Introduction Of Aegilops Sharonensis HMW-GSs Into Wheat And Characterization Of Their Genetic Variation

The quality of Chinese endemic wheat is unreasonable in varied types, and the wheat with special-purpose and high quality is limitted. High-molecular-weight glutenin subunits (HMW-GSs) are one of the most important storage proteins in seed endosperm because of their significant effects on wheat processing quality. The lower ratios of HMW-GS associated with good quality have led to the poor flour in most wheat varieties in China. Therefore, identification and characterization of novel variant HMW-GS could benefit for exploring new genetic sources for wheat quality improvement. In our previous study, we cloned two novel HMW-GS genes from Aegilops sharonensis of Aegilops section Sitopsis, the cloned novel variants possess unusually large molecular weights which were regards as associated with good quality and could be used as special genetic resources for wheat quality improvement.In this study, we made up 62 cross combinations with Aegilops sharonensis as the pollen parent to hybridize with different varieties of tetraploid and hexaploid wheats, and 6 of them obtained hybrid seeds without using other assistive technology. The F1 hybrids were verified as intergeneric hybrids by their display of characteristics in morphology, cytology, microsatellite marker and SDS-PAGE profiles. In addition, the specific molecular marker of Ae. sharonensis HMW-GS and the genetic variations of HMW-GS in hybrids seeds have been developed and analyzed. The results are as follows:1. The average crossability of the F1 hybrids from 6 cross-combinations obtained in the years of 2011 and 2012 ranged from 0.86% to 4.1%. As female parent, the F1 hybrids were backcrossed with the commercial wheat varieties and the average crossability ranged from 3.82% tol9.23%. The F1 hybrid plants were morphologically intermediate between parents and had more tillers than those of either parent. The traits of plant height, number of spikelets per spike, length of flag leaf and length of awn on F1 hybrid plants were similar to those of female parent. However, the color of internode and leaf sheath, stem and floss in auricle were close to those of Ae. sharonensis. The shapes of seeds from Si and BC1 exhibited intermediate characteristics and similar to male wheat, respectively.2. Cytological observations on root tips indicated that the F1 (T. durum×Ae. sharonensis) and F1 (T. aestivum×Ae. sharonensis) hybrid plants were triploid (2n=21) and tetraploid (2n=28), respectively. Chromosome pairing at metaphase I in Fi and Si PMCs and the parental strains were counted, and the chromosome configuration and chiasma frequency of Fi (T. aestivum×Ae. sharonensis) was 2n= 25.941+1.03 II and 1.03, for F1 (T. durum×Ae. sharonensis) that was 2n= 15.44 I+2.78 II and 3.14. Whereas it was 2n= 1.72 I+20.14 II and 33.14 in S1 (T. durum×Ae. sharonensis), which indicated that S1 (T. durum×Ae. sharonensis) chromosomes had been successfully and spontaneously doubled.3. From a total of 898 pairs of SSR primers, eight were screened out and can be used to identify the presence of genes from Ae. sharonensis in the F1 hybrids. These eight pairs of primers are codominant for the Ssh genome of Ae. sharonensis and were amplified in the same bands in the F1 hybrids and Ae. sharonensis. Out of them, two can be used to identify the presence of the genome of Ae. sharonensis in hybrids of T. aestivum×Ae. sharonensis, four can be used in T. durumxAe. sharonensis, and the other two in T. dicocconxAe. sharonensis.4. The specific markers, R7XF1-R7XR1 and R7YF1-R7YR1, were designed based on the sequences of x-type and y-type HMW-GS gene of Ae. sharonensis. These two markers can be used to select the HMW-GS gene of Ae. sharonensis in all hybrids and backcross hybrids, and will be helpful to molecular marker-assisted selection for the strains with target gene.5. For the expression differences between the x-type HMW-GS of Ae. sharonensis in F1 and S1 seeds of T. durum×Ae. sharonensis, the alignments of nucleotides and amino acids sequences indicated that the mutations of insertions and deletions of repeat motifs led to that S1 has 33 residues less than F1.6. To be concluded, four types of genetic variations of HMW-GS from all hybrids seeds by SDS-PAGE were present:1) the seeds of cross and backcross progeny fully integrated the HMW-GS of their parents, and there are no variations in HMW-GS.2) In hybrids and backcross progenies seeds, the HMW-GS of Ae. sharonensis have some mutations, such as the variation of molecular weight and silent or lost of subunits.3) In hybrids and backcross progenies seeds, the HMW-GS of tetraploid and hexaploid wheat have some mutations, such as the variation of molecular weight and silent or lost of subunits.4) With the success of the hybrid between T. dicoccoides and Ae. sharonensis, the expression of certain subunit changed and an extra band appeared in F1 and S1 hybrids compared with parents and other BC1 seeds, this may be regulated by interaction of HMW-GSs gene.