Genetic Stability Of Potato Somatic Hybrids And Their Pedigrees Derived From Backcrossing

Plant somatic hybridization that can transfer desirable genes from wild species into cultivates and recombine nuclear DNA and organelle DNA between sexually incompatible species has been adopted to create novel germplasms for plant breeding. One hundred and forty-nine potato somatic hybrids had been previously obtained from two fusion combinations in our laboratory. To provide with theoretical base for further use of the somatic hybrids, we analyzed the genetic stability, the meiotic chromosomal behavior of the pollen mother cells, pollen viability and variation in agronomic traits and the genome components of the somatic hybrids, as well as the genetic variation of the hybrids and inbreds derived from them. The main results were as follows:1. Flow cytometric analysis demonstrated that68out of108somatic hybrids had their ploidy level changed to be uniform and euploidy after successive in vitro subcultures (from2003to2007), which mainly occurred in octaploids, aneuploids, and mixoploids, while74%hexaploids were still stable in their genome dosage, indicating the somatic hybrids with a sum of parental chromosomes have more ploidy stability than those with unexpected chromosome number.2. One pair of cpDNA SSR primer and4pairs of mtDNA specific primers were used to detect the cytoplasmic components of the somatic hybrids and their stability during long-term culture in vitro. There were77.6%of the143hybrids which showed the chloroplast pattern of one parent and the rest exhibited changed patterns among the three tests conducted in2003,2005and2010. Most somatic hybrids (41out of44) presented a fused mtDNA pattern of the fusion parents, although some specific bands altered in8hybrids. The results suggested that, in comparison with the chloroplast genome, the mitochondria genome has a higher recombination frequency which is more stable in in vitro subcultures.3. The meiotic behavior of the somatic hybrids different ploidy levels was examined and compared with their fusion parents. The meiotic analysis revealed that the wild fusion parent C9701had normal meiosis with abnormal rate of1.29%while the tetraploid cultivated parents3#and8#had abnormal rates of20.44%and12.12%, respectively. The chromosome meiotic behavior in most potato somatic hybrids was more complicated than in their fusion parents, a hexaploid8C30-1had the lowest abnormality (21.10%) and a mixoploid8C13-1had the highest abnormal meiosis (73.42%). The meiotic abnormity was associated with the genomic complexity. Different types of abnormal meiotic behavior were observed during development of the pollen mother cells (PMCs) such as formation of univalent and multivalent, laggard chromosomes, chromosomal bridges, unbalanced separated chromosome at different phases in a PMC or anther as well as triads and polyads. The results demonstrated that ploidy complexity resulted from somatic hybridization in somatic hybrids is a main effect influencing meiosis of pollen mother cells. The hybrids with expected ploidy level had a lower frequency of meiosis abnormity.4. There was positive relationship between pollen germination rate and the pollen fertility identified by pollen staining. The results showed that the pollen viability of C9701was99.5%while of3#and8#was47.0%and49.9%, respectively. Except for4somatic hybrids, the pollen viability of the most hybrids was lower than their cultivated fusion parents, and accordingly, the number of small pollens of the hybrids was higher than that of the parents. The statistic analysis revealed that small and sterile pollens were the direct result of the abnormal meiosis.5. Twelve somatic hybrids with normal flowers containing viable pollens were chosen to backcross with12tetraploid cultivated potatoes. There were25crosses yielded2515seeds. Among the12somatic hybrid parents,3C28-1had the highest crossing compatibility with the tetraploid parents (13as maternal parent and7as paternal parent) and18crosses yielded2258seeds. The results demonstrated that the inter-specific somatic hybrids could be compatible with cultivated potatoes and can be used in the breeding program.6. The flow cytometric analysis showed that, of the281backcrossing pedigrees of the somatic chybrids crossed with tetraploid cultivars,181were tetraploids,10were pentaploids and90were aneuploids (4x-5x). Among the17inbreeds of3C28-1there were2tetralploids,1aneuploid (4x-5x),8pentaploids,2aneuploids (5x-6x) and4hexaploids. The results implied that diverse gametes are formed in the somatic hybrids and most of them are compatible with the gametes of tetraploid parents.7. The backcrossing pedigrees of3C28-1with the tetraploid parents possessed plant height taller than either of the parent, indicating a strong heterosis of the hybrids. The stem number of the backcrossing hybrids was less than the cultivated parent but close to the somatic hybrid parent, showing an inheritance of stem number towarding to the high parent. The leaf index of the hybrids were the average of the parents indicating a genetic trend of mid-parents, whereas stolen length, number of tubers produced per plant, tuber weight per plant and mean tuber weight varied remarkably indicating a possible multigene-control of the traits.8. Eleven SSR primers were chosen to analyze the genetic components of96backcrossing pedigrees between3C28-1and the tetraploid cultivare E1and the40inbreeds of3C28-1. The11primers amplified33unique alleles, of which C9701had13specific bands,3had4unique bands, E1had7specific bands, and3and El had9common bands. The results showed that all the33specific alleles detected in the three parental materials appeared in the backcrossing pedigrees with some variations among individuals, proving that it is inheritable of the genetic components of the somatic hybrids and a diverse segregation could occur during the sexual crossing. In40inbreeds of3C28-1, the unique alleles of the fusion parents were71.7%and59.0%for C9701and3#respectively, while some novel bands were also detected in the inbreeds. The findings reinforced the gamete complexity of the somatic hybrids and proved the events of chromosomal exchange or site-specific recombination during the meiosis.