| Citrus is one of the most important fruit trees in the world and plays a pivotal role in the economy in southern china. Seedlessness is an important characteristic in citrus on the fresh fruit market because consumers do like seedless fruits. Since the 1980 s, many allotetrploid somatic hybrids, generated from the fusion of two elite scion cultivars, have been generated by protoplast fusion technology all over the world because of their important value as potential parents to produce citrus triploids. In this study, some new flowering allotetraploid somatic hybrids were employed as pollen parents to pollinate some seedy diploid varieties, aiming to generate triploid progenies with three different genomes. Furthermore, the tetraploid progenies derived from the four crosses using ‘Nadorcott’ tangor as seed parent were utilized to analyze the genetic machnism underlying the 2n gamete production in ‘Nadorcott’ tangor. We also studied the meiotic inheritance pattern in somatic hybrid NH by cytological observing of the meiosis and SSR segregation analysis by genotyping a triploid population derived from the cross between ‘Nadorcott’ tangor and NH. The results of this study are as follows:1. In successive three years from 2010 to 2012, 12 interploidy crosses using eight monoembryonic diploid cultivars as seed parents and four allotetraploid somatic hybrids as pollen parents, were carried out with a total of 3243 flowers pollinated. With 651 fruits harvested, an average fruit set ratio of 26.95% was obtained. As a result, 952 plants were recovered from 10144 seeds cultured in vitro, which derived from 478 fruits. By flow cytometry analysis and chromosome counting, a total of 693 triplods and 19 tetraploids vere verified. After transplantation, 574 triploids and 16 tetraploids were survived at last. The results of SSR markers showed that all the triplods and tetraploids from the crosses of Huanong red pummelo × NH were of hybrid origin.2. In successive three years from 2010 to 2012, to obtain triploid citrus hybrids, eight elite polyembryonic diploid cultivars as seed parents were crossed respectively with five allotetraploids and one autotetraplod. A total of 4083 flowers were pollinated form 13 crosses, in which 1429 fruits were set, with an average fruit setting rate as 35.0%. As much as 11938 immature seeds from 1020 young fruits were cultured in vitro. After shooting and rooting induction, 2576 plants were regenerated, in which 395 and 119 plants proved to be triploids and tetraploids respectively by flow cytometry analysis. Up to 343 triploids and 96 tetraploids survived in the greenhouse after transplantation into greenhouse. The SSR markers analysis showed that all the triploids derived from the cross of ‘Nadorcott’ tangor × NH were hybrids of both parents.3. 54 tetraploid progenies, which were unexpectively obtained previously from four 2x × 4x crosses using polyembryonic ‘Nadorcott’ tangor as seed parent, were analyzed by 22 SSR markers, aiming to reveal their genetic origin and the mechanism underlying 2n megagametophyte formation. The results showed that 13 tetraploids from all these four crosses were doubled diploids as indicated by their identical SSR allelic profile with their female parent, while the remaining 41 tetraploids apparently exhibited paternally derived alleles, which confiemed their zygotic origin. Furthermore, the genotyping of all hybrids indicated that all of them arose from 2n megagametophytes. Based on the genotypes of 2n megagametophytes, the analysis of maternal heterozygosity restitution(HR) for each marker showed that it varied from 0.00 to 87.80% with a mean value of 40.89%. In addition, it was observed that 13 markers displayed a lower rate than 50%. On the basis of the above results, it can be speculated that the second division restitution(SDR) in the mechanism underlying the 2n megagametophyte formation in ‘Nadorcott’ tangor.4. Cytological observation of meiosis of pollen mother cells(PMCs) of somatic hybrid NH was carried out to evaluate its meiotic inheritance pattern. It showed that a high rate of chromosome abnormalities including univalents(0.7%), trivalents(0.7%), quadrivalents(31.6%), hexavalents(8.7%) and octavalent(0.9%) was observed in this somatic hybrid although bivalents were predominat(57.6%). Furthermore, the meiotic chromosome pairing behavior was analyzed by genotyping a triploid population derived from ‘Nadorcott’ tangor × NH using 18 SSR markers, from which the genotypes of diploid male gametes were inferred. In agreement with the expectation of cytogenetic analysis, a mixed of diploid, tetraploid and intermediate inheritance patterns was deduced due to the co-existence of preferential, non-preferential and intermediate pairing models among all analyzed loci. In this study, parental heterozygosity transmission was further analyzed among 14 loci, where each chromosome in allotetraploid could be clearly tracked. It showed that the mean value of 23.8% intra-parental and 76.2% inter-parental heterozygosity was transmitted. |
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