Dr ahmed fawzy elkot marker assisted transfer of alien genes for biotic stress resistance in field c

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