Studies On Molecule And Physiology Of S Gene Interaction In Apple Cultivars

Self-incompatibility is an important mechanism to prevent inbreeding and maintaing continuity of species , it was wide spreaded in the plant wold. Apple(Malus domestica)has gametophytic self-incompatibility which was controlled by a single multiallelic S-locus. In order to obtain the expected yield and fruit quality, It must be configured with different apple S genotypes as pollinated tree or different S genotypes apple pollen for artificial pollination. It has now become urgent issue to identify apple S genotype of pollen and pistil rapidly, to find out style physical activity changing after self-pollination and cultivate homozygous genotype apple cultivars are also important. We identified S genotype of twenty-eight apple cultivars which were SI, S genotype of SC apple Zaohongxiang also was identified, physical mechanism of self-pollination apple cultivars was studied, the result showed that:1. Self-pollinated fruit set percentage was 90.17% in Zaohongxiang apple which was fixed on self-compatible apple cultivar. Self-pollinated fruit set percentage was under 30% in Gala, Xiaguang, Shansha, Golden Delicious, Fuji and Dounan which were confirmed self-incompatibility apple. Styles semi vitro culture of with different pollinated combination showed that: Zaohongxiang was weak SI apple cultivar, Golden Delicious, Fuji and Dounan were strong SI apple cultivars, this result was consistent with pollination in the field. Pollen germination rate of SC apple Zaohongxiang and SI apple Golden Delicious, Fuji and Dounan was abave 92.62%, and there had no differences in pollen germination rate, but they were all higher than Dounan apple which pollen germination rate was only 3.20%.2. Style in situ printing analysis of Zaohongxiang apple before and after blossom showed that: style S-RNase activity was lower before anthesis , style S-RNase activity was higher in blossom day. Style in situ appeared white printing at different times after pollination, style printing was weaken from stigma to base with pollination time prolonging; Pollen tube fluorescent microscope observing after self-pollination showed that: pollen tube was growing normally after self-pollination 0 ~ 24h, self-incompatible apple cultivars stopped growing after self-pollination 48～72h when the pollen tube reached the 1/2 of the style to stigma, but , self-compatible cultivars could grow normally.3.According to PCR-RFLP and conjunction with its parent S genotype method, S genotypes of Zaohongxiang, Jingxiang, Huiminduanzhi, Hongwangjiang, Chiling, Erling, Changtian, Changhong, Apple A, Aozhouqingping, Gongqi Fuji, Qiuxing, Guohong, Anglin, Songbenjin, American8, Huaguan and Nanfangcui were S1S9, S1S9, S1S9, S1S9, S30S30, S30S30, S30S30, S30S30, S30S30, S30S30, S1S9, S30S30, S1S9, S1S9, S1S9, S9S27, S9S9 and S2S9. One of S gene of apple cultivars Yanga1, Hongjingala, Yanga2, Asi, Huangjiagala and Gaoling could be ensured, in turn were SXS27, SXS27, SXS27, SXS27, SXS27 and SXS9.4. 10 new S gene were identified from 11 apple cultivars of Sangsa, Yanga1, Hongjingala, Yanga2, Qing9, Asi, Hangjiagala, Jingxiang, Gaoling, Hongao and Dounan through DNA sequencing. C1 and C2 area of DNA and deduced amino sequence of new S gene were conserved, there were no phenomenon of base pair or amino acid were substituted, the main variable area was HV and intron area. Displace rate of deduced animo acid sequence reached 17%~50% in HV area. The 10 new S gene were marked S33-,S35-,S36-,S37-,S38-,S39-,S40-,S41-,S42-allele, which accession number were DQ219464,EU391605,EU391606,EU391607,EU391609,EU391610,EU391611,EU391612,EU391613.the S gene of 11 apple cultivars were identified through PCR-RFLP and DNA sequencing method, the S genotype were S40S4, S38S27, S39S27 ,S38S27, S41S42, S36S36, S37S27, S1S9S34, S38S9, S35S35, S33S33.5.Style S gene of self-compatable apple cultivar did not change, amino acid sequences which were deduced from pollen S gene SLF1?had 19 variable site beside two HV area, but SLF9?was only 8 variable site, DNA sequence similarity was only 83% between pollen S gene SLF1?and MdSLF1 by BLAST, the disagree base pair were distributed in the whole DNA sequence, there by, it was possible that pollen S gene of SLF1?would likely change into MdSLF2.6. The study result of style physiology activity after self-pollination showed that: The style SOD, POD and CAT activities of self-compatible cultivar Zaohongxiang was relatively stable after self-pollination 0 ~ 48h. The style SOD, POD and CAT activities of self-incompatible cultivars were relatively stable after self-pollination 0~24h when the pollen tube culd grow normally in the style. MDA content of self-compatible apple Zaohongxiang was highest but the whole changing trend was small, MDA content of self-incompatible apple cultivars changing trend was bigger than self-compatible apple, also, MDA content of self-incompatible apple cultivars was ascending after self-pollination 48~72h. Through SOD, POD, CAT activities and MDA cotent we could conclude that: whether apple self-incompatibility or not had no relashtionship with SOD, POD, CAT activities and MDA cotent high or lower, but had close relationship with the stability of them. Style protein content of self-compatible apple significantly higher than self-incompatibilty apple cultivars; the weeken degree of style R-Nase activity was different on different apple cultivars, thereinto, the changing degree of style R-Nase activity was gently after self-pollination from blossom day to self-pollination 72h on self-compatible apple Zaohongxiang, but, Dounan apple changing degree of style R-Nase activity was weaken derectly, the weaken degree of R-Nase in Fuji lower than Zaohongxiang; GA3 content was significantly higher than other self-incompatble apple cultivars after self-pollination 24h. It was testified that GA3 play a leading role in pollen tube growth. Higher (IAA + GA3) / ABA values conducive to the growth of pollen tube, ZT content had little effect on pollen tube growth; IAA oxidase activity appeared slow upward with the time of self-pollination extension which was reverse to IAA content, but, IAA oxidase activity of self-compatible apple appeared decline after self-pollination 24h.7. Apple style protein of different density had restrain effect to self-pollen tube germination, restrain degree of the self-pollen germination was enhanced with the chroma increasing. Style protein had a gentle restrination to pollen germination and pollen tube growth to self-compatible apple, pollen germinate rate was above 76.46% and pollen tube length was above 770.16μm; Apple style were trated with suitable chroma Spm, Spd and Put could increase the percentage of pollen tubes from style bottoms and the number of pollen tubes, the suitable chroma of Spm to treat style was 0.1mM on the self-incompatible apple of Hongao, Gala, Dounan, Fuji, Golden Delicious and Xiaguang, the suitable chroma of Put to treat style was 0.01mM on the self-incompatible apple besides Golden Delicious which suitable chroma was 0.25 mM; the suitable chroma of Spd to treat style was 0.01mM on the self-incompatible apple Honghao and Fuji, the suitable chroma of Spd to treat style was 0.5mM on the self-incompatible apple Dounan and Golden Delicious, the suitable chroma of Spd to treat style was 0.25mM on the self-incompatible apple Xiaguang.8. The result of embroy cultivation in seeds of self-pollination in the field showed that: the seedling which hold 1 cotyledon before euphlis growing was good, one cotyledon was taken second place, and no cotyledon was of a sort; Self-incompatible gene was accord with mendel's law, and different S genotype homozygote could be got, which established basement to build apple S genotype homozygote and accelerate breeding course.