GENOMIC ANALYSIS AND MAP-BASED CLONING OF A UG99-RESISTANCE GENE DERIVED FROM WILD GRASS
Location: Cereal Crops Research
Project Number: 5442-21000-037-25
Specific Cooperative Agreement
Start Date: Aug 01, 2012
End Date: Jul 31, 2017
The objective of this cooperative research project is to map and clone the goatgrass (Aegilops speltoides) gene Sr47, which confers resistance to Ug99 lineage races of stem rust in wheat.
Global wheat production is currently threatened by Ug99 lineage races of stem rust. The identification and deployment of Sr genes effective against Ug99 races is vital to protecting the world’s wheat supply. Sr47 is one of only a few Sr genes that confer a highlevel of resistance to Ug99 and other races. This gene was originally transferred from Aegilops speltoides (2n = 2x = 14, SS) to the durum wheat line 47-1. It was located on a T2BL-2SL•2SS translocation chromosome in which the distal 2BL segment comprised less than 10% of the long arm, with the remainder of the chromosome originating from Ae. speltoides. The translocation chromosome was recently transferred into LMPG6, which is a common wheat line with spring growth habit and is near-universally susceptible to stem rust. In addition, a set of seven ‘Chinese Spring’ (CS)-Ae. speltoides disomic substitution (DS) lines were recently established. Because LMPG6-Sr47 and CS-Ae. speltoides 2S(2B) DS showed a different infection types (; versus 2) to stem rust, they can be used for population development in the mapping and cloning of Sr47.
To map Sr47, a large F2 population (~8,000 individuals) will first be developed from the cross between LMPG6-Sr47 and CS-Ae. speltoides 2S(2B) DS. A subset of 150 F2 plants will be used for the initial mapping of the Sr47 gene with the markers recently identified in the new translocation lines with a shortened Ae. speltoides segment. The second phase of mapping will include 480 homozygous recessive F2 plants. The markers most closely flanking Sr47 based on the population of 150 F2 plants will be used to screen the population of 480 F2 plants to identify recombinants. The sub-population of recombinants will be screened with all cosegregating markers and will be used to identify additional markers tightly linked to Sr47. Once this is accomplished, we will perform high-resolution mapping in a population of 2,000 homozygous recessive F2 plants by screening the population with markers tightly flanking Sr47 to resolve the order of co-segregated markers. Colinearitywith the rice and Brachypodium genomes will be used to develop additional markers. The closely linked or co-segregated markers will be refined to make them suitable for marker-assisted selection.
To facilitate cloning of Sr47, a BAC library will be constructed from nuclear DNA of Ae. speltoides PI 369590 using BamHI in the pECBAC1 vector. Themarkers most closely flanking Sr47 will be used to screen the BAC library to identify the BAC clones containing the flanking markers. Ideally a single BAC harboring both flanking markers will be identified. If not, positive BACs will be fingerprinted, assembled into contigs, and sequenced. Candidate genes will be identified from the BAC sequences using bioinformatics, and validated by comparative sequence analysis of stem rust susceptible mutants and by complementation by transformation.