Creation And Genetic Analysis Of Cucumber (Cucumis Sativus L.) Materials With Different Ploidies

Cucumber, which is one species of cucurbitaceous, is an important vegetable crops worldwide. It can accelerate process of breeding through using haploid or doubled haploid. Meanwhile, its commercial value could be enhanced, and further molecular-cytology studies could be promoted by selecting different ploidy germplasm in cucumber. In this study, unpollinated ovary from different genotypic materials were cultured to produce haploid or doubled haploid, further to esbtalish an efficient protocol for haploid induction. Autotriploid were obtained from reciprocal crosses between diploid and autotetraploid, which obtained from ovary culture. Further, primary trisomic and a non-segregation F2 population derived from reciprocal crosses between autotriploid and diploid. In additional, cytogenetic and molecular analyses have investigated and confirmed precisely these unique germplasms, in order to support basic for ploidy breeding in cucumber. The details results as following:1. Production haploid and doubled haploid from unpollinated ovary culture in cucumberSeveral factors, i.e. the duration of thermal shock pretreatment at 35℃, the concentrations of TDZ and the silver nitrate were investigated for their effects on embryo formation in cucumber ovary culture. The results showed that embryo formation after thermal shock treatment at 35℃was significantly higher than control (0 d), and highest embryo formation was found in treating two days. TDZ had a positive effect on the embryo formation. Highest embryo formation frequency (72.7%) was recorded by adding 0.04 mg/L TDZ into the induction medium. The results found that addition of AgNO3 to induction medium had significant effect on frequency of embryo formation, and shortened embryo sprouting period and improved number of embryos formed in each ovary slice. In total, forty plantlets were regenerated from three genotype cucumber. Two were observed as haploid (2n=x=7), five were autotetraploid (2n=4x=28) and other thirty-three were diploid after chromosome counts. Homozygous of these diploid plants were identified using simple sequence repeat (SSR) analysis, and seventeen were considered as doubled haploid.2. Production and molecular cytogenetical analyses on autotetraploid in cucumberAutotetraploid plants originated from unpollinated ovary culture were identified as homozygote after simple sequence repeat (SSR) analysis. Genetic stability of the progenies from self-crossing of these autotetraploid was studied, no ploidy variation occurred among these progenies after morphology and chromosome observation. These autotetraploid plants have relatively higher pollen fertility after observation stainability of pollen grains, viability of pollen grains and number of seeds per fruit.Meiosis of pollen mother cells (PMCs) and male gametophyte development in autotetaploid cucumber was studied, and results showed that the PMCs meiosis of the autotetraploid was similar to the diploid. Various chromosome configurations, e.g. multivalent, quadrivalents, trivalents, bivalents and univalent were observed in most PMCs of the autotetraploid at metaphase I. Chromosomes'lagging was frequently observed at anaphase in both meiotic divisions. In addition, chromosomes segregations were not synchronous and equal in some PMCs during anaphase II and telophase II. Dyad, triad, tetrad with micronucleus and polyad could be observed at tetrad stage. The frequency of abnormal behavior in each stage of meiosis was counted, and the average percentage was 37.2%. Most microspores could develop into fertile gametophytes with 2 cells and 3 germ pores through the following stages:single-nucleus early stage, single-nucleus late stage,2-celled stage after pollen mitosis. The germination rate of pollen grains was noted, and it was 46.9% in autotetraploid.In additional, genetic variation among two kinds of autotetraploid with different origin (Tetra-5 and Tetra-Jinlv originated from unpollinated ovary culture and cochicine treatment) and their original diploid were studied using molecular cytogenetical analyses. From the cytogenetics analysis, we found the frequency of abnormalities at the different stage from metaphase to tetrad of meiosis was lower in autotetraploid Tetra-5 than that of in Tetra-Jinlv, especially the number of lagging' chromosome at anaphase. Comparing the chromosome configuration at metaphaseⅠin these two kinds of autotetraploid, there are relatively more quadrivalents, and relatively less univalents and trivalents in Tetra-Jinlv. On the other hand, AFLP analysis detected the genetic differences among two kinds of autotetraploid with different origin and these original diploid.2214 bands ranging with 60-500bp were amplified with 38 pairs of primers. Only 50 polymorphic loci were identified which accounted for 2.26%. The expression of polymorphic loci showed that the differential bands remained present in the diploid and autotetraploid, which account for 0.50% and 0.18%, respectively. We also detected 26 novel bands present in Tetra-5, which has much more genetic variation than Tetra-Jinlv. These findings indicated that extensive genetic variation occurred during ploidy change or origin in cucumber. Meanwhile, autotetraploid Tetra-5 showed extensive genetic variation at level of DNA which caused by somaclone variation.3. Production and cytogenetical analyses on autotriploid in cucumberThe effects of fertility of parents, pollination combination and genotype of parents on seed-setting in hybridization between autotetraploid and diploid were analyzed with conventional cross method in this study. The results showed:(1) The low seed-sets (13.0%-14.5%) of the autotetraploidy combinations may be attributed to the presence of more abnormal pollen in the anthers or length of pollen tube. (2) The seed-sets of cross combinations between autotetraploid and diploid were very low (0.26%-1.02%), but there were obvious different seed-settings between the two mating methods. If the autotetraploid cucumber was used as the male parents and the diploid as the female parents, higher seed-sets were found in the hybridized combination. However, the seed-sets in the combinations were lower if the diploid cucumber was used as male parents. (3) Besides, the effect of genotype of parents on production of autotriploid was biggish; the average percentage of autotriploid production was higher when autotetraploid and diploid were belonging to same genotype.Meiosis of pollen mother cells (PMCs) and male gametophyte development in autotriploid cucumber (Cucumis sativus L.) was studied, and results showed that the PMCs meiosis of the autotriaploid was similar to the diploid with particularities. Variable chromosome configurations, e.g. multivalents, quadrivalents, trivalents, bivalents and univalent were observed in the most PMCs of the autotriploid at metaphaseⅠ. Chromosome lagging and bridges at anaphase in both meiotic divisions resulted from irregular chromosome separation and asynchronization were frequently observed as well, which led to formation of micronucleus and inviable gametes. The frequency of normal PMCs in autotriploid at stage of tetrad was only 40.6%. Among those normal microspores, most of them (91.2%) could develop into normal gametophytes with 2 cells and 3 germ pores. The stainability and germination rate of pollen grains were noted, and they were only 18.8% and 13.5%, respectively.4. Production and cytogenetical analyses on primary trisomics in cucumberThe reciprocal crosses were made between autotriploid and diploid for selecting the primary trisomics. Many viable F1 seeds were obtained from reciprocal crosses between autotriploid and diploid. The number of chromosomes of 56 surviving progenies varied from 14 to 28, with plants having 2n=15 occurring at the highest frequency (51.8%). Primary trisomics were firstly obtained in this study. Four types of primary trisomics were isolated and they could be distinguished from each other, as well as diploid. Variable chromosome configurations, e.g. univalent, bivalent and trivalent were observed in many pollen mother cells (PMCs) of the autotriploid at metaphaseⅠ. Binomial chromosome distribution was observed at anaphaseⅠand frequency of 8/13 was 6.25%. The meiosis of autotriploid, especially the class of gametes with eight chromosomes, gave the cytological evidence of producing 2x+1 type gamete and could be induced into primary trisomic plants from progeny of autotriploid-diploid crosses. These studies have established a ground work for selecting a series of primary trisomics, and further using them for associating linkage groups with specific chromosomes in cucumber.5. AFLP analysis of genetic differences among cucumber materials with different ploidiesGenetic variation for the response to ploidy change, haploid, diploid, triploid, tetraploid and trisomic polymorphisms of cucumber variety'Jinlv No.4'were compared by AFLP. Fifty-one pairs of primer combination were employed on cucumber materials with different ploidy and a total of 2922 bands between 60-500 bp were observed. Results showed only 152 polymorphic loci were found which accounted 5.2% and AFLP fingerprints amplified from the materials with different ploidies with 13 of the primer pairs did not remarkably differ. The expression of the polymorphic loci showed that the bands remained present in trisomic and tetraploid and absent in other materials in most case, which accounted for 2.293%. In contrast with diploid material, the trisomic, triploid and tetraploid materials got their specific bands. These results indicated that extensive genetic variation occurred among these cucumber materials with different ploidy.6. Identification and analysis of F2 stable population derived from the cross of autotriploid diploid in cucumberAutotriploid (Tri-Jinlv) obtained from cross between autotetraploid (Tetra-1) and DH-Jinlv, which regenerated from unpollinated ovary culture. It was then used as parent reciprocal crossing with a doubled haploid DH-Cuiyu. From its F2 generation we obtained a genetic-stable population. To prove the uniformity of such a population, SSR markers were used to survey the F2 individual plants. The results showed that F2 individuals carried only one parental molecular marker at each polymorphic locus, and their genotypes were identical with F1 progeny. Based on the above experiments, we consider that this F2 population is definitely an early-generation stable population.