The Ecological And Genetic Study Of A Thermo-Sensitive Self-incompatibility Line HE97 In Maize And Cloning Of A Related Gene

In this study, the ecological and genetic mechanism of a novel ecological self-incompatibility (SI) line, HE97, in maize was studied, and a self-compatible related gene was cloned and analysed.For validating the main environmental factors that affecting the characteristics of SI, the inbred line was identified in the field through sowing in different stages, and the leaf number was investigated in the field. The results showed that the sensitive period of self-compatible conversion for the ecological SI line was the floret differentiation of staminate inflorescence, and it was mainly affected by daily minimum temperature. The result proved that the ecological SI line, HE97, belonged to the thermo-sensitive genic self-incompatibility (TGSI) line. The temperature boundary of self-compatible conversion was 20-24°C. When the daily minimum temperature was below 20°C in the floret differentiation of staminate inflorescence, it exhibited complete self-incompatibility; whereas plants exhibited self-compatibility when the daily maximum temperature was higher 24°C, and the SI has not significant correlation with sunlight.A set of F2 and the corresponding F2:3 populations, which derived from two inbred line, HE97 and Z58, were evaluated for two years to dissect the inheritance of the TGSI line. There was one main QTL was detected with the seed setting rate of the F2 populations as the input data when using the comfindance interval mapping method, accounting for 21.37% self-incompatibility genotypic variance. This QTL was localized to chromosome 2 between the SSR markers umc1635 and nc133. This QTL was also detected in the F2:3 population, accounting for 16.8% self-incompatibility genotypic variance. Classical genetic analyses and QTL mapping results all revealed that the self-incompatibility of HE97 was governed by a single main recessive gene, named tgsi1. The tgsi1 gene was mapped to chromosome 2 between SSR markers nc131 and bnlg1633 using bulked segregation analysis (BSA) method, with a distance of 2.40 cM from nc131 and 2.44 cM from bnlg1633.A CIPKs-like gene, which has homologous to calcineurin B-like protein-interacting protein kinases (CIPKs) in Oryza sativa L., was cloned by rapid amplification of cDNA ends (RACE) and bioformatics(GenBank accession number: EU 424058). The full-length cDNA of the candidate gene was 1918bp and contained an 1332 bp open reading frame, which encoded 443aa, and the deduced amino acid sequence contained an acidic Ser/Thr rich region toward its N-terminus and a conserved NAF domain. The results of Blastn and Blanstx all showed that it had highly homologous to Oryza sativa, Populus trichocarpa and Persea americana. From the phylogenetic tree, we know the CIPKs-like gene in maize has tightly homologous to Oryza sativa.The Real time RT-PCR method was used to analyze the expression of the CIPKs-like gene in various tissues of the TGSI line between self-incompatible and self-compatible stages. The result showed that the gene had high expression quantity in self-incompatible pollen, and there was little difference between self-compatible silk and self-incompatible silk. The candidate gene CIPKs-like which had high expression quantity in the self-incompatible pollen of the TGSI line, urged the Ca2+ concentration in the self-incompatible pollen and prevented pollen germination in the silk. These results demonstrated that the CIPKs-like gene played an important role in regulation of the characteristics of the TGSI line in maize.