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

GSystems

From gels to chips and sequencing (GBS)

ThroughputFrom singles to millions

Resolution10-30 cM to many markers per gene

Cost (per data point)Several US dollars to 1/1000 cent

Evolution of Genotyping (1980-2010s)

Marker type

Morphological Cytological ProteinDNA

RFLPRAPDAFLPSSRSNP

Xu 2010Molecular Plant BreedingCABI Publisher

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)

Xu 2010Molecular Plant BreedingCABI Publisher

Revised from:

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

Presence/Absence Variation (PAV)

Sample

A

B

Chromosome distribution

Presence

Absence

Presence/Absence Variation (PAV) results in many genes that cannot be mapped based on regular linkage mapping with SNP markers

单倍型的概念及其发展

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

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.

SNP1 SNP2 SNP3

Chromosome 1 AACACGCCA …. TTCGGGGTC….AGTCGACCG ….Chromosome 2 AACACGCCA …. TTCGAGGTC….AGTCAACCG ….Chromosome 3 AACATGCCA …. TTCGGGGTC….AGTCAACCG ….Chromosome 4 AACACGCCA …. TTCGGGGTC….AGTCGACCG ….

Individual 01 CTCAAAGTACGGTTCAGGCAHaplotype 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

SNPs

从 SNP到单倍型和标签 SNP Haplotype

Winner?

# Markers

Throughput

Cost

Data deliverry

Service

SNP Genotyping Platforms

● 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 kbCovering 19,540 genes, 2 SNPs/geneFunctionally tested with over 30 diverse

maize linesDeveloped by Illumina in collaboration

with TraitGenetics, INRA, and Syngenta

Genotyping by Arraying (Chips)

SNP genotyping by Array Tape

Douglas Scientific Array Tape 平台包括：Nexar Inline Liquid Handling SystemSoellex Thermal CyclerAraya Inline Fluorescence ScannerCentrifugeKraken 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

Genotyping By Sequencing (GBS)

GBS technology enables the detection of a wider range of polymorphisms: SNPs plus small indels

No pre-discovery or validationApplicable 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

Genotyping-By-Sequencing GBS

Created for high-throughput, semi-automated genotyping

Sequencing adaptor

BarcodeSticky ends

Genomic DNA

Images: Qiagen, Illumina, Elshire et al 2011, PLoS ONE

Restriction digest

SequenceLigate adaptors

Isolate DNA

Pool & amplify

Sample plants

• Advantages• One step SNP discovery + genotyping• Simple protocol; no reference required• Large numbers of SNPs found cheaply• Broadly applicable

• Drawbacks• False SNPs from

sequencing errors• Missing data from

stochastic sampling

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

GBS: Competitive Landscape

From P. S. Schnable

1 Commercialized by Floragenex Inc.2 Not disclosed; Data2Bio’s proprietary technology

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.

Resequencing to 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.

Sequencing Everything !!

Genotyping by Whole Genome Sequencing

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

① Soaking ② Sampling ③ Grinding

⑥ Tracking back and planting

④ DNA extraction ⑤ PCR and genotyping

Seed DNA-based Genotyping in Maize

Gao et al 2008 Mol Breed 22:477–494

Automaticseed chipping

Laser-assisted seed selection

20% 15% 10% 5% 3% 1%15

3050

100

20% 15% 10% 5% 3% 1%15

3050

100

20% 15% 10% 5% 3% 1%15

3050

100

20% 15% 10% 5% 3% 1%15

3050

1000

102030405060708090

100

0102030405060708090

100

0102030405060708090

100

0102030405060708090

100

QTL effect

Tail

size

Po

wer

(%

)

QTL effect

Tail

size

Po

wer

(%

)

QTL effect

Tail

size

Po

wer

(%

)

QTL effect

Tail

size

Po

wer

(%

)

N = 200 N = 500

N = 1000 N = 3000

Selective Genotyping: QTL Effects and Population/Tail Sizes

Sun et al 2010 Mol Breed 26:493–511

Bulked or Pooled DNA Analysis

PCR markers Chip genotyping DNA sequencingRNA sequencing

0.0

0.5

1.0

0.0

0.5

1.0

Population distribution

Selection

DNA Pools

GenotypingLinked

Unlinked

High tail

Lowtail

0.0

0.5

1.0 Linked

Linked

Linked

Unlinked

R plants S plants

A B

Alle

le f

requ

enc

y

Xu, 2010, Molecular Plant Breeding, CABI