Development Of Vernalization Gene ZCCT-2 Marker In Common Wheat And Analysis Of Relationship Between Gene Composition And Heading Date Of Wheat Vernalization In China

The temperature characteristics of wheat are mainly controlled by vernalization genes,which not only affect the growth and yield of wheat,but also determine its environmental adaptability.The study of vernalization genes,and their allelic variation and effects,have guiding significance for the introduction of wheat and ecological breeding.In this study,the full-length sequences of ZCCT-A2,ZCCT-B2 and ZCCT-D2 were obtained by homologous cloning,and detection markers were designed.Through the use of molecular markers from predecessors and from this study,we detected the vernalization genes composition of 370 wheat varieties(lines)in different wheat regions of the country,analyzed and compared the distribution characteristics of vernalization genes in different wheat regions.Through identification the field and green house heading date,analysis of the relationship between gene composition and heading phenotypes.The main results are as follows:1.Cloning of wheat vernalization gene ZCCT-2 and molecular marker development.In order to understand the function of wheat vernalization gene ZCCT-2,the full-length sequences of three homologous genes of wheat variety J411 ZCCT-2 were obtained by homologous cloning.The coding region of the three homologous genes of ZCCT-2 was found to consist of two exons and one intron.The identity of the proteins encoded by ZCCT-A2,ZCCT-B2,and ZCCT-D2 was as high as 91.74%,and the domain was highly conserved,indicating that the three homologous genes of ZCCT-2 may have similar functions.By comparing the ZCCT-2 gene with the donor gene,the c DNA of the cultivar J411 ZCCT-A2 was 99.22% identical to the c DNA of the Triticum urartu’s;the c DNA of the ZCCT-B2 was94.52% identical to the Aegilops speltoides’ s;the c DNA of the ZCCT-D2 was 98.44%identical to the Aegilops tauschii’s,which indicate that ZCCT-A2 is more conservative than ZCCT-B2 and ZCCT-D2 in the evolution of wheat.Since the ZCCT gene is mainly composed of null forms and non-functional forms,in order to facilitate the detection of null form mutations,the detection primer VZ2ABD-F2/R1 was designed in the ZCCT-2 promoter part,that ZCCT-A2(375 bp),ZCCT-B2(357 bp),and ZCCT-D2(382 bp)were amplified separately;in order to facilitate the detection of non-functional form,specific primers were designed in the second exon part,EX2ZCCTA2-F1 /R1,EX2ZCCTB2-F1/R1,and EX2ZCCTD2-F1/R1,which amplified the second exon part of ZCCT-A2(682bp),ZCCT-B2(891bp),and ZCCT-D2(667bp),respectively.The development of these molecular markers will facilitate the study of the ZCCT-2 gene.2.The genetic composition of wheat in China and its relationship with the heading stage were clarified.By using molecular markers and existing in this paper,370 samples of the different wheat varieties(lines)of wheat vernalization genotype composition,phenotype identification,analysis of vernalization gene and phenotypic relationship.There were 26 alleles with dominant alleles in the VRN-A1 locus of vernalization gene,accounting for 7.0%;3 new mutations,accounting for 0.8%;57 of the VRN-B1 loci were dominant alleles,accounting for 15.4%;155 of VRN-D1 loci are dominant alleles,accounting for 41.9%;3 of VRN-B3 loci are dominant alleles,accounting for 0.8%.According to the characteristics of different wheat areas,the dominant allelic variation of Vrn-A1 was highest in the Northwestern Spring Wheat Region(68.2%),and the lowest in the Northern Winter Wheat Region(0%)and Yellow and Huai River Winter Wheat Region(0%).;the dominant allelic variation of Vrn-B1 was highest in Northwestern Spring Wheat Region(68.2%),and the lowest in the Northern Winter Wheat Region(0%)and Yellow and Huai River Winter Wheat Region(0%);the dominant allelic variation of Vrn-D1 was highest in Middle and low Yangtze Valley Winter Wheat Region(92.4%),and the lowest in the Northern Winter Wheat Region(0%);VRN-B3 in the 370 wheat varieties is mainly composed of recessive allelic variation type vrn-B3(99.2%),the dominance allelic variation only detected in spring wheat area,but the distribution is very small.It can be seen that the dominant allelic variation of the vernalization genes VRN-A1 and VRN-B1 shows an trend of high altitude,high altitude,and low latitude and low altitude increasing trend;the dominant allelic variation of VRN-D1 is decreased with the increase of latitude.Through the detection of vernalization gene ZCCT promoter region,we found that ZCCT-1 and ZCCT-2 were polymorphic.The ZCCT-1 locus was mainly expressed as Null ZCCT-A1 type,accounting for 26.9%.The ZCCT-2 locus was mainly Null ZCCT-B2 type with a proportion of 51.1%.Different combination types and field heading combination of the promoter region of ZCCT gene by deletion analysis results show that under field conditions,the ZCCT gene promoter region of different deletion types on heading date of heading is very small,that is a complex physiological process under the control of multiple genes;Analysis of the combination of deletion type and field traits in the ZCCT-2 gene promoter region revealed that the correlation coefficient between the different type of deletion and the cold resistance of the ZCCT-2 gene was 0.262,which reached a very significant level,indicating that the ZCCT-2 genes related to cold resistance under natural conditions.3.There were no functional alleles of VRN2 in common wheat varieties.A new allelic variation type was discovered at the ZCCT-B1 locus by sequencing in PH1521.There are two SNPs in the second exon,both the 227 th base mutation from T to C,the 280 th base mutation from A to G,resulting in the second exon encoded protein is mutated from glycine(G)to aspartic acid(D)at position 93.Through greenhouse(16h,20℃)phenotype identification,the mutant variety of the gene can be eared in the greenhouse without vernalization,and the seedling heading period is only 50 days;Sequence analysis of ZCCT genes of XN6426 material,the new allele type was found in the ZCCT-D1 locus,the variant promoter of the existence of a SNP site,both upstream of the start codon seventy-sixth base C mutated into T,the mutant variety of the gene can be eared in the greenhouse without vernalization,and the seedling heading period is only 53 days...