Construction Of Dasypyrum Villosum Genetic Map And Characterization Of A Novel Wheat Powdery Mildew Resistant Gene Located On The Short Arm Of Chromosome 1V

The narrowing genetic basis is the bottleneck for further improvement of wheat yield,unearthing superior alleles from wild relatives is an important way to increase the wheat genetic diversity.Dasypyrum villosum(2n=14,VV)is the tertiary gene pool of common wheat known for its potential as a source of biotic and abiotic stress resistance genes that could be used to increase the wheat genetic diversity.It is necessary to construct V chromosome genetic map to transfer and utilization of superior gene in Dasypyrum villosum.High-density genetic map is useful to determine the chromosomal relationships between D.villosum and wheat,and facilitates to tap into beneficial genes from V genome into wheat improvement programmes.In the present study,the wheat 55 K single nucleotide polymorphism(SNP)array and chromosome engineering technology were used to construct V genome linkage map.A novel wheat powdery mildew resistant gene located on 1V was charactered.The results are shown as following.1.Construction of V chromosome genetic map of Dasypyrum villosumIn the present study,two T.durum-D.villosum amphiploids NAU1801 and NAU1802 were employed to obtain F2 population.After cytological analysis(GISH),115 F2 individuals with 14 D.villosum chromosomes were used to construct the genetic linkage map.With the aim to anchor genetic linkage groups to D.villosum individual chromosomes,seven co-dominant intron targeting(IT)markers between amphiploids NAU1801 and NAU1802,selected from the D.villosum chromosome-specific markers and assigned their chromosomal locations by using 1V to 7V disomic addition lines were also used to genotype the F2 plants.The current genetic linkage map of D.villosum contains 361 SNPs and seven IT markers presented in all seven linkage groups covering 1218.9 cM.The average chromosome map length ranged from 111.4 cM(2V)to 257.4 cM(4V)and the average distance between adjacent markers was 3.9 cM.The maps identified the homoeologous relationship with wheat and revealed that the D.villosum and wheat genomes are collinear and relatively conserved with the exception of 4V which shared multiple chromosome segments of group 4,5,and 7 of hexaploid wheat,but did not contain the reciprocal translocations such as 4L/5L and 4L/7S seen in wheat and other Triticeae.In addition,some small chromosome fragment rearrangements were observed throughout the V genome compared with the wheat genome.This genetic map provides an excellent starting point for more in-depth studies on V genome.2.Characterization of a novel wheat powdery mildew-resistant gene located on Dasypyrum villosum 011140 chromosome arm 1VSTwo T.aestivum-D.villosum translocation lines NAU1814(T5AS·1VS)and(T1DS·1VL),disomic substitution line 1V(1D),and deletion line1VL·1VS-del were developed from the progeny of(ZY1286×01I140)F1×NAU0686 BC4F1 and BC4F2.The mixed races of powdery mildew collected in Nanjing region were used to infect the four lines.Results showed that the lines lVL·1VS-del and T1DS·1VL were susceptible in whole growth stage.However,the lines T5AS·1VS and 1V(1D)gave immune reaction in the seeding stage,but partial resistance on leaves and immune reaction on sheath after tiller stage.As a result,chromosome arm 1 VS carries a novel powdery mildew resistance gene which was temporary named Pm1V.This resistance type broadens our knowledge on the fungi disease resistance gene,and which may confer the novel molecular mechanism.Using 47 pairs of IT markers specific to D.villosum 1VS to screen the four chromosome 1V alterations,15 1T markers were mapped in the physical bin 0.75-1.0 with Pml V,which could be used to identify this resistance gene in the breeding programmes.RNA-seq was performed on sheath and leaf from adult stage material substitution line 1V(1D),deletion line 1VL·1VS-del.Result showed that 60 differentially expressed genes in the suspect-resistant sheath,6 differentially expressed genes in the suspect-resistant leaf were screened out in the first linkage group of short arms of wheat.Among them,the differentially expressed gene TraesCSIA01G104300 was located in the interval of IT marker location.It is up-regulation in resistant sheaths and leafs simultaneously.Besides,the gene contains a peroxidase domain.So,it is speculated that the gene TraesCS1A01G104300 might relate to powdery mildew resistance.And RNA-seq results were verified by qRT-PCR.