水稻F1花粉不育基因的精细定位及其遗传分化研究

水稻籼粳亚种间杂种的不育性限制了亚种间的遗传交流和杂种优势利用。本研究通过发展位置特异性的微卫星标记将F1花粉不育基因S-6座位进行了精细定位；通过分析近等基因系中代换片段的遗传效应，鉴定出了F1花粉不育基因S-d座位，利用位置特异性的微卫星标记将S-d进行了定位；根据基因组的序列资料和利用较大的作图群体对S-6和S-d两个座位进行了物理作图；通过分子标记辅助选择培育了一批复等位基因近等基因系，对育性基因的遗传分化进行了研究。取得了如下主要结果：1、根据S-6座位初步定位的结果发展位置特异性的微卫星标记，将F1花粉不育基因座S-6进行了精细定位。结果表明多态性标记均与S-6座位紧密连锁，其中R830STS、PSM7、PSM8、PSM9、．PSM59和PSM60位于S-6座位一端，与S-6座位遗传距离分别为1．5cM、1．2cM、0．9cM、0．9cM、0．9cM和0．9cM，而PSM202、PSM206、PSM208、RMl3、R2213SSTS和RM413位于S-6座位的另一端，与S-6座位的遗传距离分别为0．9cM、2．1cM、3．8cM、4．1cM、4．4cM和5．3cM。2、根据S-6座位精细定位的结果，从IRGSP下载了S-6座位所在区域克隆的序列，将克隆的序列进行了拼接，同时将与S-6座位紧密连锁的分子标记与序列拼接图进行了电子整合。根据整合的结果发展位置特异性的微卫星标记和STS标记，利用500株的作图群体，最终将S-6座位界定在PSM8与PSM215之间182．2kb的范围，其中PSM214、T17、T18和T19与S-6座位完全连锁。3、通过对近等基因系E11-5中代换片段遗传效应的分析，在第1染色体的代换片段上鉴定出一个新的F1花粉不育基因座S-d。根据基因组的序列发展位置特异性的微卫星标记将S-d座位进行了定位。结果表明多态性标记均与S-d座位紧密连锁，其中PSM27、PSM24、．PSM26、PSM23、PSM31、PSM25、PSM37、PSM41、PSM42、PSM43、．PSM44、．PSMl2和PSMl3位于S-d座位的一端，与S-d座位的遗传距离分别为10．6cM、7．2cM、7．2cM、6．8cM、6．8cM、6．4cM、6．4cM、4．8cM、4．8cM、3．2cM、1．6cM、0．4cM和0．4cM，而RM84、RM86、RM323、RMl和RM283位于S-d座位的另一端，与S-d座位的遗传距离分别为3．8cM、4．6cM、6．7cM、7．5cM和8．7cM。4、根据S—d座位定位的结果，从IRGSP下载了S-d座位所在区域克隆的序列，将克隆的序列进行了拼接，同时将与S-d紧密连锁的分子标记与序列拼接图进行了电子整合。根据整合的结果发展位置特异性的微卫星标记和STS标记，利用2160株的作图群体，最终将S-d座位界定在67．8kb的范围内，其中PSM93和PSM74位于S-d座位的两侧且各与S-d仅有一个重组，而PSM95、PSM96、T1和T2与S-d座位完全连锁。RiceGAAS注释分析表明在此区段有17个ORF。，其中3个ORF可能与杂种不育性有关。BLAST分析表明此段序列籼粳之间的同源性较低，这也可能是杂种不育的一个原因。5、通过分子标记辅助选择，在F1花粉不育基因s-n、s-6、s-c和s-d座位各培育了一批复等位基因近等基因系，测交分析表明各不育基因座位上的不育基因不仅分化为相对的S^i和S^j，而且基因型类型相同的复等位基因的遗传分化也达到了显著差异的水平。携带有多个复等位基因的近等基因系的测交分析表明，复等位基因近等基因系的遗传效应为各基因座位遗传效应的累加，各基因的遗传效应相互独立，彼此间无相互作用。     

The origin of polyploids via 2n gametes in Vaccinium section Cyanococcus

Summary The production of 2n pollen (pollen with the sporophytic chromosome number) was evaluated in 4x and 6x taxa of Vaccinium section Cyanococcus. Mean frequencies of 2n pollen producers were 17.1% and 8.3% in natural 4x and 6x populations, respectively. The frequency of 2n pollen producers in the 4x species ranged from 8.6% (V. angustifolium) to 23.8% (V. pallidum). Level of 2n pollen production was genotypically variable (1% to 37.4%). The widespread occurrence of 2n pollen in 2x, 4x and 6x taxa suggests that sexual polyploidization was widespread and responsible for the origin of the polyploid species found in this genus. The frequency of 2n pollen producers was not significantly different between the 4x species and their putative 2x ancestors. These results support the origin of 4x and 6x taxa as a consequence of sexual polyploidization. Polyploids derived from sexual polyploidization would be expected to have increased fitness and flexibility due to the mode of 2n pollen formation. In blueberry species the predominant mode of 2n pollen formation is genetically equivalent to a first division restitution mechanism (FDR). FDR 2n pollen transmits a high percentage of the heterozygosity and a large fraction of the epistasis from the 2x parent to the 4x offspring.     

Effect of genotype,environment and carbohydrate on anther culture response in head cabbage (Brassica oleracea L. convar. capitata (L.) Alef.)

Summary The effect of genotype, growing conditions for donor plants and type and concentration of carbohydrate in the culture medium was investigated for anther culture of head cabbage (white cabbage, savoy cabbage, pointed-headed cabbage). Strong genotypic effects on embryo formation from the cultured anthers were shown as well as superior embryo formation from anthers of field grown donor plants compared to plants grown in the greenhouse. When comparing 7, 10 and 13% sucrose in the medium, embryo response increased with increasing sucrose concentration. With maltose, which was generally inferior to sucrose as carbohydrate source for anther culture, the embryo response did not increase with maltose concentration above 10 per cent.     

Pollen and pollination experiments. I. the contribution of stray pollen to the seed set of depetalled,hand-pollinated flowers of apple

Summary Using the finger nails to remove most of the petals is a fast method of preparing apple flowers for hand pollination in the balloon stage. Such flowers still appeared quite attractive to insects; left to open pollination, the seed set per flower averaged two thirds of that of similarly treated, but hand-pollinated flowers. With the aid of marker pollen of scab or mildew resistant donors and the subsequent screening of the seedling progenies for resistance, it was shown that the stray pollen was responsible on average for one of every three seeds formed by unbagged depetalled and hand-pollinated flowers.     

In vitro pollen germination and tube growth of tomato (Lycopersicon esculentum Mill.) and its relation with plant growth

Summary In vitro pollen germination at 10°C, 14°C and 22°C of four groups of two pure line tomato varieties was compared with their plant growth at 19°C D/10°C N under controlled environmental conditions. Generally, pollen germination was slow at 10°C but after 6 h the percentage of germination was similar to that at 22°C. Maximum germination was obtained at 14°C already after 4 h. The longest pollen tubes occurred at 22°C. The two varieties within each group differed significantly from each other for percentage of pollen germination. For one group, this difference was greater at 10°C than at 22°C. The varieties in two groups also differed significantly for pollen tube growth. These differences in pollen tube growth rate were greater at 22°C than at 10°C. There was no clear relationship between pollen germination, pollen tube growth and plant growth in any of the four pairs of varieties. The results are discussed with regard to the possibility of pollen selection at low temperature in order to improve the efficiency of breeding for growth at low temperature.     

Barriers to hybridization of Solanum bulbocastanum Dun. and S. Verrucosum Schlechtd. and structural hybridity in their F1 plants

Summary Results of reciprocal crosses between Solanum verrucosum (2n=2x=24) and S. bulbocastanum (2n=2x=24) are described in terms of pollen tube behaviour in styles, of berry and seed set, of fertility and of meiotic behaviour of the F₁ hybrids. Pollen tube growth of S. verrucosum is strongly inhibited in styles of S. bulbocastanum, whereas no inhibition is observed in the reciprocal cross. Therefore S. bulbocastanum x S. verrucosum fails to set berries or seeds, whereas the reciprocal cross produces both berries (54.4% berry set) and seeds (0.3 per berry). Only 14.6% of the seeds germinate. Both the diploid and corresponding tetraploid hybrid plants are vigorous, flower abundantly, have a rather regular meiosis (mainly rod bivalents), but show a high degree of cytoplasmic-genic male sterility. Crossability of the diploid hybrid plants is nil when used as pollen parents and near to zero when used as pistillate parents. The barriers to hybridization of the parent species (unilateral inhibition of pollen tube growth, somatoplastic sterility, cytoplasmic-genic male sterility and structural differences of the parental chromosomes) are discussed and methods are suggested to overcome these barriers.     

Chemically induced sterility in wheat for hybrid seed production

Summary The gametocidal effects of RH-531, RH-532, and RH-2956 were studied with three rates and times of foliar application on two spring wheat (Triticum aestivum L.) varieties, Anza and Yecora 70. RH-531 and RH-532 applied at or before booting stage caused rather large reduction in plant height while RH-2956 had a small effect on height. Application of any of the three chemicals on Anza at meiotic stages reduced self-pollination (SP) fertility by 97–99%. If 90% SP fertility reduction is acceptable, the application time could vary from premeiosis to postmeiosis for Anza. Yecora 70 was less sensitive than Anza to gametocide treatments. The lowest SP fertility obtained in Yecora 70 with RH-531, RH-532, and RH-2956 was by treatment at booting (4 kg/ha), at premeiosis (2 kg/ha), and heading (8 kg/ha), and the corresponding SP fertility reductions were 74, 89, and 71%. Pollen stainability-determined by aceto-carmine, IKI, and Alexander's stains-was not affected by gametocide treatment and was not useful in evaluating sterility induced by these chemicals. For the RH-531-treated Anza and Yecora 70 and the RH-532-treated Anza, more than 94% of the seeds produced by open-pollination on treated plants were from self-or sib-pollination as determined by progeny testing. However, RH-532-treated Yecora 70 and RH-2956-treated Anza and Yecora 70 did result in more crossed seeds, with a maximum of 20% outcrossing. It was believed that spike compactness induced by the chemicals and the related poor flower opening were the major limitations for outcrossing. Artificial pollinations made to the test varieties showed that RH-531 induced both male and female sterility and was undesirable for practical use. RH-532, however, could be applied at the booting stage to avoid severe female sterility, and RH-2956 clearly was the best of the three chemicals, with the female fertility very close to those of the untreated checks of both Anza and Yecora 70. Of the three chemicals, RH-2956 gave the highest percentage of hybrid plants in the progeny of hand-or open-pollinated spikes. Its practical use for hybrid seed production, however, will depend largely on improvement of methods to increase cross pollination in wheat.     

Incidence and molecular markers of 2n pollen in Populus tomentosa Carr.

Microscopic examination, amplified fragment length polymorphism (AFLP) and SCAR (sequence-characterized amplified region) molecular markers were employed to determine the incidence of 2n pollen (unreduced pollen) in Chinese white poplar (Populus tomentosa Carr.) and to identify related molecular markers. The presence of a parallel and tripolar spindle at metaphase II and the absence of cytokinesis at telophase II were found to be determining factors in 2n pollen formation. A group of 298 clones that originated from their indigenous areas were investigated for the production of 2n pollen based on pollen size differences, both within a clone and between n and 2n pollen. Pollen grains were collected from 224 of the clones, six of which were subsequently determined to produce only normal pollen; the remainder produced 2n pollen at different frequencies (0.6–21.9%). The frequency at which 2n pollen was produced was significantly and highly significantly different among and within indigenous populations, respectively. Clones produced by the six normal and twenty-two 2n pollen clones were selected for AFLP analysis. Following an initial screening with 55 primer combinations, the E50-M38 (CAT/ACT) primer was identified: it generated a PCR fragment (246 bp) from the normal clones, but not from the 2n pollen producers. In addition, the E31-M50 (AAA/CAT)-amplified DNA fragment (204 bp) was present in 2n pollen producers, and absent in normal clones. These two discriminating AFLP markers were developed into easily detectable SCAR (sequence characterized amplified region) markers which can be used in combination with previously developed AFLP markers to distinguish between normal and 2n pollen clones.     

A gamete abortion locus detected by segregation distortion of isozyme locus Est-9 in wide crosses of rice (Oryza sativa L.)

Summary A locus for male gamete abortion in hybrids for Japonica and Indica rice was identified with the aid of marker genes Rc and Est-9 on chromosome 7. In an Indica-Japonica cross, AKAMAI 1/IR50, the Indica allele Est-9 ² was transmitted via the male gamete with a ratio of 0.29 instead of the normal 0.5, whereas no segregation distortion was observed for the Rc locus. The recombination value (p) for Est-9 and Rc was estimated to be 0.38 by a least square method after adjusting Mendelian segregation ratios with the male transmission ratios of 0.29 (Tr) for Est-9 ² and 0.71 (1-Tr) for Est-9 ¹. The recombination value (q) for the new locus for male gamete abortion, ga-11, and Est-9 was estimated to be 0.23 by using 56 F₃ lines from F₂ plants which were heterozygous for the Est-9 locus. No linkage for Rc and ga-11 was found. Therefore, the two markers and ga-11 were located in the order of ga-11-Est-9-Rc. Using the estimated recombination value (q), the male transmission rate (k) of ga-11 ^a was estimated to be 0.11 with the F₂ data and-0.07 with the F₃ line data. Thus, it was apparent that male gametes possessing ga-11 ^a were frequently aborted in the Indica-Japonica hybrid.     

Doubled haploid production of sunflower (Helianthus annuus L.) through irradiated pollen-induced parthenogenesis

Induced parthenogenesis as a possible method of haploidization in sunflower (H. annuus L.) was tested in previous investigations (Todorova et al., 1994) and conditions for reproducible regeneration of gynogenic doubled haploids were established by the present work. Forty eight treatments were studied, involving four pollen donors and four recipient hybrids. Pollen was irradiated with doses of 300 Gy, 600 Gy and 900 Gy. In total, 2279 embryos were cultivated in vitro of which 1107 plants were obtained and 582 of them produced seeds after selfing. The ploidy level of the regenerants was evaluated at the two – three leaf stage and 296 of the plantlets obtained were haploids. Some of them underwent spontaneous diploidization, the others were treated with colchicine solution for chromosome doubling. The diploid plantlets were checked for their gynogenic origin by genetical and biochemical methods. The effectiveness of the method expressed as the number of agronomically useful DH lines to the number obtained that were fertile and resistant to downy mildew, branched and unbranched plants was 8,6% on average. This revised version was published online in July 2006 with corrections to the Cover Date.