Comparative Analysis Of Tb1 Gene And 22kD Prolamin Gene Family In The Genera Zea

Teosinte is the most related wild ancestor of modern maize. But the architectures of maize and teosinte are strikingly different. However, how the ancient agriculturists domesticate maize into teosinte in such a short time was still a mystery. In this study, we choose the major domestication gene, teosinte branched1 and the important storage protein gene family, 22kD prolamin gene family, and carry on comparative analysis to study the evolution and domestication of modern maize.Teosinte branched1 encoded a TCP family transcriptional factor and controls maize basal branching and ear phenotype. In maize, tb1 has a relatively high expression level in axillary meristems and stamens of ear primordial thus suppress the development of corresponding organs. Previous studies revealed the domestication region located 58-69kb upstream of tb1. 58-64kb upstream region controls the ear phenotype while 64-69kb upstream region controls the basal branching. But the exact sequences that alter tb1 expression remain unknown.In this study, we use comparative genomics and association analysis to figure out the causative mutation for tb1. We analyzed the conserved noncoding sequences (CNSs) of tb1 upstream region among maize, sorghum, rice and brachypodium. We found most CNSs located 58-72kb upstream of tb1, suggesting this region might be important for tb1 regulation. Then we isolated and sequenced the tb1 region of Zea mays ssp. parviglumis, Zea diploperennis, maize haplotype W22 and Yu87-1. Comparative analysis revealed that PVG was highly conserved with maize, while Zea diploperennis show much less conservation mostly due to retroelements insertions. We find the major difference between maize and teosinte that might cause tb1 mutation were one LTR-retrotransposon and one MITE insertion in maize tb1 regulatory region (58-69kb upstream of tb1 coding region). We detected the presence of these two transposable elements in 589 maize haplotypes and 189 teosinte accessions. The results revealed the LTR-retrotransposon and MITE were strictly associated in maize. Finally, we sequenced tb1 regulatory region in some other maize haplotypes and teosinte accessions. Sequence alignments exclude all the SNPs or InDels that might be candidate locus for tb1 mutation except for the two TEs. We concluded that the LTR retrotransposon and MITE insertions upregulate tb1 expression in axillary meristems and stamens of ear primordial, thus lead to the domestication of tb1.22kD prolamin was the major storage protein in maize kernel. It was encoded by a gene family and was a good model to study the evolution of gene family. It is composed of one gene cluster and one single gene (fl2) in maize genome. Our lab previously compared the 22kD prolamin gene cluster among maize sorghum and coix. We also predicted the formation and evolution history of the gene cluster. In this study, we isolated 22 BAC clones in Zea mays ssp. parviglumis and 40 clones in diploperennis that containing 22kD prolamin gene. According to the fingerprints, DNA blotting and flanking marker screening, we predicted the possible organization of 22kD prolamin gene cluster in teosinte genome. We found two 22kD prolamin gene cluster with flanking orthologous markers in both Zea mays ssp. parviglumis and Zea diploperennis. The gene cluster extended from 200kb to 300kb. We sequenced 5 BAC clones of Zea diploperennis. We analyzed the fl2 region between maize and Zea diploperennis. They only shared 28% homologous sequences. Like in the tb1 region, LTR retrotransposon insertion and following recombination are the major cause of variation.This study revealed that the domestication of maize tb1 gene was the result of two transposable elements insertion events. Transposable elements not only expanded the maize genome, but also had great impact on gene regulation. Through isolation and identification of 22kD prolamin gene family, we predicted the organization of gene cluster, facilitated the further study of 22kD prolamin gene evolution in the genera Zea. We isolated and sequenced teosinte large genomic sequences and compared with maize for first time. This study enhanced our knowledge of genome composition and evolution of the genera Zea.