Analysis Of Oilseed Rape Flowering Time Variation And Cloning Of Arabidopsis Late Flowering Time Gene

Flowering is an important reproduction and development characteristic of oilseed rape. Flowering time is controlled in a multiple gene form with locus variation between different varieties and affected by environment factors. Studies on flowering time may help breed varieties suitable to crop rotation and manipulate pollination control systems in hybrid breeding and production.In this paper 78 oilseed rape lines with different flowering time were studied in Wuhan and Xining in 2003-2005 and molecular polymorphisms were detected using AFLP technique. The results were obtained as follows:1. The flowering time variation of 78 lines was significant (P<0.05) between years in Wuhan, but not inXining. The significant variation between years in Wuhan could be caused by temperature differences between two years based on analysis of environment factors (daily mean temperature, accumulative temperature, daily sunlight hours and total sunlight hours).2. Path analysis showed that effective accumulative temperature from planting to flowering was the majorfactor which affected flowering time in Wuhan and the others environment factors affected flowering time through the factor.3. Twenty two pairs of selected primer combinations were used for AFLP analysis of 78 oilseed rape lines.There were total 1323 bands amplified and average 53 bands per primer pair were amplified. By ANOVA, 137 AFLP bands were found to correlate with flowering time in Wuhan and Xining in 2003-2005. The clustering analysis of 78 oilseed rapes lines divided these lines into 5 groups: the first group was those flowering from January to early February;the second group was only one line which had the least number of bands;the third group was those flowering from 10 to 25 of February;the fourth group was those flowering from late February to March;and the fifth group was those flowering from March to April and all were B.juncea and B.rapa. The grouping was consistent with that based on flowering time.The second part in the paper is to screen Arabidopsis mutants for flowering time and clone corresponding genes. There were about 51 660 T-DNA insertion mutants of the T3 generation screened and from them 4 mutants with late flowering time were identified. One of the 4 mutants, At2-4, was further studied as follows:1. Flowering time (from sowing to flowering) changes of the line derived from the At2-4 mutant was from 34 to 72 d while that of wild type from 30 to 33. (called as normal flowering time). In the line, late flowering time: normal flowering time = 3:1 based on the Chi square test. Positive plants: negative plants of PCR detection = 3:1 based on the Chi square test. Further Southern blot indicated that T-DNA insert was a single copy. These confirmed At2-4 was heterozygous with a single locus mutation.2. A target fragment was amplified using TAIL-PCR after a series of primer combinations were tested.The fragment length is 798 bp positioned in the left side of T-DNA in the Arabidopsis genome.3. Bioinformatic analysis indicated that the T-DNA inserted into gene AT5G24290 of Arabidopsis andsimilarity between the 798 bp and AT5G24290 was 100%. AT5G24290 had a putative function of integral membrane protein, but we did not find any gene functions reported experimentally for all plant DNA sequences searched with similarity against the 798 bp. However, the 798 bp has 63.9% similarity with yeast gene CCC1 which regulates Ca2+ levels of cells, therefore we predicted AT5G24290 could play function in delaying flowering through the Ca2+ signalling pathway or by regulating cell Ca2+ levels. It is a first report that the function of a gene controlling flowering time is related to Ca2+. The further experimental confirmation is under way.