS Created for high-throughput, semi-automated genotyping Sample plants Isolate DNA Restriction digest Ligate adaptors Pool & amplify Sequence • Drawbacks • False SNPs from sequencing errors • Missing data from stochastic sampling • Advantages • One step SNP discovery + genotyping • Simple protocol; no reference required • Large numbers of SNPs found cheaply • Broadly applicable Sequencing adaptor Barcode Sticky ends Genomic DNA Images: Qiagen, Illumina, Elshireet al 2011, PLoS ONE

  14. 1. 限制性酶切 2. 添加接头 3. 混池构建 4. 片段长度选择 5. 测序 6. 质量检控 7. 序列比对 8. HMM模型拟合 9. 下游分析 Andolfatto et al. 2011 Genome Research

  15. GBS:CompetitiveLandscape 1 Commercialized by Floragenex Inc. 2 Not disclosed; Data2Bio’s proprietary technology From P. S. Schnable

  16. Maize GBS 2.7 Build • Trained on 32K taxa including extensive CIMMYT material (landraces and diverse breeding materials) 45K taxa now scored with build • 960K core SNPs • Production Tags On Physical Map (TOPM) file for one step SNP calling available at panzea.org (imputation and calling in 15 min) Ed Buckler,personal comm.

  17. Genotyping by Whole Genome Sequencing Sequencing Everything !! Resequencingto discover SNPs, haplotypes and tag SNPs Tag SNPs can be developed to represent haplotypes. Each tag SNP represents one haplotype fragment. A set of tag SNPs can be developed to represent whole genome diversity.

  18. Approaches to Reduce Cost and Increase Scale in Genotyping • Seed-based DNA genotyping • Efficient sample tracking • Selective genotyping and pooled DNA analysis • Integrated diversity analysis, genetic mapping and MAS • Developing breeding strategies for simultaneous improvement of multiple traits

  19. Seed DNA-based Genotyping in Maize ① Soaking ② Sampling ③ Grinding ⑥ Tracking back and planting ⑤ PCR and genotyping ④ DNA extraction Gao et al 2008Mol Breed 22:477–494

  20. Automatic seed chipping Laser-assisted seed selection

  21. 100 100 100 100 90 90 90 90 80 80 80 80 70 70 70 70 60 60 60 60 50 50 50 50 40 40 40 40 30 30 30 30 20 20 20 20 10 10 10 10 0 0 0 0 Selective Genotyping: QTL Effects and Population/Tail Sizes N = 200 N = 500 Power (%) Power (%) 100 100 50 50 30 Tail size 20% 30 Tail size 20% 15% 15% 10% 15 10% 15 5% 5% 3% 3% QTL effect 1% 1% QTL effect N = 1000 N = 3000 Power (%) Power (%) 100 100 50 50 30 Tail size 20% Tail size 30 20% 15% 10% 15% 15 10% 5% 15 5% 3% 3% 1% 1% QTL effect QTL effect Sun et al 2010 Mol Breed 26:493–511

  22. Bulked or Pooled DNA Analysis PCR markers Chip genotyping DNA sequencing RNA sequencing Xu, 2010, Molecular Plant Breeding, CABI

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