Construction Of Radiation Hybrid Panel/Map Of Common Wheat Using Asymmetric Somatic Hybridization

Asymmetric somatic hybridization by protoplast fusion has become a successful means to transfer donor genetic materials into recipient and create hybrids across sexual borders. Many intra- and inter-specific, intergeneric, intertribal, and even inter-familial somatic hybrid plants have been obtained. Whole Genome Radiation Hybrid (WGRH) mapping via asymmetric somatic hybridization has played a key role in gene mapping and genome analysis of mammalian species. The presence or absence of markers in a panel of hybrid cells provides a direct way of ordering loci at a resolution not easily obtained by other mapping methods. WGRH panels have now been used to create such maps in a number of model and other species, e.g. human, rat, mouse, horse etc. However, there are a few reports on the research of radiation hybrid panel and map in plant, which has been interested in plant with big genome (including common wheat) in the world.In the present study, somatic hybridizations between common wheat (Triticum aestivum L. c.v. Jinan 177) and Bupleurum scorzonerifolium Willd were carried out. We investigated the possibility of the utilization of wheat/Bupleurum scorzonerifolium asymmetric somatic hybrids for the construction of radiation hybrid panel and map. And then we compared the effects of ultraviolet light (UV) withγray on the genetic constitution of radiation hybrids of wheat. Based on the results mentioned above, two wheat WGRH panels with different numbers of hybrid lines were constructed. Calculating the retention frequencies of molecular markers in two radiation hybrid panels respectively, we constructed a comprehensive radiation hybrid map of wheat chromosome 5 A, located ESTs of three unigenes of wheat 5A and ordered previously deletion-binned but unordered 25 ESTs on the 5A via radiation hybrid mapping approach. The goal of our study was to explore the feasibility of construction of the wheat WGRH panel and map and location of genes using the method of in vitro rescue. 1. Possible utilization of the asymmetric somatic hybrids of common wheat for the construction of radiation hybrid panel /mapFor wheat breeding, we initially tried to transfer gene(s) related to a medicinal ingredient of B. scorzonerifolium to wheat through the introgression of its chromosome fragments induced by UV. Therefore, the protoplasts of B. scorzonerifolium were irradiated with UV and fused with protoplasts of wheat without UV treatment. However, the genetic constitution of the hybrids obtained was unexpected. Instead of generating wheat hybrids carrying chromatin of B. scorzonerifolium, we found the reverse. GISH karyotyping and DNA marker analysis of the hybrids demonstrated that wheat chromatins were introgressed into the nuclear genome of B. scorzonerifolium. Although the wheat genome itself was not irradiated directly, the indirect effect of UV on wheat chromatin breakages and introgression to B. scorzonerifolium genome worked. Wheat SSR loci in asymmetric hybrid cell lines remained stably over a period of 2.5 to 3.5 years and the average retention frequency of wheat specific SSRs in the asymmetric hybrids was 20.50%, the same as most radiation hybrid panel in animal reported. This result provided us an opportunity to assist in the construction of a radiation hybrid panel and map of wheat, which is the first wheat WGRH panel induced by UV instead ofγray. We suggested that asymmetric somatic hybrids between B. scorzonerifolium and wheat treated by radiation would be more suitable for the construction of WGRH panel and mapping of wheat, therefore it was investigated in following studies.2. Effects of UV andγray on wheat chromatin introgression to radiation hybridsTo compare the effects of UV andγray radiation in asymmetric somatic hybridization, 7 asymmetric combinations with wheat irradiated by UV orγray and symmetric combination (control) were performed between B. scorzonerifolium and wheat. A total 155 and 8 hybrid was obtained from 7 asymmetric somatic combinations and symmetric hybridization respectively. The average differentiation frequency was 22.0%. Genomic in situ hybridization revealed that wheat chromatins were located on the B. scorzonerifolium chromosomes. Eight hybrid clones were picked out randomly from each of the 7 asymmetric somatic hybridization and the control combinations, respectively. In all, 64 hybrid clones were selected and constituted 8 hybrid populations, which represented 8 different hybridization combinations with different radiation and radiation dose. Genetic diversity of the 8 hybrid populations and their parental species were analyzed.Random Amplified Polymorphic DNA (RAPD), Inter-SSR Amplification (ISSR) and Simple Sequence Repeats (SSR) markers were used to investigate the genetic constitution of the somatic hybrids. A total 124 RAPD bands; 126 ISSR bands and 42 species specific SSR loci of wheat were detected in each hybrid. The dendrograms of hybrid populations were constructed using unweighted pair group method with arithmetical averages from the genetic-similarity matrixes based on the RAPD and ISSR data. Two groups with the exception (WB3K) according to the types of radiation treatments were clustered distinctly. These results, together with the wheat DNA retention frequencies in each of hybridization combinations derived from SSR analysis, revealed that neither doses nor types of radiation could effect on the regeneration frequency. Both the radiation treatment and phylogenetic relationship of the parents could affect the asymmetry of the genome of radiation hybrid, but both factors were not independent and their effects did not be simply overlaid each other. Actually, it was a complex and interactive process. The relationship of equivalently biological doses was constructed modestly between UV andγray. In asymmetric somatic hybridization with remote phylogenetic relatedness (maybe interfamiliar), the ultimate effects of different ranges of UV radiation on wheat chromatin retention in hybrid panel were similar to those ofγray radiation dose (3Krad), and higher than those of 5Krad and 7Krad. Therefore, UV can efficiently be used to induce the transfer of donor chromatin to recipient genome, and more suitable for the construction of wheat radiation hybrid panel.3. Characteristics of asymmetric somatic hybrids of common wheat/B scorzonerifoliumWheat protoplasts on their own could grow just beyond small clusters; B. scorzonerifolium protoplasts could only generate green spots. Therefore, the regenerated hybrid plants were the result of complement of both parent's genomes. Those clones that had the ability to regenerate plants could be identified as hybrids primarily. Based on this characteristic and vigor growth of hybrids, we could reduce the workload of identifying hybrids and avoid the nonrandom retention of alien chromosome fragments in hybrids induced by using the selected markers. The chromosomes of B. scorzonerifolium were stable (over 90% cells being normal diploid) even it was subcultured in vitro for 12 years. The wheat/B. scorzonerifolium somatic hybrid lines used for genome coverage of wheat were also chromosomally stable. SSR analysis showed that the wheat chromatin introgressed into B. scorzonerifolium genome could still be inherited stably after one year's subculture. Thus, the stable genome of B. scorzonerifolium had the ability to rescue the chromosome fragments of wheat and construct a wheat radiation hybrid panel. In addition, based on the large difference in nucleotide sequence composition between wheat and B. scorzonerifolium because of their distant phylogenetic relationship, we could use the polymorphous, and even the non-polymorphous markers to construct a radiation hybrid map of wheat.4. Construction of two whole genome radiation hybrid panels of common wheatThe wheat WGRH panel I and panel II with different cell lines were constructed in this study. There were 92 and 184 radiation hybrids in panel I and pane II, respectively. We used 68 wheat species specific SSR markers of chromosome 5A to analyze the retention of wheat chromatin in the hybrids. The average marker retention frequency of Panel I was 33.59%; each hybrid contained at least one SSR locus. The average marker retention frequency of panel II was 33.98%; out of 184 hybrids, 10 had lost all 68 SSR loci. Using the command mapocb of software CarthaGene, we found that there were 1557 and 2034 obligate chromosome breaks in panel I and panel II, respectively. The approximate size of wheat chromosome 5A was 781Mb by estimate, so the mapping resolutions of two panels were 501.6Kb和383.9Kb respectively.5. Construction of the radiation hybrid map of chromosome 5A of common wheat and location of ESTs of three 5A unigenes in this mapUsing the software CarthaGene, a radiation hybrid map of wheat chromosome 5A was constructed based on the panel II because it had higher mapping resolution (383.9Kb). This analysis produced a comprehensive map with a total length of 2103cR covered by 68 SSR markers of wheat chromosome 5A. The approximate size of wheat chromosome 5A was 781 Mb by estimate, thus 1cR was equal to 371Kb in this map. It is known that the numbers of both radiation hybrids and markers of mapping could impact the resolution of a map. If the number of markers using for this mapping was 378, the resolution of this map would be 89Kb theoretically. Then we located the ESTs of three unigenes on the chromosome 5A based on this map. BQ172091 and CA616563 were co-retained in the hybrids and they linked closely with Xwmc524 with the distance 13.7cR. A603697 and Xbarc92 linked closely with the distance 15.2cR. These results also suggested that somatic hybridization could rescue the radiation-segmented genome of wheat irradiated with suitable dosage. This advantage of in vitro rescue process could allow generation of much more retention models of wheat genome in hybrid panel and map with much higher resolution.6. Ordering the 25 ESTs within the bin C-5AL10-0.57~*Bin C-5AL10-0.57* comprised the portions of centromere and long arm of chromosome 5A. Using the software CarthaGene, we ordered 25 ESTs within the bin C-5AL10-0.57~* based on the panel II. Twenty-five ESTs were divided into two linked groups and 15 individual ESTs. The first linked group consisted of BE591152. BE423288, BE442763, BE403443, BE446342, BE423213, BE443745, BE637989 and the second one consisted of BE443755 and BF202930. The total length of this map was 2700.5cR calculated by the command build of software CarthaGene. The results showed that radiation treatment could induce the breakages anywhere along the chromosome. The radiation hybrid mapping approach had the ability to order the markers within a single bin in the region of centromere even the breakages were lacked in this region in the deletion lines of wheat.