Cytogenetic And Molecular Identification Of High-molecular-weight Glutenin Subunit Glu-R1 Variation In Wheat-Rye Hybrid

Wide hybridization plays a crucial role in evolution and speciation of species, and has been considered as an important tool for crop improvement. Rye (Secale cereale L.,2n= 2x= 14, RR), the tertiary gene pool of wheat, harbors a plethora of valuable genes for wheat breeding. Hybridization of wheat with rye might not only introduce the elite rye gene pool into common wheat but also induce novel genetic variation in hybrids and many of that are potential valuable. Therefore, the study of these genetic variations induced by wide hybridization is very important for wheat improvement.High-molecular-weight glutenin subunits (HMW-GS) are one class of seed storage proteins synthesized in developing wheat endosperm. The amount and composition of these proteins are important determinants of the processing and bread-making quality of wheat flour, and has been regarded as the important quality and agronomic traits of wheat.In the current study, we investigated the HMW-GS variation hybrids, derived from a hybridization of common wheat Shinchunaga (Triticum aestivum L. cv. Shinchunaga) with Qinling rye (S. cereale L. cv. Qinling), using SDS-PAGE, cytological analysis, gene cloning, and prokaryotic expression. The results are as follows:1. The SDS-PAGE analysis revealed that the subunits composition of HMW-GS in F1 hybrid 100 belong to a wild type. Two different subunits composition of HMW-GS were identified in F5 seeds. One was wild type. Another was mutant type that absent one rye HMW-GS subunit. Both of them can be transmitted into F6 and F7 seeds by selfing.2. The chromosome composition of root-tip cells in F7 progenies which derived from the selfed seeds of Felines 100-5 (wild type) and 100-8 (mutant type) were studied. The cytological analysis showed that 100-5-1 (F7) had 42 chromosomes, including 14 rye chromosomes,11 A genome chromosomes (loss of one 7A and a pair of 1A chromosomes), 14 B genome chromosomes (including a pair of 4BL.6BS/6BL.4BS reciprocal translocation chromosomes), and three D genome chromosomes (one 7D and a pair of 1D). The offspring of 100-8 showed two different types:(i) the 100-8-1 (F7) had 42 chromosomes, including 14 rye chromosomes,12 A genome chromosomes (missing a pair of 1A chromosomes),14 B genome chromosomes, and two D genome chromosomes (a pair of 1D chromosomes); (ii) the 100-8-2 (F7) had 40 chromosomes, including 14 rye chromosomes,11 A genome chromosomes(loss of one 7A and a pair of 1A chromosomes), 13 B genome chromosomes (loss one of 4B chromosome), and two D genome chromosomes (a pair of 1D chromosomes). Our results indicated that these materials were secondary hexaploid triticale.3. The coding regions of Glu-Rx and Glu-Ry were amplified from gDNA of F1 hybrids 100, and F6 plants 100-5 and 100-8 using two pairs of gene specific primers. The results showed that Glu-Rx specific bands can be amplified from 100,100-5 and 100-8. Cloning and sequencing analysis indicated that all of them were same and 2304 base pair (bp) in length. Sequence alignment with the Glu-Rx in a wheat translocation line 7841 (GenBank number:AF216868) showed that the sequence identity is 99.91%, and only two variable sites at the position 777 nucleotide and 1130 nucleotide were identified. However, the specific products for Glu-Ry can be only amplified in 100 and 100-5, suggesting that the disappeared rye HMW-GS subunit in 100-8 might be the Ry subunit. Sequences analysis revealed that both of the Glu-Ry sequences were complete identical, the full length was 2286bp. Sequence alignment with the Glu-Ry in Qinling rye (GenBank number: GU373814) found that the similarity is 96.96%, and 34 SNPs as well as a 18bp insertion at position 1339-1356 nucleotide, and a 18bp deletion at position 2013-2030 nucleotide were detected. Both Glu-Rx and Glu-Ry have an entire open reading frame (ORF) and could be active HMW-GS genes. The deduced amino acid sequences of Glu-Rx and Glu-Ry exhibited typical structure similar to HMW-GSs with a predicted molecular weight of 81387.8Da and 81794.2Da, respectively. Based on the different molecular weight of Glu-Rx and Glu-Ry, we assume that the electrophoretic mobility of Ry subunit will be slower than that of Rx subunit in SDS-PAGE system.4. Glu-Rx and Glu-Ry isolated from 100-5 were successfully expressed in E. coli. SDS-PAGE analysis indicated that the electrophoretic mobility of Ry subunit was slower than that of Rx subunit. These results further confirmed that the rye HMW-GS subunit absent in 100-8 is Ry subunit.