Genome-wide Identification And Evolutionary Analyse Of Flowering-time Genes In Brassica Species

Flowering is an important transition from vegetative growth to reproductive growth of higher plants, which influence the timing of duration period, the pollination and the seed development. Researches on model plant Arabidopsis showed that there are at least 4 pathways controlling the flowering time:the photoperiod pathway, the vernalization pathway, the autonomous pathway and the gibberellin pathway. Brassica napus L., one sibling specie of Arabidopsis thaliana, was allotetraploid obtained by natural crossing of the Brassica rapa (AA,2n=2x=20) and Brassica oleracea (CC,2n=2x=18) followed by chromosome reduplication. Recent comparative genomics researches on Arabidopsis and Brassica plants found that the differentiation happened 20 million years ago. Moreover, ancestors of Brassica plants tripled their genomics about 16 million years ago, in other words, the diplontic modern Brassica rapa and Brassica oleracea are evolutive from the hexaploid ancestor. Analysis on the genomes of Brassica rapa and Brassica oleracea showed that there were plenty of genes lost during the triploid process; Meanwhile, researches on lots of plants showed that some special kinds of genes lost relatively less and multi-copy genes were reserved. So, what about the flowering-time genes from Brassica plants after the genome triploid? Are they reserved prior? Do the multi-copy genes have the expression differentiation? This study compared flowering-time genes from Brassica rapa, Brassica oleracea and Brassica napus (unpublished) and analyzed the data. Moreover, CONSTANS gene (CO) in the photoperiod pathway was analyzed, and the expression level of CO in different duration period, timing and tissues are determined respectively. The main results contain:1. The identification and the evolution of the flowering-time genes on the genome level of Brassica rapa, Brassica oleracea and Brassica napus1.1 The identification of the flowering-tune genes on the genome levelAccording to previous studies, we collected 211 flowering-time genes in Arabidopsis thaliana.And according to the gene domain,383 flowering-time genes were identified from B.rapa,385 flowering-time genes from B.oleracea and 667 flowering-time genes from Brassica napus respectively. The flowering-time genes in B. rapa were 1.95 fold of Arabidopsis thaliana, while they were 2.01 in B. oleracea and 3.30 in Brassica napus.These results indicated that from Arabidopsis thaliana to Brassica rapa, Brassica oleracea then Brassica napus,Some flowering-time genes have been lost in the triplicated process of the genome. The retention of 2 or 3 paralogous is far more than genome, and this indicated these genes is important in the evolutional process of species.1.2 The distribution of the flowering-time genes in Brassica rapa and Brassica oleracea73%-80% of the assembled scaffolds on Brassica rapa and Brassica oleracea genome were anchored onto the genetic map, which formed 10 and 9 pseudo-chromosomes respectively. It was found in this study that 373 of 383 flowering-time genes of B.rapa were located on 10 chromosomes; while 312 of 385 flowering-time genes of B. oleracea were located on 9 chromosomes. And it was obvious that chromosome 3 had the most genes in both B. rapa and B. oleracea. Most of the genes on Arabidopsis, Brassica rapa and Brassica oleracea genome were divided in 24 blocks (A to X) according syntenic analysis between spices. And 314 genes of B. rapa and 307 genes of B.oleracea distributed on 24 different blocks. The distributions of these genes on Block U, R and J were more than other blocks. These results implied the flowering-time genes were conservative and necessary in evolutional process.1.3 Gene Ontology and GO term enrichmentThere are 85 categories of GO terms of flowering-time genes in Brassica rapa (933 GO terms),93 categories of GO terms in Brassica oleracea (978 GO terms),83 categories of GO terms in Brassica napus (2022 GO terms) involving cellular component, biological process and molecular functions three main areas.Analysis on GO enrichment shown that regulation of transcription, DNA-dependent, transcription factor activity, nucleus and lyase activity were enriched. And the richment of genes function is same in Brassica rapa, Brassica oleracea and Brassica napus.1.4 Analysis on structural variation of the reserved multi-copy genesStructural variations between paralogous were observed in Brassica rapa and Brassica oleracea, the differences showed up in the numbers of exons and domains. And the expression were also different. Structural variations were also observed between paralogous whose GO term have differences. It was showed that structural variation happened in multi-copy genes and they may be evolutively new genes with important functions.1.5 Phylogenetic analysis of flowering-time genesKa,ks and Ka/ks between Arabidopsis and Brassica rapa.Brassica oleracea indicated that flowering-time genes were selected negatively during evolutional process. The four metabolic pathways regulated flowering time were selected by different pressure.But the dfference was not significantly. Evolution regularities of the two were consistent with each other.2. The expression differentiation and evolution of flowering-time genes from Brassica rapa and Brassica oleracea375 of the 383 flowering-time genes were found expressing in at least one of the six tissues in Brassica rap; while 373 of the 385 genes were found expressing in at least one of the six tissues in Brassica oleracea. And the expression showed the tissue specificity and the functional differentiation and there were three types of expression models: single-copy mainly expression model, multi-copies co-expression model and complementary expression model.Alternative splicing (AS) differentiations was one of main forms of the expressing differentiations between the paralogous. This variation was observed in Brassica rapa and Brassica oleracea. There have 2-3 ASs per flowering-time gene. According these results we inferred there was functional differentiation between paralogous.3. The cloning and expressing analysis of CONSTANS gene from B. napusOne CO homologous was cloned from early line and late line named A-CQ-1and B-CO-1 respectively, they have 1101bp CDS sequences. The result of sequencing analysis was shown that they have a high homology with the known CO gene. There have differences in three nucleotide bases and one amino acid site between A-CO-1 and B-CO-1, cloned from early line and late line respectively. Whether the differences can affect the gene function, we need further studies.Expression analysis of CO gene in different tissues:there were high expression levels in bolting period and initial flowering period, so we inferred that CO gene related to the flowering in Brassica napus L.. The highest relative expression levels of CO gene in different tissues show that the early line was higher than the late line.Expression analysis of CO gene in different growth period:The highest expression was detected at leaf In squaring period, bolting period Ⅱ,initial flowering period and flowering end period respectively;In bolting period Ⅰ,the highest expression was detected at leaf in line D125-5,but at bud in early line D626-6; In full-blossom period, the highest expression was detected at leaf in the early line D626-6, but at bud in the late line D125-5; A highest CO gene expression detected at leaf in all growth period maybe because the leaf was a photosensitive parts.The highest relative expression of CO gene was detected in different growth period both of early line and late line in AM or PM.Expression analysis of CO gene in different materials:At AM of bolting period Ⅱ (before the flowering) and initial flowering period, the early line D626-6 has a significant highest CO gene expression than the late line in leaf; At NOON, the early line D626-6 has a higher expression than the late line D125-5; But, at PM, the early line D626-6 has a significant lowest expression than the late line D125-5.