Marker Assisted Transfer of Alien Genes for Biotic Stress Resistance in Field crops
Ahmed Fawzy Elkot Presented at Agriculture Research Station, ARC, Ismailia, Egypt: March 26, 2015
Marker Assisted Transfer of Alien Genes for Biotic Stress Resistance in Cereals
Importance of Single Genes in Saving Crops from Epidemics
Importance of Single Genes in Saving Crops from Epidemics (‌‌.cont.)
Another gene from India that provided American sorghum resistance to greenbug insect had resulted in millions of dollars of annual benefit to American farmers
Genetic bottlenecks imposed on crop plants during domestication and through modern plant-breeding practices
Tanksley and McCouch, 1997
Our objective is to re-enforce variability from wild to cultivated species
Field View of the wild species garden at PAU - 2014
Wheat Gene Pools Tertiary gene pool
Secondary gene pool
Primarys gene pool
All diploid and tetraploid Triticum & Aegilops species with A, B, & D genomes landraces, early domesticates
All non-progenitor diploid & tetraploid species
Tetraploid species with one genome common with wheat Diploid S-genome species from Aegilops section Sitopsis
Fine mapping of PM resistance genes -Survey Sequence data of 7AL-IWGSC
7AL contigs aligned against Brachypodiu m Genic sequences
SSRs designed from genic contigs
7AL genic contigs identified
Resistance Gene Analog markers designed from NBS/LRR domains
Contigs annotated for NB_ARC domains to identify NBSLRR disease resistance genes
Fine mapping of Triticum boeoticum PM genes using wheat NGS data
40 SSRs, 73 RGAs designed 40 RGAs amplified in TM/TB 10 SSRs, 20 RGAs mapped as dominant/ codominant/ CAPS markers 12 SNP markers mapped through HRM analysis
Integrated linkage map of 7AL consisting of markers from NGS data; Markers in red colour are RGA-STS, in magenta SSRs and in blue are those mapped as SNPs. Black markers represent the ones from Singh et al. (2007) and updated in Chhuneja et al. (2012)
Comparative partial linkage maps of chromosome 7AL based on SSR, DArT, STS and RGA markers (a) SSR, STS (b) SSR, STS and DArt markers (Chhuneja et al. 2012) and (c) SSR, STS, DArT, RGA and SNP markers showing chromosomal location of significant QTL for powdery mildew resistance in T. boeoticum/T. monococcum RIL population.
Comparative mapping of Chinese Spring RGAs mapping on T. monococcum chromosome 7AL with Brachypodium and Oryza genes
Comparative mapping of Chinese Spring RGAs mapping on T. monococcum chromosome 7AL with Brachypodium and Oryza genes
Primer sequences and amplification conditions of the RGA markers suitable for MAS of T. boeoticum powdery mildew resistance genes Marker ID
Primer
Primer Sequence
Ann. (째C) / Ext. (s)
Marker Type
Restriction Enzyme
Ta7AL-4556232_rga F
TTTCAAATAACGGCTTCTGG
55/150
19 bp InDel# DraI CAPS$
Ta7AL-4556232_rga R
GAGACGAGCAAATAGATATGG
Ta7AL-4426363_rga F
GAATCCTCCAAAGCCTCCAC
55/150
CAPS$
HphI
Ta7AL-4426363_rga R
GGCATATCTCATGTGAAGAACTG
Ta7AL-4544237_rga F
CACTACAATGATGGTAAGCGA
55/150
CAPS$
TaqI
Ta7AL-4544237_rga R
GCAAGAAGAAACAAGGAGAG
The sequence alignment of the PCR products of RGA marker Ta7AL4556232_rga from T. monococcum, T. boeoticum and Chinese Spring. 19 bpInDel in T. monococcum and T. boeoticum resulted in size polymorphism
The sequence alignment of the PCR products of RGA marker Ta7AL4556232_rga from T. monococcum, T. boeoticum and Chinese Spring. 19 bpInDel in T. monococcum and T. boeoticum resulted in size polymorphism
Validation of markers linked to the powdery mildew resistance gene and identification of polymorphic markers on the carrier chromosome Marker assisted transfer of the powdery mildew resistance genes from T. boeoticum to T. aestivum Evaluation of phenotypic expression of the powdery mildew resistance genes in hexaploid wheat
Plant Materials • The material used in the present study comprised of: •
Donor parent: Triticum boeoticum accession pau 5088
• Bridging species: Triticum durum cv. PBW114 • Recipient parent: Leaf rust and stripe rust resistant PBW343 introgression lines (PBW343-IL) and PBW 621 , a recently released
high yielding cultivar
T. durum (2n=28, AABB)
×
F1 (2n=21, AbAB)
T. boeoticum (2n=14, AbAb)
PBW343-NIL, PBW621 (2n=42, AABBDD)
×
F1 complex (2n=35, ArABBD)
×
BC1F1 (2n=40-42, ArABBDD)
PBW343-NIL, PBW621 (2n=42, AABBDD)
×
PBW343-NIL, PBW621 (2n=42, AABBDD)
BC2F1 (2n=40-42, ArABBDD)
Crossing strategy adopted for transferring Powdery mildew resistance genes
Cytological analyses A
B
(A, B) Pollen mother cells showing pollen sterility in F1 (2n=21, AbAB) of the cross T. boeoticum acc 5088/ T. durum cv PBW114
A
B
(A, B) Pollen mother cells showing pollen fertility in BC2F1 (2n=40-42 , ArABBDD) of the cross T. boeoticum acc 5088/ T. durum cv. PBW114///PBW621,PBW343-IL
Meiotic analysis in selected BC2F1 plants a) PBW114/ T. boeoticum pau5088 //3*PBW343-IL with 2n=40 (18''+4') and b) PBW114/T. boeoticum pau5088//3*PBW621 with 2n=42 (19''+4')
Summary of the material generated for transfer of PmTb7A and Pm1Tb from T. boeoticum to hexaploid wheat Cross
Generation and year
Total seeds obtained Total plants survived
PBW114/T. boeoticum pau5088
F1 (main campus; 2011-2012)
24
14
PBW114/T. boeoticum pau5088//PBW343-IL
Complex F1 (main campus; 2012-13)
61
36
PBW114/T. boeoticum pau5088//PBW621
Complex F1 (main campus; 2012-13)
65
42
PBW114/T. boeoticum pau5088//2*PBW343-IL
BC1F1 (offseason nursery 2013)
1756
118
PBW114/T. boeoticum pau5088//2*PBW621
BC1F1 (offseason nursery 2013)
1316
121
PBW114/T. boeoticum pau5088//3*PBW343-IL
BC2F1 (main campus; 752 2013-14)
282
PBW114/T. boeoticum pau5088//3*PBW621
BC2F1 (main campus; 639 2013-14)
245
Number of pollinated florets and number of seed set for generating complex F1 progenies from the cross PBW114/T. boeoticum //PBW343IL, PBW621
Cross-ID PBW114/T. boeoticum //PBW343-IL PBW114/T. boeoticum //PBW621 Total
No. of pollinated florets 7009
No. of seed
5308
65
12317
126
61
BC1F1 plants 2012-2013
A
B
C
PCR amplification profile for parental polymorphism survey between parents, donor parent T. boeoticum acc. pau5088, Bridging species PBW114, Recipient parent: PBW343 introgression lines (PBW343-IL) and PBW 621 using RGA-EST markers. a) 7AL-4556232_rga marker linked to PmTb7A, b) 7AL-4426363_rga (CAPS) marker linked to Pm1Tb after restriction with Taq1, c) 7AL-4544237_rga (CAPS) marker linked to Pm1Tb after restriction with Hph1 restriction enzymes.
Marker analysis for the powdery mildew resistance genes PmTb7A and Pm1Tb in pentaploid F1, BC1F1 and BC2F1 Generation
Total plants analysed
No. of plants positive for T. boeoticum allele(s) of PmTb7A Pm1Tb PmTb7A + Pm1Tb PBW114/T. boeoticum//PBW343-IL F1 pentaploid 36 11 3 9 BC1F1 117 34 5 25 BC2F1 121 47 13 6 PBW114/T. boeoticum//PBW621 Pentaploid F1 42 8 7 21 BC1F1 98 22 26 21 BC2F1 93 21 16 14 Total plants Pentaploid F1 78 19 10 30 BC1F1 215 56 31 46 BC2F1 214 68 29 20 Note: This total is not correct
Marker assisted foreground selection
In vitro amplification profile of 7AL-4556232_rga markers linked to PmTb7A with a random set of BC2F1 plants. The PBW343-IL shows null allele for this marker
In vitro amplification profile of 7AL-4426363_rga markers linked to Pm1Tb with a random set of BC2F1 plants
WL711 –NIL Control
PBW343-NIL
PBW114
PBW621
PBW343
WL711
TB
BC2F1
BC2F1
In vitro amplification profile of 7AL- 4445409_rga ,Xcfa2257 markers of BC2F1 selected plants and parental T. boeoticum, WL711, PBW343, PBW621, PBW114, PBW343-IL and WL711
Introgression profile of 40 (Forty) selected BC2F1 for chromosome 7A using SSR and RGA-STS markers. Blue region indicates T. boeoticum specific introgression and red region indicate recurrent parent specific regions. The chromosomal locations of different markers were according to Chhuneja et al (2014).
Marker screen for Stem rust
In vitro amplification profile of the 40 selected BC2F1 plants carrying PmTb7A and/or Tm1Tb with Sr22 specific marker Xsr22: XcsIH81-BM/ XcsIH81-AG
Selection for homozygous plants Plant ID
Cross
Total plants analysed
CBT3-6
PBW114/T.boeoticum //4*PBW343-IL
32
23
9
PBW114/T. boeoticum //4*PBW343-IL
69
44
25
PBW114/T. boeoticum //4*PBW621
20
4
6
10
Total plants analysed
Homozygous
Heterozygous Resistant
Homozygous Susceptible
38
6
15
17
PmTb7A CBT15-9
PmTb7A
CBT72-1
PmTb7A Plant ID
Cross Pm1Tb CBT38-1
PBW114/T. boeoticum//4*PBW621
Homozygous
Resistant
Resistant
Heterozygous Resistant
Homozygous Susceptible
Powdery mildew reaction of the parents and introgression lines developed from the cross: T. durum cv. PBW114//T. boeoticum acc 5088 //3*PBW343-IL and PBW621 at the adult plant stage against powdery mildew and strip rust under field condition
Evaluation of phenotypic expression of the powdery mildew resistance genes in hexaploid wheat
Phenotypic evaluation of BC2F1 progenies for powdery mildew resistance at adult plant stage PM gene PmTb7A
PmTb7A-
Total plants
Pm1Tb
analyzd
Pm1Tb
T. durum cvPBW114/T. boeoticum acc. Pau5088 //3*PBW343-IL Resistant to PM
41
13
6
60(121)a
Resistant to powdery
37
9
4
50
mildew and stripe rust T. durum cvPBW114/T. boeoticum acc. Pau5088 //3*PBW621 Resistant to PM
21
16
14
51(93)a
Resistant to powdery
20
15
10
45
mildew and stripe rust aNumbers in
parentheses indicate the number of plants that were analyzed with markers linked to PM resistance genes
Acknowledgements Advisory Committee
Dr. Kuldeep Singh (Major Advisor)
Dr. Parveen Chhuneja
Name
Designation and Department
Dr. Kuldeep Singh (Major advisor)
Sr. Molecular Geneticist School of Agricultural Biotechnology
Dr. Parveen Chhuneja
Molecular Geneticist School of Agricultural Biotechnology
Dr. NS Bains
Senior Plant Breeder Dept. of Plant Breeding and Genetics
Dr . Mohammed Javed
Associate Professor Dept. of Mathematics, Statistics and Physics
Dr. Yogesh Vikal (Dean PGS Nominee)
Geneticist School of Agricultural Biotechnology
Faculty Members
Dr. Satinder Kaur Dr. Priti Sharm
Dr. Kumari Neelam
Indian council of Agriculture research (ICAR) for
financial support and fellowship grant School of Agricultural Biotechnology – PAU Indo-Swiss collaboration in Biotechnology (ISCB) Ministry of Agriculture – Egypt , Agriculture research center Prof Dr: Naglaa Ashray
Thank you