| Using the specific primers,39sequences of5’untranslated region (5’UTR) of Waxy genes were obtained from33accessions of Triticum L. and Aegilops L. species by PCR cloning. All sequences together with other42sequences of Waxy genes from30Triticum L. and Aegilops L. species and8sequences of Waxy genes from Hordeum vulgare L., Oryza sativa L., Zea mays L., Solanum tuberosum L.and Arabidopsis thaliana L. from NCBI, were estimated through analysising of polymorphism of Restriction enzyme site, single nucleotide polymorphisms (SNPs), simple sequence repeat, Indels, and uORFs. In order to investigating the relationship between species, types, and sequences, neighbour-joining trees and Population analysis were also performed. Our results provided further information to understand the functional mechanism and regulation of Waxy gene, and deeply utilitize the gene resource of Triticum L. and Aegilops L. species in wheat improvement. The main results are summried as followed:1. Using the specific primers, only one band was amplified from diploid wheat, tetraploid wheat, and Aegilops L. species, whereas two bands from hexaploid wheat. A total of39sequences were obtained after cloning and sequencing of all bands. The length of sequences from7D locus are longer than those from7A and4A loci. Based on the sequence variation, all the sequences of tetraploid and hexaploid wheat could be grouped into Type A and Type B from tetraploid wheat with genome AABB; Type Ⅰ and Type Ⅱ from hexaploid wheat with genome AABB; Type GⅠ and Type GⅡ from wheat with genome AAGG and AAAAGG.2. These sequences of Triticum L. and Aegilops L. species contained170singleton variable sites and484polymorphic. There was abundant variation of Indels, and the average length of Indels was8.5bp in all sequnences. The restriction enzyme site Pst I was detected at the end of all seuqnces of5’terminal of Waxy gene. The concentrated region of restriction enzyme site was located between250bp to350bp. The type and distribution of restriction enzyme site in Triticum L. and Aegilops L. was similar with barley, maize, and rice, and different with Arabidopsis and potato. The variation of two simple sequence repeats,"GAA" and "CTGA", was observed in all sequences. A total of65uORFs were detected and could be classed into37types, longth from66bp to546bp, with the variation in uORFs mainly due to single nucleotide polymorphisms (SNPs), and also to the presence of Indels.3. The comparision of restriction enzyme sites, Indel and uORFs among sequences, indicated that Type I was more similar to Ae. tauschii, whereas Type II was more similar to Type B, and both of them were more similar to Ae. longissima. Interestingly, Type A was more similar to Ae. speltoides. On the other hand, comparied with the diploid wheat, the sequence of Waxy gene5’UTR of tetraploid and hexaploid could be keep the conservative region from ancestors.4. Population analysis of sequence type and diploid further confirm that T. boeoticum was very similar to T. monococcum and both similar to T. urartu, Ae. tauschii was very similar to Type I and Type B similar to Type Ⅱ, while Ae. speltoides was similar to Type A. Otherwise, the different relationship between tetraploid and hexaploid was observed in neighbour-joining trees, implied that only partial tetraploid could join the process of polyploidization of hexaploid wheat. The ancestors of T. urartu may provided the AA genome of tetraploid wheat(AAGG);The ancestors of Ae. speltoides, Ae. longissima and Ae. tauschii, could provided respectively the basic genomes of hexaploid, whereas Ae. longissima laterly join the process of hexpaloid appearance, compared with Ae. speltoides. The BLAST cluster of5’UTR of Waxy gene in NCBI, demonstrated that the sequences from rice and maize were clusted into one group, and diploid wheat was one group, which also contained sorghum and partial maize. Tetraploid and hexaploid wheat were clusted into one group. In addition, barley was one group, which also contained partial Aegilops L. sequences. |
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