The Identification Of Somatic Hybrids Chromosome Consititution And Agronomic Traits

To transfer the excellent genes from wild species and closely related-species to cultivated potatoes, our lab had obtained some somatic hybrids by protoplast fusion of Solanum tuberosum L. and Solanum melongena L. and SSR molecular markers screening. This research identified the ploidy level of the somatic hybrids by chromosome counting. Also we observed the somatic hybrids agronomic traits and evaluate the degree of resistance to bacterial wilt. We identify the genome constitution of the somatic hybrids and the chromosome rearrangement. The main results are as follows:1. The identification of somatic hybrids chromosome counting. The root tips slides were prepared by enzymatic hydrolysis, then chromosome counting were performed on32asymmetrical fusion somatic hybrids, which potato used as receptor while eggplant used as donor, and12potato-eggplant symmetrical fusion somatic hybrids. The results showed that, the ploidy of somatic hybrids of symmetrical fusion were complex, there were8euploid and4aneuploid. Though the parents were diplod, there were3tetraploid,3hexaploid and2heptaploid of the8euploid.4aneuploid were from the same callus, and the number of chromosome was between68to95.There were24tetraploid,4hexaploid,3aneuploid and1mixoploid of the32asymmetrical fusion hybrids. The tetraploid accounted for75%of the hybrids.2. Analysis of somatic hybrids chromosome constitution. We performed GISH on hybrids plant root tips slide using potato and eggplant genomic DNA as probes. The results showed that, the chromosome origined from potato-eggplant symmetrical fusion hybrids could be clearly distinguished via GISH. For the further study, we found that the hybrids from same callus had similar chromosome constitution. The3hybrids from Callus3were hexaplois, which consist of48potato chromosomes and24eggplant chromosomes. The2hybrids from Callus4were heptaploid, consisting60potato chromosomes and24eggplant chromosomes. The6hybrids from Callus60were aneuploid, including54-60potato chromosomes,15-23eggplant chromosomes and unequal amounts of rearrangement chromosomes. The number of rearrangement chromosomes in the6hybrids of PE-60-1, PE-60-2, PE-60-9, PE-60-10, PE-60-12, PE-60-13were2,3,1,6,2and1, respectively. The results indicated that, most parental chromosomes could exist separately, and it also can exist by rearranging chromosome in distantly-related somatic hybrids. The plats PE-29-1, PE-29-4, PE-57-4were detected to be hybrids via molecular markers, and they were tetraploid via chromosome count, and GISH indicated that48chromosomes were from potato, no clear eggplant signal was detected. The hybrids may consist small segment of eggplant chromosome, but it was hard to be detected by GISH. The GISH results of asymmetrical fusion somatic hybrids showed that, all the hybrids had potato chromosome hybridization signals, and some dispersed punctuate signal existed on potato chromosome. While potato and eggplant are homologue, the signal might be eggplant genome signal or that of the homologous sequences.3. Analysis rearrangement chromosomes of hybrids. Previous research has reported that chromosomal rearrangement was prone to take place on the chromosome contained rDNA. The rDNA-FISH analysis was performed on6somatic hybrids, using25s rDNA and5s rDNA as probes. The results showed that, the symmetrical fusion somatic hybrids PE-60-4and PE-60-10were detected to have16and1125s rDNA sites, and some signals existed on the central section rather than the terminal of the chromosome short arm as parents. PE-29-4was tetraploid, detected to have no eggplant chromosome, containing525s rDNA sites, one more site than normal tetraploid. Hybrid P-4-4had46chromosomes, and it contained625s rDNA sites, two more sites than normal tetraploid, and one of sites was transferred from the end to the central section of the chromosome. P-13-1was hexaploid, expected to have625s rDNA sites and65s rDNA sites, but results showed75s rDNA sites and525s rDNA sites. The results indicated that chromosomal rearrangement took place at rDNA site regions.4. Analysis of aneuploid chromosome. We tried to figure out which chromosomes lead to aneuploidy via potato centromeric specific repeats FISH associated with GISH in hybrid PE-60-10. We chose3centromeric specific repeats (ST3-58, ST18, ST57) as probes to perform FISH on slide combined with GISH, the results showedthat ST3-58,and ST57which located on chr2and chr7seperatedly were euploidy, but there were5chromosomes where ST18located (chr9).5. Observation of the character and evaluation of resistance of hybrids.34asymmetrical fusion hybrids and5symmetrical fusion hybrids were planted and grown in greenhouse, then investiged and measured the growth potential, plant height and leaf traits of hybrids. The results showed that5symmetrical fusion somatic hybrids died young and asymmetrical fusion hybrids could grow normally. In obtaining somatic hybrids via somatic cell fusion, asymmetrical fusion was an efficient way. The character of the somatic hybrids was similar to potato parent, and all had ability to form tuber, but the tuber were smaller than that of8#. The results of evaluation of resistance to bacterial wilt were that only2hybrids, P-22-1, P-4-3were resistant.