Using Suppression Subtractive Hybridization To Find Apomixis Related Genes

Among a set of monosomic addition lines of Beta corolliflora Zoss in sugar beet, a line, designated as M14, that constitutes of the normal 18 B. vulgaris chromosomes plus B. corolliflora chromosome 9, was found to have a chromosome transmission frequency up to 97% through our investigation in successive four generations. According to the results of genetics and embryology of M14, suppression subtractive hybridization (SSH) was taken to set up subtractive cDNA libraries in three key periods of M14 development including forward and reverse subtractive libraries of meiosis period, forward subtractive library in synergid pre-degeneration period, and also including forward subtractive library in autotomization period of egg cell and secondary nuclear of central cell. 276 recombinant clones were obtained from forward subtractive cDNA library of meiosis period, 229 of which were positive insertional fragments with the average sizes of 100~1000 bp by PCR identification. Then, 43 clones which were randomly selected from 229 clones were made reverse Northern Blot hybridization screening using M14 cDNA and diploid cDNA as probes with DIG-labeled. As a result, 24 fragments differentially expressed in M14 were obtained. These fragments provides a reliable source and guarantee to clone the correlative genes of apomixis.