Development Of Diverse Primary Wheat-Rye 1RS.1BL Translocation Lines And Molecular Biology Of Expression Of Alien Genes And Accompany Mutations In The Wheat Genome

Wheat is one of the most important crops of the world.Introduce the chromatin of close relatives of wheat into wheat' genome,is the most important way to improve the wheat hereditary basis.Rye(Secale cereal L.,RR,2n=14)is the most valuable relative genera for the wheat genetics improvement.The 1R chromosome arm of rye was introduced into common wheat through rye-wheat 1RS.1BL chromosome translocation.Many useful genes transferred into common wheat,such as genes against to stripe rust(Puccinia striiformis f.sp.Tritici,Pst)and powdery mildew(Blumeria graminis f.sp.Tritici,Bgt),genes enhanced the yield potential and wide range of environmental adaptability.Therefore,the rye 1R chromosome was used worldwide in wheat breeding programs.However,the significant weakness of translocation wheat lines with the 1R chromosome derived from Petkus rye is its narrow genetic base,which is due to its single origin.Since the 1990s,the Yr9 gene has not provided protection against these pathogens,due to the prevalence of virulent pathotypes.Moreover,IRS chromosome arm is also known to have detrimental effects on grain processing quality.These undesirable dough properties are considered to be partially caused by the presence of co-secalins,which encoded by the genes at the Sec-1 locus on the IRS arm.Thus,introducing a large amount of new genetic variation from many different rye sources into wheat,increase the genetic diversity of 1RS.1BL translocation,should be a good method to increase the genetic diversity of wheat.Also,it has important means for the study of genome structure and evolution of polyploid plant.In this study,we identified several new primary 1RS.1BL translocation lines which were originated from the cross of wheat variety A42912 and Chinese local rye Weining,the cross of wheat cultivar Mianyang11 and Chinese local rye Aigan,the cross of Mianyang11 and Chinese local rye Baili by the methods of GISH,FISH,ND-FISH,A-PAGE and molecular markers.We also analyzed the genetic diversity among several 1RS.1BL translocation lines by SSR markers.The mutant of the LMW-GS was analyzed by SDS-PAGE.The new primary 1RS.1BL translocation lines' quality,agronomic traits,resistant to disease,and pre-harvest traits were also analyzed.All the results were described as below:1.8 regular 1RS.1BL translocation lines T917-15,T917-26(Expression deletion of gene Sec-1),T1245-5,T857-7,T852-29,RT1163-4,RT1217-1 and RT1249,1 1R(1B)substitution line RS1200-3,3 particular 1RS.1BL lines T1245-6(1R satellite deletion),T857-8(1R satellite deletion)and T852-31(6B satellite deletion),were identified by GISH,FISH,ND-FISH,A-PAGE and molecular markers.Also,2 common wheat lines with chromosome deletion were identified:the 1B satellite deletion line 15-262 and the IBS deletion line 15-263.2.T1245-6 and T857-8 were 1RS.1BL translocation with different level of 1R satellite deletion.Both lines were no expression of the Sec-1 locus by A-PAGE,and no bands amplified by PCR.It was indicated that Sec-1 was located on the satellite of 1R chromosome.Also the break point of T1245-5 was confirmed between SCM9 and TSM587;the break point of T857-8 was confirmed between Nor-R1 and TSM123.T852-31 was a 1RS.1BL translocation line with 6B satellite deletion.And several ?/?-gliadin were missed in the A-PAGE.For the wheat lines 15-262 and 15-263,several ?-gliadin and LMW-GS were missed in the A-PAGE and SDS-PAGE.And also,there were no amplified products by the primers GliB1 and GluB3,it indicated that the gene Gli-B1 and Glu-B3 were located on the satellite of 1B chromosome.The break point of 15-262 was confirmed between Xbarc8 and Xgm273.3.Two new primary 1RS.1BL translocation lines were developed from the cross of wheat variety A42912 and Chinese local rye Weining,which named as T917-15 and T917-26.Both lines carried a pair of intact and homogeneous 1RS.1BL chromosomes.T917-26 also harbored an expression deletion of some genes at the Sec-1 locus,which originated from a mutation that occurred simultaneously with wheat-rye chromosome translocations.These results suggest that the accompanying mutations of the evolutionarily significant translocations are remarkable resources for plant improvement.Comparison of translocation lines with its wheat parent showed improvements in the end-use quality parameters,which included protein content(PC),water absorption(WA),sodiumdodecyl sulfate sedimentation(SDSS),wet gluten(WG),dry gluten(DG)and dough stickiness(DS).whereas significant reduction in gluten index(GI)and stability time(ST)were observed.These findings indicate that IRS in wheat has produced a higher amount of protein,although these comprised worse compositions.However,in theT917-26 line that harbored an expression deletion mutation in the Sec-1 genes,the quality parameters were markedly improved relative to its sister line,T917-15,especially for GI and DS(P<0.05).These results indicated that expression deletion of Sec-1 genes significantly improves the end-use quality of wheat cultivars harboring the1RS.1BL translocation.We sequenced the CDS of Sec-1 in the Weining rye,T917-15 and T917-26,respectively.The results indicated that the CDS of these 3 lines were completely same.It was suggested that a mutant with expression deletion of gene Sec-1 in T917-26.4.A stable wheat-rye substitution line and 3 primary 1RS.1BL translocation lines were selected from the progeny of the crossing of the Chinese local rye Aigan variety and wheat cultivar Mianyang11.The substitution and translocation lines were identified by molecular cytogenetic analysis.PCR results,fluorescence in situ hybridization and acid polyacrylamide gel electrophoresis indicated that there were pair of 1R chromosomes in the substitution line which have been named RS1200-3,and a pair of 1RS.1BL translocation chromosomes in the other 3 translocation lines,which have been named RT1163-4,RT1217-1,and RT1249.When inoculated with stripe rust isolates,these 4 lines expressed high resistance to several Puccinia striiformis f.sp Tritici pathotypes that are virulent on Yr9.Moreover,the different response pattern of resistance among them suggested that the diversity of resistance genes for wheat stripe rust exists in the rye.These 4 lines also showed better agronomic performances than their wheat parent.The GS indices also showed the genetic diversity of the IRS which derived from same rye variety.The present study indicates that rye cultivars may carry untapped variations that could potentially be used for wheat improvement.5.In the study of resistance of pre-harvest sprouting among the 4 pairs of sister 1RS.1BL lines,all 1RS.1BL lines were significant lower in SGR(p<0.05)when compare with their wheat parent,except T917-25.The activity of a-amylase of all translocation lines were decreased when compared with their wheat parent.Especially,1245-5,T1245-6,T857-7,T857-8,T852-29 and T857-31 were significant lower than their wheat parent(p<0.05).The translocation lines were inconformity in the test of GI.The previous studies showed the expression of LMA increased when 1RS.1BL translocation occurred,it will increase the activity of a-amylase.But in our experiment,the new 1RS.1BL translocation lines we identified showed an opposite phenomenon,some of them even showed a significate decrease in activity of a-amylase.We deduced the new 1RS in those new 1RS.1BL translocation lines lead this.6.Frequent aberrations of wheat and rye chromosomes have been observed,which were induced by the monosomic addition of rye chromosomes into that of wheat.We called those as accompany mutations,whose parents have same genetic background,but mutations still occur for wheat-rye chromosome translocation.HMW-GS analysis of 4 pairs of 1RS.1BL translocation sister lines showed mutations appear in all the 1RS.1BL translocation lines compared with their parent wheat MY11,and there are also some mutations between sister lines T917-15 and T917-26,T852-29 and T852-31.Loci Glu-B1 had the most mutation number,7 translocation sister lines had mutations except T917-15;Glu-Al had the second number,6 translocation sister lines had mutations except T917-26 and T852-31;Glu-D1 had the least number,only T852-29 had mutation on this loci.We deduced that accompany mutations occurred in wheat genome when wheat-rye chromosome translocated,and it triggered HMW-GS genes locus on Glu-1 mutated.When 1RS.1BL translocated,Glu-1 is an high incidence loci of accompany mutations,the rate of mutations on Glu-B1 is the highest,Glu-Al took the second place,Glu-D1 had the lowest rate.1BL is the only chromosome in wheat which combined with the alien chromosome 1RS,so it had the highest rate of mutations.At the same time,deletion of 1BS triggered instability of B group in wheat,in other hand,it also triggered the mutations occurred in B group.So we deduced that accompany mutations is one of the important reasons of evolution in wheat genome.