INTRODUCTION Substantial breeding efforts in the last century led to - - PDF document

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Truong Quoc Anh Department of Biotechnology Institute of Agricultural Sciences for Southern Vietnam

Ho Chi Minh City, September, 16th, 2011

MOLECULAR MARKER, QTL MAPPING AND MARKER-ASSISTED SELECTION FOR CROP IMPROVEMENT

PRESENTATION

INTRODUCTION

• Substantial breeding efforts in the last century led to the

improvement of agronomic traits of many crops including yield, grain quality, heading date and stress tolerance…

• Agronomic traits are influenced by a number of genes, gene

x gene interaction and gene x environment interaction. Consequently, continuously phenotypic variation

• ccur

in hybrid progenies

• The genes are generally referred to as quantitative trait loci

(QTL).

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• For the past ten years, molecular markers have been used

successfully in gene mapping and QTL analysis based on high density linkage map for identifying chromosomal regions that controlling quantitative traits.

• Genetic markers represent genetic differences between

individual organisms or species.

• There are three major types of genetic markers:

(1) Morphological markers (2) Biochemical markers (3) Molecular markers

INTRODUCTION MOLECULAR MARKERS

Development, distribution, function and application of SSR molecular markers

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QTL ANALYSIS

Anthesis silking interval (ASI)

10 20 30 40 50 60 4-5 5-6 6-7 7-8 8-9 9-10 10-11

Number of plants

The frequency distributions of 194 F2 plants of maize for ASI

DAYS

QTL ANALYSIS

Grain yield (g) 10 20 30 40 50 60 70 80 300-400400-500500-600600-700700-800800-900 Number of Plants

The frequency distributions of 194 F2 plants of maize for grain yield

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QTL ANALYSIS

The genetic linkage map contains 85 SSR markers and covers 1350 cM, using 194 F2 plants derived from a cross between D12 and CLM161

C1

NS T -PR

T-PR T-PR NS C1

NS T -PR

T-PR T-PR NS C1

NS T -PR

C1 C1

GY ASI

T-PR ASI ASI GY

QTL ANALYSIS

Condit ion QTL Chro moso me Interval markers Nearest marker (cM) Additive effect Dominant effect Phenotype variation (%) LOD Water stress GY 1 bnlg1429-bnlg1811 bnlg1429(2.6)

• 7.26
• 3.66

10.60 2.87 ASI 1 bnlg1811-umc2228 bnlg1811(9.1)

• 0.92
• 0.01

12.10 2.6 GY 9 umc1804-umc1675 umc1804(2.0) 4.314 7.212 13.69 4.02 ASI 9 umc1078-bnlg1091 bnlg1091(3,7)

• 0.299
• 0.188

12.80 3.67 ASI 3 umc1588-umc1399 umc1399(7.3) 0.215 0.202 5.00 2.78

QTLanalysis for ASI and GY in 194 F2 plants of D12 and CML161.

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MARKER ASSISTED SELECTION

Resistant Resistant Susceptible

Bacterial blight (BB)

• f

rice is considered the serious disease in rice- growing countries worldwide. Since there is the no bactericide effective to the disease, introduction of resistance varieties is the most effective measure to control disease. In Viet Nam, bacterial blight of rice is

• ne of major rice diseases affecting to

yield, grain quality and productivity. The change

• f

rice ecosystem, cropping pattern, and intensive farming as well as susceptible varieties are significantly influenced to disease. The objective of this study to develop the restorer lines containing Xa7 and Xa21 by a combination of phenotype against Xoo diagnostic strains and marker-assisted selection.

MARKER ASSISTED SELECTION INOCULATION METHOD

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MARKER ASSISTED SELECTION

Genetic linkage map of Xa7 AND Xa21 resistance genes in chromosome 6 and chromosome 11

MARKER ASSISTED SELECTION

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 M 1 2 3 4 5 6 7 8 9 10 11 12 13

Electrophoresis result of PCR product using marker RM20590. Note, M: Marker ladder, 1: Que99, 2: IR24, 3 to 14 BC3F1 population Que99(Xa21)3*/IRBB7 Electrophoresis result of PCR product using marker RM20590 Note, M: Marker ladder, 1:MK63, 2: IR24, 3 to 13 BC3F1 population MK63 (Xa21)3*/IRBB7

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MARKER ASSISTED SELECTION

M 1 2 3 4 5 6 7 8 9 10 11 12 13 M 1 2 3 4 5 6 7 8 9 10 11 12 13 14

Eloctrophoresis result of PCR product using marker M1Xa21 To detect Xa21 gene. Note M: Marker ladder, 1: Que99, 2: IR 24 , 3 to 14 : BC3F1 population Electrophoresis result of PCR using marker M1Xa21 To identify Xa21 gene. Note M: Marker, 1: IR24 , 2: Pk63 , 3 to 13 : BC3F1 population

MARKER ASSISTED SELECTION

Soybean production in Vietnam has recently been threatened by the widespread dissemination of soybean rust (SBR) caused by the fungus Phakopsora pachyrhizi. Currently, chemical spray containing fungicides is the only effective method to control the disease. This strategy increases production costs and exposes the environment to higher levels of fungicides; introduction of resistant varieties is the most effective measure to control this disease. As first step towards the development of SBR resistance cultivars, we have used PCR based molecular markers in a backcross-breeding program to introgress Rpp5 gene of SBR resistance into HL203 from two donor lines (DT2000 and Stuart 99084B-28).

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MARKER ASSISTED SELECTION

HL203 x DT2000 HL203 x Stuart 99084B-28

F1 x HL203 F1 x HL203 MAS MAS BC1F1 x HL203 BC1F1 x HL203 MAS MAS BC2F1 x HL203 BC2F1 x HL203 MAS MAS BC3F1 x HL203 BC3F1 x HL203 MAS MAS BC4F1 BC4F1 Selfing Selfing BC4F2 BC4F2

Breeding schemes were produced for developing backcrossing population containing SBR resistant gene with genetic background of HL203

MARKER ASSISTED SELECTION

Genetic linkage map of Rpp5 locus

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MARKER ASSISTED SELECTION

M 1 2 3 4 5 6 7 8 9 10 11 12 13

PCR detection of Rpp5 gene in representative in BC4F1 plant. Marker used was Sat_275 (M: 100bp ladder, 1: HL203 recurrent parent, 2: DT2000 donor, 3-13:BC4F1 plants

MARKER ASSISTED SELECTION

a b c e d f g h i

Late blight disease of tomato

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MARKER ASSISTED SELECTION Ph-3 resistant gene on chromosome 9 MARKER ASSISTED SELECTION

Ladder 1 2 3 4 145 bp 150 bp 100 bp 127 bp 200 bp 188 bp 245 bp 250 bp 1 2 3 4 5 6 Ladder

PCR product with marker SSR383 on chromosome 9. Note: 1, 2: resistant lines, 3: susceptible line. PCR product with marker SSR69 on chromosome 9. Note 1- 2: resistant lines, 3: susceptible line

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Phenotype Population Segregation F2, BC, RILs, DH

. Continuously distribution

. 3:1 or 1:1

QTL analysis Detect QTL QTL . Effect . Position Mapping

N=100-200

Making NIL

BC3 or BC4

High resolution mapping N = 3000

• 10000

Cloning . ANOVA . SIM . CIM

THANKS FOR YOUR ATTENTION!