Establishment Of EcoTILLING Technique And Its Utilization In Mining Favourable Multiple Alleles For Cotton Breeding

Targeting induced local lesion in genomes(TILLING) technology is a reverse genetic approach used in screening the point mutation (SNP), which was originally created by the research team led by Steven Henikoff from Fred Hutchinson Cancer Research Center and Luca Comai from the Department of Botany of Washington University. Combination of random chemical mutagenesis with PCR method, it screened the mutation of the target gene region. Subsequently, they discovered that TILLING not only can detect the point mutations engendered by inducement, but also can detect gene polymorphism in natural populations. And they termed it as EcoTILLING technology (ecotypic TILLING).Cotton is an important economic crop in our country. In order to explore and take advantage of excellent genetic resources to lay the foundation for molecular design breeding of cotton, this article carried out to create the cotton EcoTILLING technology system, and with the cotton EcoTILLING system established, we designed some pairs of At-or Dt-subgenome specific primers for two fiber development-related genes, namely, GhSUS1genes and Gh14-3-3L gene, to mine favorable multiple alleles. The results are as follows:Endonuclease enzyme CEL Ⅰ was successfully extracted from celery (Apium graveloens L) using salting out method and evaluated by molecular weight and enzyme activity (Control C and Control G heterogeneous double-stranded comes with SURVEYOR Mutation Detection Kit Components). The results indicated that the molecular weight is43KD as reported and its enzyme activity is as good as the SURVEYOR Nuclease purchased from the Transgenomic, it can also cleavage the heterogeneous double-stranded in EcoTILLING study.Owing to cultivated cotton is allotetraploid, it is nessary to design At-or Dt-subgenome specific primers for the two fiber development related gene GhSusl, and Gh14-3-3L, in order to avoid disturbance of heteroduplex resulted from A and D duplicated genes. A total of50pairs of primers for these two development related gene were synthesized based on their genome sequences, of which only16can be used for EcoTILLING study.Using16pairs of primers above, the DNAs from TM-1and the remaining320samples of cotton germplasm were amplified by PCR, and then,10μL of PCR products from TM-1and equal volume of each other320samples, respectively, were mixed, denatured and annealed. From which,8μL of mixture were taken out to mix with1μL buffer and1μL CJE, and then keep them at42℃for40min for digestion. If SNP existed leading to heteroduplex, the digestion products would be detected by PAGE electrophoresis. The results have also been confirmed by sequencing. At last, a stable Ecotilling system suitable for high-throughput mining gene diversity of cotton have been established and can be used to discover desirable alleles.Using the Ecotilling technique established, diversities of the two fiber development-related candidate genes in320cotton germplasm resources were analysized and discovered4SNPs and9genotypes of the Susl gene in At subgenome,4SNPs and six genotypes in Dt subgenome;1SNPs and2genotypes of the14-3-3L gene in At subgenome, one SNPs and2genotypes in Dt subgeneome.The relationships between single marker and fiber quality traits were analysized by SPSS Statistics17.0software. The results showed that Susl genes has a close relationship with cotton fiber quality traits, the Susl gene in At subgenome, genotypes3,7,8showing longer, stronger, better fineness and uniform fiber than the genotypes1,2and4, and genotypes3and7having higher lint percentage and bigger boll weight, while in Dt subgenome, genotype4being better in all fiber traits. The14-3-3L gene in At subgenome, genotype1shows better fiber quality than genotype2, but there is no difference of fiber quality between two gentypes in Dt subgenome.Association analysis was performed by using the STRUCTRE software and the TASSEL software. The results indicated that there was highly significant association between SNP S1, S6and fiber length, fiber strength, micronaire and uniformity; S7, S8, S9and micronaire:significant association between S2and fiber length uniformity; S4and fiber strength; S5and boll weigh. The results could be further used for molecular breeding by design in cotton fiber quality improvement.