Haplotype Diversity Of Rice Blast Resistance Gene Pi-ta And Fine Mapping Of The Ptr(t) Gene

Rice blast,caused by the filamentous ascomycete Magnaporthe oryzae(formerly M. grisea),is one of the most serious diseases worldwide,and is also an important factor of threatening the high and stable yield of rice.Resistance(R) genes in rice can confer resistance to rice blast efficiently.The Pi-ta gene is a single copy R gene in rice, which locates near to the centromere of chromosome 12,and encodes a protein with a coiled coil-nucleotide binding site-leucine rich repeat(CC-NBS-LRR) motif.The Pi-ta protein can recognize the product of the corresponding AVR gene in M.oryzae inside plant cell triggering effective signal transduction cascades.The single amino acid at position 918 of the Pi-ta protein was verified to be critical for its specificity to AVR-Pita.The rice germplasm composed of accessions of Asian and African cultivated rice and their wild relatives collected worldwide were subsequently used to study structural and functional properties of the Pi-ta gene and its variants.The Ptr(t) gene, another recently identified critical plant component in the Pi-ta mediated signal pathway,was fine mapped.Specifically:1.A total of 54 accessions from Oryza sativa,O.glaberrima,O.rufipogon,O. nivara,O.barthii,O.officinalis,O.aha,O.latifolia and O.glumaepatila were sequenced to investigate the diversity present at the Pi-ta locus.The Pi-ta allele was not amplified from some accessions of O.alta,O.latifolia and O. glumaepatila(CCDD genome) suggesting that DNA sequences of the Pi-ta allele were extremely divergent among these species.Two major clades were identified,consisting of 1 original Pi-ta sequence(AF207842 in NCBI database) and 15 Pi-ta variants,from AA genome species Oryza.Sativa,O.glaberrima,O. rufipogon,O.nivara and O.bathii and CC genome species O.officinalis. Alignment of DNA sequence assemblies revealed DNA similarity ranged from 91.9%-99.9%among Pi-ta sequences.A total of 105 nucleotide polymorphism plus 21 InDels was identified with most of polymorphisms in the intron region. TheÏ€value of Pi-ta within O.sativa is 0.00345.2.Pi-ta is a constitutive expressed gene in rice.The 15 Pi-ta variants were translated into 9 proteins highly similar to the Pi-ta protein.Computation modeling using similar protein revealed that alanine-918 is critical in theβ-sheet of the 3-D structure of the Pi-ta protein while serine at 918 would formα-helix. Analysis of the Pi-ta variants and disease reactions revealed that only the original Pi-ta protein carried alanine at position 918 confers resistance to the race that contains AVR-Pita,and the other variants can not do it.3.A total of 1790 accessions were characterized for the Pi-ta gene.Among them, 164 accessions from 56 countries distributing in Africa,Asia,Southeast Asia, Balkans,Central,North and South America,Europe,Mideast,North and South Pacific,and Indian Subcontinent carrying Pi-ta were identified via a combination of marker assisted selection(MAS) using Pi-ta-Indel marker,and sequencing the two critical genomic regions,intron and LRD of Pi-ta.These accessions were then evaluated using 46 SSR markers distributed on 12 rice chromosome for genetic diversity.A total of 7 groups were identified by cluster analysis of the SSR data.75 accessions were positively identified to contain the Pi-ta gene.However,83 accessions were predicted to contain Pi-ta independent R gene because they were resistance to the race IE-1k(without AVR-Pita).These accesions can be used to identify novel R genes.4.The Ptr(t) gene in rice has been reported to involve in Pi-ta mediated pathway. Rice germplasms,Amane,hatadawee,Patchaiperumal and Nema have spontaneous mutations at the Ptr(t) locus that were identified.A F₂ population of Amane/katy was used for fine mapping Ptr(t).Two SSR markers(XW195/196å’ŒXW249/250) were identified to delimit the Ptr(t) gene within a 50 kb region on chromosome 12.