The Development Of Haplotype Construction Technique System Of Common Wheat 7DL Homologous Chromosomes

Haplotype is the combination of a class of variant sites on a single chromosome that has a statistically significant association.Each chromosome has its own unique haplotype.During meiosis,the recombination between non-sister chromatids of homologous chromosomes can generate new haplotypes,which facilitates the widespread recombination of genetic information and provides impetus to genome evolution.As a common data analysis method,the analysis of haplotype is effective for the localization of heterozygosis SNPs on single chromosome,the excavation of disease genes and the search of maladies treatments.Common wheat(Triticum aestivum L.,2n = 6X = 42,AABBDD)is a hexaploid composed of three chromosomal groups AA,BB and DD,and is one of the most widely grown food crops.The 7DL chromosome is about 340 Mb in size and contains many genes that control important agronomic traits.The haplotype analysis technique of single chromosome can be used to analyse the difference between homologous chromosome and give important information to the tracing of individual genetic relationship and the analysis of allelic variation and genetic recombination hot spots.At present,the effective single chromosome haplotype analysis technique has not been established.In this study,7DL homologous chromosome of common wheat was used to explore the technique system of plant single chromosomes haplotype construction.7DL homologous chromosomes were microdissected from a cell with well spreaded chromosomes in a mitosis chromosome sample made using root tips and their DNAs were separately amplified by the MDA(multiple displacement amplification)and MALBAC(multiple annealing and looping-based amplification cycles)technology.The amplification products were detected regarding for the concentration and size.The product distribution was characterized by FISH(Fluorescent in situ hybridization).For the MDA products,the results showed that the DNA concerntration was up to 2,000 ng/?L,the DNA size was mainly about 15 Kb,and the products distributed on every chromosomes and any parts of chromosomes of wheat,which indicates the amplification has no significant bias.Whereas,the DNA concerntration was 600 ng/?L,the DNA size was mainly about 200-5,000 bp,and the products distributed on every chromosomes and there was no FISH signal on distal end of most chromosomes of wheat,which indicates the amplification has significant bias.Therefore,The MDA method was obviously superior to the MALBAC method for the 7DL amplication.So the MDA amplification products were used for subsequent experiments.In order to test the possible contamination of the amplified product with bacteria DNA,organell DNA and other DNAs from non 7DL chromosomes,the DnaN and DnaA of E.coli,the chloroplast genes PsbC and PsbD and other genes on different wheat chromosomes such as 5AL,7BS,5BL,4DS and 7AL were selected for PCR amplification using the MDA amplification products of 7DL as the templates.The results showed that MDA amplification products have a small amount of bacteria DNA and chloroplast genome DNA contamination,but without other chromosome DNA contamination of wheat.In order to further verify the integrity of MDA amplification products,22 SSR markers and 10 genes on 7DL were selected for PCR test.The results indicated that most of markers and some genes can be amplified,showing the 7DL MDA amplification products can be used in subsequent experiments.Finally,different SNP genotypes of 7DL homozygous chromosomes(1-1,1-2,5-1 and 5-2)from different seeds(different generation)were detected by 660 K SNP and the 7DL homologous chromosomes SNP map was developed.The results indicated that the number of SNP sites in homologous chromosomes 1-1 and 1-2 accounted for about 50% of the 1-1 total SNP sites and 42% of those of the 1-2.The number of SNP sites in 5-1 and 5-2 accounted for about 57% of their own total number of SNP sites.Analysis of the 1-1,1-2,5-1 and 5-2 SNP differential sites revealed that the 5-1 and 5-2 SNP sites same as 1-1 were approximately 30% of their own total SNP sites,respectively.The 5-1 and 5-2 SNP sites same as 1-2 were approximately 37% of their own total SNP sites.It suggested that there should be significant differences between the homologous chromosomes of 7DL in a cell,and more differences between 7DL in different offspring cells.In this study,7DL homologous chromosome haplotype analysis system was investigated,which was of great value in finding differences among homologous chromosomes,digging elite alleles and analyzing gene recombination,chromosome evolution,heterosis.And it also lays the foundation for the establishment of a set of universal chromosome haplotype analysis system in plant.