Genetic And Physical Mapping Of Powdery Mildew Resistance Genes Pm41 And MlHLT In Wheat

Powdery mildew,caused by Blumeria graminis f.sp.tritici(Bgt),is one of the severest fungal wheat diseases worldwide.Breeding and using resistant cultivars is the most profitable and environmental friendly method to prevent disease losses.Discovering novel resistance genes and identifying the linked molecular markers will contribute to pyramiding multiple resistance genes and developing wheat cultivars with durable and broad-spectrum disease resistance.Wild emmer wheat and Chinese wheat landraces harbor abundant powdery mildew resistance genes,providing valuable germplasms for wheat genetic improvement.In present study,a high-density genetic linkage map of powdery mildew resistance gene Pm41,originated from Triticum dicoccoides,was constructed through comparative genomics analyses among wheat,Brachypodium,rice and sorghum.Combining comparative genomics analyses,bulked segregant analysis and RNA-Seq(BSR-Seq),Aegilops tauschii physical map and draft sequences,as well as haplotype analyses,the fine physical and genetic linkage maps of powdery mildew resistance gene MIHLT,derived from Chinese wheat landrace Hulutou,were constructed.The main results are as following:1.The orthologous genomic regions of the Pm41 locus were identified on Brachypodium chromosome 2,rice chromosome 1 and sorghum chromosome 3 through comparative genomics analysis.Orthologous wheat EST sequences,common wheat cv.Chinese Spring 454 shotgun contigs and International Wheat Genome Sequencing Consortium(IWGSC)chromosome 3B survey sequences were used as templates to design primers.Finally,19 polymorphic markers,XWGGC1501-XWGGC1519,were developed and intergrated into the Pm41 genetic linkage map.The Pm41 gene was mapped in a 0.6 cM genetic interval flanked by markers XWGGC1505 and XWGGC1507,and co-segregated with marker XWGGC1506.This region corresponds to an 11.7 kb genomic region(Bradi2g60650-Bradi2g60670)in Brachypodium,an 19.2 kb genomic region(0s01g72090-Os01g72120)in rice,and an 24.9 kb genomic region(Sb03g045760-Sb03g045780)in sorghum,respectively.Detailed comparative genomics analysis of the markers flanking the Pm41 locus in wheat and the putative orthologous genes in Chinese Spring 3B chromosome,Brachypodium,rice,and sorghum suggests Brachypodium is even more closely related to wheat than rice and sorghum.2.Chinese wheat landrace Hulutou is highly resistant to powdery mildew.Genetic analysis indicated that the powdery mildew resistance of Hulutou is controlled by a single dominant gene,provisionally designated MlHLT.By applying comparative genomics analysis,3 new polymorphic markers,Xwggc33,Xwggc3026 and Xwggc3148,were developed using wheat EST sequences on chromosome IDS,Chinese Spring 454 contig sequences and International Wheat Genome Sequencing Consortium(IWGSC)Chinese Spring survey sequences,as well as the draft genome sequences and extended single nucleotide polymorphism(SNP)marker sequences of Aegilops tauschii.MlHLT was mapped in a 3.6 cM genetic interval between markers Xwggc3026 and Xwggc3148,which corresponds to a 13.4 Mb genomic region(AT1D0107-AT1D0135)on IDS of Aegilops tauschii,a 369.8 kb genomic region(Bradi2g37770-Bradi2g38130)in Brachypodium,a 380.8 kb genomic region(Os05g02990-Os05g03530)in rice and a 298.4 kb genomic region(Sb09g002040-Sb09g002270)in sorghum.3.Markers Xwggc3026 and Xwggc3148 could be anchored on assembled BAC contigs ctg220 and ctg1065 in the physical map of Aegilops tauschii,respectively.This region contains 7 BAC contigs,ctg220,ctg4623,ctg1063,ctg5929,ctg3163,ctg699 and ctg1065.The MTP BACs of the 7 contigs were sequenced using a Mi-Seq platform and were subsequently used as templates to develop 5 new polymorphic markers,Xwggc10233,Xwggc9952,Xwggc10410,Xwggcl0576 and Xwggc9819,tightly linked to MIHLT.Further more,by applying bulked segregant analysis and RNA-Seq(BSR-Seq),two polymorphic SNP markers were identified and converted to dCAPS markers,Xwggc10531 and Xwggc10532.Thus,the powdery mildew resistance gene MIHTL was mapped within 0.232 cM genetic interval between markers Xwggc9952 and Xwggc9819,and co-segregated with markers Xwggc10531,Xwggc10532,Xwggc10410 and Xwggc10576.4.Haplotype analysis was performed in the powdery mildew resistance gene MIHLT candidate genomic region using 27 susceptible Aegilops tauschii accessions and 92 susceptible wheat core collections.The powdery mildew resistance gene MIHLT was further delimited into a 532.6 kb genomic region between markers Xwggc9952 and Xwggc10532,Gene annotation revealed that a resistance gene analog(RGA)with coiled-coil(CC),nucleotide binding site(NBS)and leucine rich repeats(LRR)domains was presented and could be considered as candidate of MIHLT.