20/09/2011 PRESENTATION MOLECULAR MARKER, QTL MAPPING AND MARKER-ASSISTED SELECTION FOR CROP IMPROVEMENT Truong Quoc Anh Department of Biotechnology Institute of Agricultural Sciences for Southern Vietnam Ho Chi Minh City, September, 16 th , 2011 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 occur in hybrid progenies The genes are generally referred to as quantitative trait loci (QTL). 1
20/09/2011 INTRODUCTION 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 MOLECULAR MARKERS Development, distribution, function and application of SSR molecular markers 2
20/09/2011 QTL ANALYSIS 60 50 Number of plants 40 30 20 10 0 4-5 5-6 6-7 7-8 8-9 9-10 10-11 DAYS Anthesis silking interval (ASI) The frequency distributions of 194 F 2 plants of maize for ASI QTL ANALYSIS 80 70 Number of Plants 60 50 40 30 20 10 0 300-400400-500500-600600-700700-800800-900 Grain yield (g) The frequency distributions of 194 F 2 plants of maize for grain yield 3
20/09/2011 QTL ANALYSIS C1 C1 C1 C1 C1 NS NS NS GY T -PR ASI T -PR T -PR T-PR T-PR ASI T-PR ASI T-PR T-PR NS NS GY The genetic linkage map contains 85 SSR markers and covers 1350 cM, using 194 F 2 plants derived from a cross between D12 and CLM161 QTL ANALYSIS QTLanalysis for ASI and GY in 194 F2 plants of D12 and CML161. Chro Phenotype Condit Nearest Additive Dominant QTL moso Interval markers variation LOD ion marker (cM) effect effect me (%) GY 1 bnlg1429-bnlg1811 bnlg1429(2.6) -7.26 -3.66 10.60 2.87 Water stress 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 4
20/09/2011 MARKER ASSISTED SELECTION Bacterial blight (BB) of 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 Resistant Susceptible Resistant to control disease. In Viet Nam, bacterial blight of rice is one of major rice diseases affecting to yield, grain quality and productivity. The change of 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 5
20/09/2011 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 M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Electrophoresis result of Electrophoresis result of PCR product using PCR product using marker RM20590. marker RM20590 Note, M: Marker ladder, Note, M: Marker ladder, 1: Que99, 2: IR24, 3 to 1:MK63, 2: IR24, 3 to 13 14 BC 3 F 1 population BC 3 F 1 population MK63 Que99(Xa21)3*/IRBB7 (Xa21)3*/IRBB7 6
20/09/2011 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 Eloctrophoresis result of Electrophoresis result of PCR product using PCR using marker M1Xa21 marker M1Xa21 To detect To identify Xa21 gene. Xa21 gene. Note M: Marker, Note M: Marker ladder, 1: 1: IR24 , 2: Pk63 , 3 to 13 : Que99, 2: IR 24 , 3 to 14 : BC 3 F 1 population BC 3 F 1 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). 7
20/09/2011 MARKER ASSISTED SELECTION HL203 x DT2000 HL203 x Stuart 99084B-28 F 1 x HL203 F 1 x HL203 MAS MAS BC 1 F 1 x HL203 BC 1 F 1 x HL203 MAS MAS BC 2 F 1 x HL203 BC 2 F 1 x HL203 MAS MAS BC 3 F 1 x HL203 BC 3 F 1 x HL203 MAS MAS BC 4 F 1 BC 4 F 1 Selfing Selfing BC 4 F 2 BC 4 F 2 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 8
20/09/2011 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 BC 4 F 1 plant. Marker used was Sat_275 (M: 100bp ladder, 1: HL203 recurrent parent, 2: DT2000 donor, 3-13:BC 4 F 1 plants MARKER ASSISTED SELECTION a b c e f d g h i Late blight disease of tomato 9
20/09/2011 MARKER ASSISTED SELECTION Ph-3 resistant gene on chromosome 9 MARKER ASSISTED SELECTION 1 2 3 4 5 6 Ladder Ladder 1 2 3 4 245 bp 188 bp 145 bp 127 bp 250 bp 150 bp 200 bp 100 bp PCR product with marker PCR product with marker SSR69 on SSR383 on chromosome 9. chromosome 9. Note 1- 2: resistant Note: 1, 2: resistant lines, lines, 3: susceptible line 3: susceptible line. 10
20/09/2011 Phenotype Population F2, BC, Segregation RILs, DH . Continuously distribution . 3:1 or 1:1 Mapping QTL analysis . ANOVA N=100-200 . SIM BC3 or . CIM Making NIL Detect QTL BC4 High resolution mapping QTL N = 3000 . Effect -10000 Cloning . Position THANKS FOR YOUR ATTENTION! 11
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