Genetic Characteristics And Genomic Analysis Of Somatic Hybrid Offspring Of Wheat And Agropyron Elongatum

There were few systematic studies on the heredity theory of somatic hybridization, although lots of works upon somatic hybridization have been done since the first somatic hybrid plants were regenerated in 1972. In our laboratory, various progenies from asymmetric somatic hybrid were obtained via protoplast fusion between wheat cv. Jinan177 and Thinpyrum ponticum (Podp.) Barkworth&Dewey, The protoplast of Th. ponticum with superior quality and salt-tolerance was UV- treated. In order to understand the traits and heredity and the genome compositions of some hybrid progenies, three inbred lines: â…¡-2, â…¡ - â…  -8 (F₂^F₆) and 8-1 (F₃^F₆) ( segregated from F₂â…¡ - â…  -8 ), which were derived from a same asymmetric somatic hybrid cell, were chosen to be the typical materials studied. The analysis for morphological and agronomical traits, components of HMW (high molecular weight) glutenin subunit, cytology, salt-tolerance, GISH (Genome in situ hybridization), FISH (Fluorescent in situ hybridization) for 18s -5.8s-26s rDNA loci, RAPD (Random amplified polymorphic DNA) as well as SSRs (simple sequence repeats) for nuclear and chloroplast genomes were preformed. The results were as following:I . The basic characteristics of somatic hybrid lilies 1. Observation of morphological and agronomical traitsDuring propagation, the three lines showed no obvious changes in basic morphological traits in all of the lines from F₂ (F₃) to F₆ : â…¡-2 had taller stems (80 cm on average) with largest ears and grains, and exhibited potential for enhanced yield; â…¡ -â…  -8 had shortest stems (55 cm on average) with a strong tillering ability, medium ears and grains; and 8-1 had about 80 cm stems and the ear size in between.2. SDS-PAGE analysis for HMW gluteninThe SDS-PAGE was employed to analyze the subunit components of HMW glutenin and the results obtained were that the hybrid lines â…¡-2 and 8-1 have the same patterns of HMW glutenin subunits ("2+12" and "7+9"), while the â…¡-â…  -8 possessed the significant pattern of HMW glutenin subunits ("5+12" and "13+16") of wheat-like, which contributed tothe high quality of this line. There were no changes in the composition of HMW glutenin subunits during propagation.3. Analysis for salt-toleranceIn the hyponic salt-tolerance tests of two generations, such items as morphology, growth and other physiologies (contents of Na+, K+, free proline and Na+/K+ratios) showed that the somatic hybrid lines II -2, II - I -8 exhibited stronger salt-tolerance than parental wheat. Two generations of somatic hybrid lines were cultivated in the natural saline soils of Dongying and Dezhou in Shandong province, compared with the native cultivated salt-tolerance cultivars (Dekang 961). The results showed that the two hybrid lines especially the II -2 exhibited obvious salt-tolerance.II. Cytological analysis1. The chromosome numbers and karyotypes of hybrid lines and their parental wheat cv. Jinan 177The chromosome number of root tips in F5 hybrid lines was counted. All of the three hybrid lines possessed 40—42chromosomes. The karyotype analysis showed that some differences existed between hybrid and parental wheat, also among the hybrid lines. The chromosome structure changes occurred mainly in B group in hybrids2. Analysis for number and behavior of chromosome in the pollen mother cell (PMC) Over 88% of PMC possessed normal numbers of chromosome (2n=20H ²1 II). Highfrequency of bivalent( II) showed a normal behavior of chromosome in hybrids during meiosis.III. GISH and FISH analysis for hybrid F5 1. GISH analysisUsing the total DNA of Th ponticum as probe, the results of GISH in the three hybrid lines showed that the chromatin fragments instead of entire chromosome or entire chromosomal arm of Th ponticum existed in a few of chromosomes of all three hybrid lines.The Th. ponticum chromatin fragments were inserted into different loci in different hybrid lines.2. FISH analysis for 18S-5.8S-26SrDNA lociUsing the/?Ta 71 plastid (contained 18S-5.8S-26SrDNA coding repeated sequences) as probe, the results of FISH were as following: the parental wheat possessed four pairs of NORs loci including IB (located in satellite region), 6B (located in satellite region), 1A (located in the terminal region of short arm) and 5D (located in the terminal region of short arm); Th. ponticum possessed ten pairs of terminal NORsloci; while all of the three hybrid lines lost one terminal region locus. The results indicated that the three hybrid lines were derived from the same somatic hybrid cell.IV. RAPD analysis for nuclear genomeVisible amplified bands were obtained from 45 of the 60 random primers selected. Of them, stable polymorphism bands were only detected in 7 primers, but only three of them amplified different Th ponticum specific bands in different somatic hybrid lines. The amplified bands of other primers were most like parental wheat except for a few different bands among the three hybrid lines. RAPD assay in different generations showed that somatic hybrid lines were relatively stable on most loci in different generations. Although not so many primers have been used, in combination with other results analyzed from these hybrid lines, it suggested that changes might only happen in a few loci during generation passage.V. SSRs analysis for Chloroplast genomeThere were consistent amplified bands in the 3 hybrid lines of Fe on the 7 sites selected, of which both parents and the 3 hybrid lines all exhibit consistency on the sites of WCt6 (trnC-rpoB), WCt7 (ropC2), WCtll (atpF) and WO20 (in/A). The band patterns from the 3 hybrid lines were consistent with parental wheat on sites of WQ9 (atpI-atpH) and WCtl3 (trnF-ndhJ). Amplification band profiles of the three hybrid lines were from both parents on the site of WCt23 (rpll4-rpll6). Results of F2, F4 and F6 were consistent with the WCt23 {rpll4-rpU6) primer screened. It is suggested that integration and rearrangement occurred inthis locus since there were no changes on the other three loci with biparental polymorphismVI. SSRs analysis for nuclear genomeThe 182 primers (191 loci screened) were employed to test the genomes of three hybrid lines (F2, and Fe) and their parents (wheat cv. Jinanl77 and Th. ponticum),.as well as wheat calli mixed with different times of subculture. The results showed that there were significant differences among hybrid lines. Different numbers of Th. ponticum DNA fragments were inserted into different loci among the different hybrid lines. Compared with the amplification profiles of mixed calli, almost all of the missing loci either were replaced by Th. ponticum DNA fragments or occurred rearrangement of biparental DNA in the hybrid lines, which may be the hypervanation region in wheat genome. It was suggested that there were several hypervariation regions (or hot spots) in the wheat genome. These hypervanation regions may be lost easily during the process of tissue culture and became the hot spot attacked easily by alien DNA fragments or changed through DNA integration and rearrangement of both parents. This work demonstrated that there were more hypervariation loci existed in the genome of wheat than in that of Arabidopsis thaliana. This was related to the magnificent genome (2n=6x=42) of wheat with 3 homoeologous genomes of A, B and D, which make it possible to possess better tolerance or plastic than that of A. thaliana with single and small genomes. It is the first report that the SSRs were used to analyze the hypervariation regions of wheat. The hybrid plant regeneration via complementation of both parent genomes in somatic hybridization of wheat with grass was reported. But the genetic mechanism of which was still not known. Upon this result, it suggested that it might be related to the chromatin inserted into the lost loci of wheat culture cell, and then the following diploidization of hybrid genome by further exclusion of donor DNA and also the rearrangement of biparental DNA.The hybrid II -2 and II - I -8 lines were derived from one cell, but there were obviously differents in traits and heredity, which indicated that their genomes are not stable in the early term of hybrid cell culture and regeneration. According to the analysis upon several generations, these hybrid lines became stable in sexual generation from F2U) F6,e.g. Stable...