Development Of Wheat-haynaldia Villosa 4VS Structure Aberration Library And Fine Mapping Of Wheat Yellow Mosaic Virus Resistant Gene Wss1

Wheat have rich wild relatives,which are vast genetic resources for broadening genetic basis of cultivated wheat.Haynaldia villosa（or Dasypyrum villosa）is a diploid wild relative of wheat and belonging to the third gene pool due to its distant relationship with common wheat.To transfer and utilize useful genes controlling resistances to pests and diseases,tolerances against various abiotic stresses,high tiller numbers etc.of H.villosa,various alien chromosome lines have been developed.Murray et al（1994）reported that the disomic substitution line 4V（4D）was resistant to eyespot;Zhang et al（2005）found the 4V（4D）substitution and T4VS·4DL whole arm translocation lines were highly resistant to wheat yellow mosaic virus,and the resistance gene was mapped to 4VS and designated as Wss1.Zhao et al（2013）created chromosomal aberrant involving 4VS and narrowed the Wssl to the 4VS region FL0.78-1.00.To fine map and clone Wss1,in the present research,we firstly obtained the 4VS genome sequence by chromosome sorting and NGS sequencing.By sequence comparison,4VS specific molecular markers were developed.The 4VS structural aberration library and a Bin map of 4VS were constructed,which allowed us to map Wssl into a smaller region.A ph1b-based strategy was further employed to identify translocations having smaller 4VS fragments for fine mapping Wssl into less than 1Mb genomic region.Finally,to evaluate the potential value of Wssl in wheat breeding,the T4VS·4DL was introduced into five different wheat varieties.To summarize,the aims of this research are:（1）to dissect the genome of 4VS;（2）to fine map Wss1;（3）to develop new breeding lines having Wss1.The main results obtained are as follows:1.Genome sequence of 4VSThe chromosome arm 4VS were sorted through flow cytometry using T.aestivum-H.villosa ditelosomic addition line Dt4VS.The genome sequences were obtained by the Illumina NGS method and assembled.The obtained 4VS sequences were～170.6Mb in size and composed of 201,193 scaffolds.The maximum and minimum length of the scaffolds were 521,059 bp and 111 bp,respectively.The mean length was 848 bp and N50 was 59,654 bp.The sequence analysis indicated in the assemble 4VS sequences,about 64.6%were repetitive elements,and the coding fraction represented 1.5%of the arm,corresponding to 1977 annotated genes.Chromosomal synteny analysis suggests the homology regions of 4VS were corresponding to Bd₁ and 4 of Brachypodium chromosome,R3,11 and 12 of rice chromosome,and Sb1,5 and 8 of sorghum chromosome.A total of 893 out of 1977（45.2%）4VS evidenced genes have blastn hits in wheat homoeologous group 4,with the number of homologous genes of 574,773 and 525 in wheat 4AL,4BS and 4DS,respectively.This indicated,during evolution,there has been chromosomal rearrangements in 4A or 4V.2.Development and assignment of 4VS specific molecular markersThe sequence of H.villosa 4VS was aligned to the sequence assemblies of wheat chromosome arms 4AL,4BS and 4DS and Ae.tauschii 4DS.The introns within coding genes,which have more than 10%differences in intron sizes between 4VS and wheat 4AL/4BS/4DS were identified for developing intron targeting（IT）markers.A total of 359 IT based primer pairs were designed and used for amplification in the T.aestivum cv.Chinese Spring（AABBDD）,T.durum-H.villosa amphiploid（AABBVV）,translocation line（T4DL·4VS and T4VL·4DS）,substitution line（DS4V（4D））and 4VS ditelosomic addition line（Dt4VS）.Among of them,232 could clearly distinguish V from A,B and D sub-genomes of common wheat,and can be considered as 4VS specific markers.The marker development efficiency was up to 64.26%.Based on the amplification patterns in sub-genomes V,A,B and D,these IT markers could be classified into six different types.The type Ⅰ could differentiate all the four subgenomes V,A,B and D,there are 21 markers in type Ⅰ,accounting for 9.05%of the total markers.The type Ⅱ,Ⅲ,Ⅳ could differentiate three sub-genomes（V,A,D,V,B,D;or V,A,B）,there are 29,5 and 21 markers and they account for 12.5%,2.15%,9.05%of the total markers,respectively.The type Ⅴ only distinguish the two sub-genomes（V and D）,there are 17 markers and they accounted for 7.3%of the total.The last type Ⅵ has the most number（139,account for 59.9%）,they can identify 4VS from wheat background,while cannot differentiate the sub-genomes A,B and D.3.Dissection 4VS and mapping Wss1 by the development of wheat-Haynaldia villosa structural aberration libraryTwo approaches,iron irradiation and Triticum aestivum cv.Chinese Spring（CS）ph1b mutant（CSph1b）,were used to induce chromosomal aberrations or translocations.By fluorescence in situ hybridization and marker analysis,various structural aberrations involving 4VS were identified and characterized.The 4VS structural aberration library and the Bin map of 4VS were constructed.The library consisted of 57 homozygous structural aberrations,including 39 which were induced by CSph1b and 18 were induced by irradiation.The structural aberration types included terminal translocation,interstitial translocation,deletion and complex structural aberration.A total of 199 4VS-specific markers were assigned to 39 bins,named binl to bin39,in the 4VS cytological Bin map.In average,each bin contained five markers,with bin-1 having the most numbers of markers（34 markers）and nine bins（bin-3,bin-9,bin-12,bin-13,bin-22,bin-24,bin-27,bin-29 and bin-37）having only one marker.By using BLASTn search of the marker sequences against the Ae.tauschii 4DS reference genome sequences,we further assigned each bin to corresponding 4DS genome regions.The average physical distance of each bin was 4.7Mb.The largest bin was bin-39 near the centromere,which had two markers and represented a physical distance of approximately 49.1 Mb in 4DS of Ae.tauschii.The bin-3 was the smallest,which had only one marker and represented 16.1 kb physical distance in 4DS.The 57 aberrant were evaluated for WYMV resistance,and the Wss1 was narrowed into the corresponding physical region of bin1,i.e.,0-14.3 Mb physical distance in Ae.tauschii 4DS.4.Fine mapping and candidate gene prediction of WYMV resistance gene Wss1The ph1b gene was further used for further fine mapping of the Wss1.Five homozygous translocations（NAU421,NAU440,NAU460,NAU429,NAU431）having different sizes of 4VS were selected to cross to（CSph1b）,and five BC1F2 populations including 3,824 individuals were obtained.Screening using markers flanking the 4VS segments in the five female translocations identified 83,39,40,46 and 52 recombinants,respectively.Among these recombinants,118 recombinants in binl were identified using binl flanking markers CINAU623 and CINAU687.These recombinants could be classified into six haplotypes by genotyping using 34 4VS specific markers in bin1.The haplotypesⅡ,Ⅲ,Ⅳ and Ⅴ were susceptible to WYMV,and the haplotypes Ⅰ and Ⅵ were resistant.Combined the phenotyping and genotyping results,Wss1 was fine mapped to 781.1 kb interval in 4VS flanked by two markers 4VS-426 and 4VS-465,covering 719.805kb in Ae.tauschii 4DS.There are 13 4VS specific molecular markers allocated in this region,covering 781.1 kb genome region of 4VS.Referring to the Chinese Spring reference sequence,this interval contains 41 genes,including 27 high confidence genes（HCG）and 14 low confidence genes（LCG）.Three of the 27 HCG were specifically present in genome V,two HCG had copies in two wheat sub-genomes,and 22 had copies in all wheat three subgenomes.Gene annotation showed among the three V specific genes,which encoded putative protein of unknown function,a zinc-binding in reverse transcriptase and a plant invertase/pectin methylesterase inhibitor,respectively.5.Genetic effects of T4VS-4DL in background of five wheat varietiesThe T4VS·4DL translocation was introduced into Zhengmai 9023,Zhoumai 9823,Yangmai 15,Shi 4185 and Mianyang 26 by molecular marker assisted backcrossing.The backcrossing lines were evaluated for resistance WYMV,and all had improved resistance compared with their recurrent parents,respectively.Their agronomic traits,i.e.plant height,spike length and grain yield were compared in 2018-2019 and 2019-2020 two growth seasons in two locations.The spike length of lines ZRH1 and ZRH26 were significantly longer than their recurrent parent Zhengmai 9023 in two years and two locations,their grain yield was significantly higher in 2018-2019;The line ZRH54 showed significant increase for plant height,while no obvious change for grain yield comparing with recurrent parent Zhoumai 9823 in two years and two locations;The line ZRH19 and ZRH45 showed significant increase for grain yield comparing with recurrent parent Yangmai 15.There was no significant change for analyzed traits in the other lines.These indicated the introduction of T4VS·4DL in different wheat background could improve WYMV resistance without obvious linkage drag for agronomic traits and yield.