Development Of Probes Specific To Chromosomes Of Dasypyrum Villosum And Identification Of Triticumaestivum-Dasypyrum Villosum Translocations Involving Chromosome 2v

With the gradual simplification of wheat varieties,the genetic basis of wheat has already been increasingly narrowed,and it is difficult to make major breakthroughs in the improvement of important characteristics such as yield,resistance and adaptability through cross-breeding.In breeding work,wild hybridization can effectively break the isolation between species and genus,and produce new types and varieties by recombining two or more species through long-term evolution and accumulation of nature.Using the potential disease resistance genes,nutrient-efficient genes,adaptive and high-yielding genes coming from wild species,new germplasms could be obtained through distant hybridization.These new germplasms can further broaden the genetic basis of cultivars,increase their production potential,and cultivate multi-resistance and nutrient efficiency.Therefore,the introduction of alien chromosomes or fragments by distant hybridization such as alien additional lines,alien substitution lines and alien translocations are an important part of chromosome engineering breeding.Wheat wild relative species,Dasypyrum villosum(L.)(Candargy,Dv;2n=14,VV)Schur(syn.Haynaldia villosa(L.)Schur)is an important tertiary genetic resource for genetic improvement of common wheat.The accurate and efficient identification of alien chromatin in wheat background is an important step in chromosomal engineering breeding.Until now,the resolution of the FISH kaiyotype of D.villosum is still insufficient due to the limited number of probes that can be used to identify the chromosomes of D.villosum.Therefore,it is urgent to develop a system to quickly and accurately identify the chromosomes of D.villosum.At the same time,the induction of alien small fragment translocation lines and introgression lines has a low degree of unfavorable gene redundancy,and can better exploit the beneficial genes of wild species.The 2V chromosome of D.villosum carries many potential genes,such as the gene controlling the ridge bristles(Bgr-V1),the gene controlling the photoperiod(Ppd-V1),and the genes controlling the agronomic traits such as spike length.Therefore,the transferring of the superior genes from the 2V chromosome of D.villosum into the common wheat background can greatly improve the genetic diversity of common wheat and promote the wheat breeding process.In this study,we first analyzed the repetitive sequences by software based on the second-generation sequencing data of the 6VS chromosome of D.villosum,and developed 10 oligonucleotide probes based on these repetitive sequences.The results showed that 7 probes were chromosome-specific oligonucleotide probes of D.villosum;the CSph1b mutant was used to induce the 2V chromosome structural variant of common wheatD.villosum.The variants were screened and identified in the self-crossing and backcross progenies using in situ hybridization technique and molecular marker-assisted selection,and 32 different types of structural variants were obtained.The transmission rate of 2VS chromosomes and the effects on agronomic traits were analyzed in the T2VS·2DL of the common wheat-D.villosum chromosome arm translocation line and the positive and negative BC1F1 and self-crossing populations(F2,F2:3,F2:4)of Yannong 19,Yangmai 22 and Ningmai 9,respectively,using the molecular markers and the morphological marker of the 2V chromosome of D.villosum.1.Development of repeated sequence oligonucleotide probes using sequencing information of 6VS chromosome in D.villosum.In the present research,based on the sequences of 6VS of D.villosum,ten oligo probes were designed according to the new tandem repeats.Seven of the ten oligo probes,which were specific to chromosome of D.villosum,produced strong and distinct signal bands on D.villosum 91C43 chromosomes,T.durum-D.villosum amphiploid(AABBVV)and Chinese Spring.oligo-6VS-CL1,oligo-6VS-CL35,oligo-6VS-CL57,oligo-6VS-CL44,oligo-6VS-CL56 and oligo-6VS-CL73 probes generated hybridization signals on every chromosome of D.villosum 91C43,respectively.oligo-6VS-CL18 probe produced hybridization signals on 1V,2V,3V,4V and 6V chromosomes of D.villosum 91C43.The signals of probes oligo-6VS-CL1 occurred on the telomeric,subtelomeric,centromeric and pericentromeric regions of individual chromosomes of D.villosum 91C43.This oligo-6VS-CL35 probe produced clear signals almost the same as those from the oligo-pSc119.2 probe,but oligo-6VS-CL35 probe produced much stronger and richer signals than that of oligo-pSc119.2 probe.We established an oligo-based FISH karyotype for D.villosum using oligo-6VS-CL35 and oligo-6VS-CL1,which can be used to identify individual chromosomes of D.villosum.2.The transmission effect on agronomy traits of translocation chromosome T2VS·2DL in different varieties background.Using the molecular markers and the morphological markers of the 2V chromosome of D.villosum,the T2VS·2DL chromosome arm translocation line and the positive and negative BC1F1 and self-crossing populations(F2,F2:3,F2:4)of Yannong 19,Yangmai 22 and Ningmai 9,respectively,were analyzed.The transmission rate of the 2VS chromosome and its effect on agronomic traits were analyzed.It was found that the T2VS·2DL chromosome in the translocation line was indeed able to participate in pairing and isolation normally,and could be normally transmitted through the male and female gametes,but the efficiency of each combination transmitted through the male and female gametes was different.In different wheat genetic backgrounds,the genetic effects of T2VS·2DL translocation chromosomes were different.In general,T2VS·2DL translocation chromosomes had positive effects on yield traits such as spike length and spikelet number.3.Induction of structural variants involving 2V chromosome of T.aestivum-D.villosum by Chinese Spring ph1b mutant.In this study,in order to induce the structural variants involving 2V chromosome,the CSph1b mutant was used to cross with the T2VS·2DL homozygous arm translocation line and alien substitution line DS2V(2D).Using cytological identification technology(GISH/FISH)and molecular marker-assisted selection,a total of 32 structural variants involving 2V chromosome were screened,including 7 homozygous termial translocations,10 heterozygous terminal translocations,15 telomeric and ditelomeric chromosome.Using the oligonucleotide probes oligo-pAs1 and oligo-pSc119.2,the identity of translocation chromosomes was identified,and found that the chromosomes belong to the second groups of wheat 2A,2D and 2B,further indicating that the CSph1b mutants lead to compensatory translocations.