Precise Identification And Genetic Analysis Of New Wheat-Dasypyrum Breviaristatum Translocation Lines

Dasypyrum is one of the wild relatives of wheat,which is mainly distributed along the Mediterranean coast and the Caucasus.Dasypyrum includes the annual species D.villosum and the perennial species D.breviaristatum.So far,there have been many studies on D.villosum,such as the excellent wheat powdery mildew resistance gene Pm21 and it has the novel resistance to drought,cold,stress and salt tolerance.Therefore,Dasypyrum is an excellent genetic resource for wheat improvement.The research on D.breviaristatum is less than D.villosum,mainly because of the scarcity of materials and the difficulty of its hybridization to wheat.TDH-2（2n=42,AABBV^bV^b）was selected from the wheat-D.breviaristatum partial amphiploid in our lab.Then TDH-2 was hybridized with common wheat ML-19 and Mianyang 11 to obtain the experimental parent material D2138.The karyotype of D2138 is 2n=44,which has a pair of 6V^bS.2V^bL in the wheat background.D2138 was hybridized with three different wheat（Chuanmai 62,Mianyang 11,and Jimai 22）to construct three F2-F3 hybrid populations.The precise identification of chromosomes,genetic transmission and analysis of related genes and traits were carried out by using these three populations.The main results are as follows:1.Karyotype analysis of D2138 and three common wheat was performed by FISH and the probe Oligo-pSc119.2、Oligo-p Ta535、Oligo-（GAA）₇.It was found that six chromosomes have the ND-FISH signal changed in different wheat background.In addition,Mc-FISH and Oligo-FISH painting analysis was carried out on D.breviaristatum.Based on the constructed karyotype of the 28 chromosomes of D.breviaristatum,a large amount of variation in FISH signals between homologous chromosomes was found,indicating a large heterozygosity in the D.breviaristatum genome.2.Cytological analysis of group D2138/Chuanmai 62,D2138/Mianyang 11 and D2138/Jimai 22 was carried out by multiple fluorescence in situ hybridization.The results showed that the three hybrid groups had different degrees of chromosome structural variation,accounting for 5.5%,12.2%and 18.8%,respectively.It indicated that the introduction of alien chromosomes had different effects depending on the wheat backgrounds,and the most affected group was D2138/Jimai22.In the three populations of D2138 and Chuanmai 62,Mianyang 11 and Jimai 22,the transmission rate of 6V^bS was 21.5-45.5%,and the transmissibility of 2V^bL was 25.6-35.6%,respectively.In the background of Chuanmai 62,the transmission rate of 6V^bS is higher than 2V^bL,which is opposite in other groups.The total transmission rate of alien chromosomes was the highest in group D2138/Chuanmai 62,followed by group D2138/Mianyang 11,and the lowest in group D2138/Jimai 22.3.A tottal of 60 pairs of 6V^bS specific molecular markers were screened,and 39pairs of specific primers between 6V^bS and 6VS were showed the different amplication between D.breviaristatum and D.villosum.The breakpoint position for four special fragments of chromosomes 6V^bS.2V^bL was identified by molecular markers and FISH.4.By specific PCR analysis of theα-gliadin,theα-gliadin gene of D.breviaristatum was located at 6V^bS in D2138,and their physical localization was also determined.The A-PAGE analysis confirmed that theα-gliadin gene encoded on 6V^bS from D2138 and its derived progeny.The 6V^bS specificα-gliadin genes was cloned,sequenced and phylogenetically analyzed.It was found that 78%of the pseudogenes existed in the sequences.Comparative analysis revealed that the proportion of the 6V^bSα-gliadin pseudogene in D2138 was higher than that in the D.breviaristatum.5.Through field character investigation of D2138 and its derived progenies,it was found that the plants with 6V^bS significantly increase the plant height and thousand-grain weight of compared with those absent of alien chromosome.At the same time,the analysis of seed quality indicated that the protein content,dough development time and grain hardness of the progeny containing 6V^bS were significantly improved,which may be affected byα-gliadin on 6V^bS.In conclusion,this study completed the precise identification of translocations and deletions of the D.breviaristatum chromosome in the wheat background,compared the molecular divergences between the 6V^bS in D.breviaristatum and the 6VS in D.villosum,cloned the 6V^bS specificα-gliadin gene and investigate agronomic traits.It provided a clue for D.breviaristatum genome evolution research and D.breviaristatum novel gene introgression into wheat for breeding purpose.