Genetic Diversity Analysis Of The Granules Associated Protein In Triticum L. And Aegilops L. Species

Wheat starch is one of the main constituent of wheat grain, and also plays an key role in wheat yield and quality. Starch synthesis is mainly controled by a series of the enzymes. Starch Granule Associated Proteins (SGAPs) is a kind of more conservative enzymes, and function through combining indirectly and directly with starch granules in the process of starch synthesis. SGAPs includes granule-bound starch synthase (GBSS Ⅰ), soluble starch synthase (SSS), and starch branching enzyme (SBE). In our present study, the genetic diversity of starch granule associated proteins (SGAPs), were estimated in 563 accessions from Triticum L. and 43 accessions from Aegilops L. by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Moreover, starch synthase geue Ⅱ (SSⅡ) gene was also cloned in some accessions, the main results are as follows:1. Starch granule associated proteins (SGAPs) of 10 accessions from Ae.tauschii, T. urartu, T. turgidum, T. aestivum and Chinese Spring nullitetrasomic lines, were investigated by SDS-PAGE. The results showed that eight protein bands had been identified in all materials. The bands SGP-1B and SGP-1D were observed, and SGP-1A was not at SGP-1 locus in T. aestivum and T. turgidum. A single band from Sichuan landrace, Chengduguangtou, was identified as SGP-1D through MALDI-TOF-TOF. Therefore, our results suggested that the method of extraction and electrophoresis of SGAPs were reliable and workable, and they could be applied in the genetic diversity evaluation of starch granule associatied proteins in Triticum L. and Aegilops L.2. The results of starch granule associated proteins (SGAPs) of 453 hexaploid wheat accessions by SDS-PAGE, showed that 10 different protein bands and 5 combinations were identified. The null band of SGP-1 A was detected in 248 new wheat cultivars (lines),21 wheat founder parents, and 9 synthetic hexaploid wheat, but not in Sichuan wheat landraces, T. sphaerococcum, T. compactum, and T. macha. We found that three cultivars (lines),06-7299, Sumai375, and SW14945, had the null SGP-2 band, whereas five accessions,2012B73, Ningchun43, PI40941, PI78639, and PI428148, had the null allele of Wx-IA. Moreover,44 and 27 accessions had the null alleles of Wx-1B and Wx-1D, respectively. The genetic diversity of starch granule associated proteins, were not detected in Sichuan wheat landraces. On the other hand, wheat varieties (lines) showed abundant variation than other hexaploid wheat materials. Our study strongly suggested that there is the genetic diversity of starch granules associated proteins in hexaploid wheat, and the materials with new alleles could be applied in wheat starch quality breeding.3. The SGAPs of 67 tetraloid wheat accessions from T, turanicum, T. polonicum, T. turgidum, T. carthlicum, T. dicoccum, T. durum, and T. dicoccoides, respectively, were investigated by SDS-PAGE. The results showed that the null SGP-1A band was observed in all materials, whereas there was not different bands among tetraploid wheat accessions. Our result suggested that starch granule associated proteins in tetraploid wheat had more conservative and less genetic diversity.4. The SGAPs of 39 diploid wheat accessions from T. urartu, T. Monococcum, and T. boeoticum, respectively, were detected by SDS-PAGE, and indicated that SGP-1A protein in T. urartu showed the same mobility with T. aestivum (Chinese Spring), but faster mobility than T. monococcum and T boeoticum. No difference of bands was observed for other SGAPs proteins in diploid wheat accessions, suggested that there was lower genetic diversity of SGAPs in diploid wheat.5. The SGAPs of 43 Aegilops accessions from Ae.tauschii, Ae.speltoides, Ae.longissima, Ae.sharonensis, Ae.searsii and Ae. Bicornis, respectively, were detected by SDS-PAGE. Five different protein bands were observed. The bands exhibited the same mobility and pattern within species, but was detected different mobility among species. The SGP-1 protein in Ae.tauschii showed the same mobility with T. Aestivum, whereas, this protein in other Aegilops species, such as Ae.longissima, Ae sharonensis, Ae.searsii, and Ae.bicornis, showed slower mobility than in T. aestivum. Our present study suggested that Aegilops speceis have the different gene resouces of SGAPs, compared with Triticum L. species.6. Using three pair of SSⅡ gene specific primers (F2/R2, F3/R3, and F4/R4),10 sequences about 1277bp,8 sequences about 904bp, and 7 sequences about 2150bp, were obtained by cloning from 11 accessions. Sequences alignment showed that Agelopsis species had the insert fragments with 103 bp in the fifth intron and 130 bp in the seventh intron, respectively, and deletion in the seventh intron around the domain of 259-283 bp and 468-531 bp, compared with Triticum species. Further sequences alignment of F2/R2 fragment indicated that 12 single nucleotide locus change in exon domain and resulted in five sense mutant. Cluster analysis showed that all sequences could be clustered into three groups. The sequences of SSⅡ gene from Barley, Aegilops and Triticum were clustered into group Ⅰ. The SSⅡ genes from rice were clustered into group Ⅱ. The group Ⅲ was composed of the sequences from maize and sorghum. In group Ⅰ, three sequences from T. boeoticum were clustered respectively into different sub-group. Our results suggested that SSII gene sequences had higher similarity and more close relationship between diploid species of Triticum and Aegilops, but they also displayed clear difference of SSII gene comparing with other species.