| In the present study, a series of experiments were carried out to determine the EPP by delayed and bud pollination, histochemical tests and fluorescence microscopic observation. And the primary factors influencing the pollination and fertilization were systematically researched, such as stigma receptivity, pollen viability, pollen germination in situ, pollen tube growth, ovule vitality, rainfall and temperature. And the effects of primary factors influencing the EPP on fruit and seed set was elucidated. Simultaneously, to further elucidate the pollen tube kinetics, an AS-PCR reaction system was established and optimized for the first time in loquat and the S-genotypes of18cultivars or lines were determined, and eight novel genes named S11, S12, S13, S15, S16, S18, S31and S41respectively were cloned from these cultivars or lines. The main results were as follows.1. Determination of EPPThe data of delayed and bud pollination, histochemical tests and fluorescence microscopic observation showed that the duration of stigma receptivity was long, lasting until five or six DAA. The stigmas had the moderate receptivity in ’Dawuxing’ and ’Longquan No.5’, then for3days after that, the stigmas had the intense receptivity, while in’Chuannong No.l’, only partial stigmas had a little receptivity at anthesis, and duration of intense receptivity was two to three DAA.2. Pollen tube kineticsThe results of pollen tube kinetics showed that there was no significant difference in pollen germination between self-pollination and cross-pollination. The rate of pollen germination was about35%in2HAP, about12%pollen tubes were observed in upper of styles in4HAP, and the pollen tubes were observed in bottom of styles in12HAP.And pollen tubes that reached upper styles, middle styles and bottom styles in cross-pollination were significantly more than those in self-pollination. Furthermore, there was significant difference in the rate of styles whose upper, middle and bottom were traversed by pollen tubes respectively. The aforementioned results showed that the upper and middle of styles were the main locations of self-incompability in loquat.3. Effects of fowler age on pollen germination and pollen tube growthThe data of the effects of flower age on pollen germination and pollen tube growth showed that the self-incompability was weak. Namely, the number of styles traversed by pollen tubes in self-pollination in big budding period or4-6DAA was more than other days. And the result was evidenced by the higher fruit set in self-pollination in big budding period or4-6DAA.4. Effects of determined factors of EPP on fruit set and seeds setThe results of correlation analysis showed that the EPP was closely related to stigma receptivity, rate of styles traversed by pollen tubes and ovule viability. The higher fruit set and more seeds per fruit in ’Dawuxing’ and ’longquan No.5’ were attributed to the longer period in which the styles had intense stigma receptivity, and more styles and ovules were traversed by pollen tubes, while the lack of seeds was attributed to the shorter period in which the styles had intense stigma receptivity, and lower rate of styles and ovules traversed by the pollen tubes in ’Chuannong No.1’. These differences in stigma receptivity and ovule longevity were not due to external factors, such as climatic conditions, for these experiments were performed simultaneously in the three cultivars. Consequently, the stigma receptivity and ovule degeneration seemed to determine the EPP in loquat. In conclusion, fruit and seed set of ’Chuannong No.1’ was genotype-dependent and can be limited either by stigma receptivity or ovule longevity, while that of ’Dawuxing’ and ’Longquan No.5’ may be limited by stigmatic receptivity.5. Effects of rain on stigma receptivity and pollen viabilityThe effects of rainfall on stigma receptivity and pollen viability were studied in ’Dawuxing’ loquat. The results showed that the stigma receptivity and pollen viability decreased with the rainfall lasting days, which should reduce pollination efficiency and had the negative effects on production of loquat. Moreover, the detection results of stigma receptivity showed that ’Dawuxing’ loquat had a longer flower receptivity period until5 days after anthesis, and the stigma receptivity could be recovered from negative effects of rainfall by a sunny day which could reduced the negative effects of rainfall on production to some degrees.6. Effects of rain on pollen-stigma adhesion and fertilizationThe results of field experiments showed that the number of germinated pollen grains and the percentage of fruit set differed significantly between the-4,-2,-0or+0treatments and the control in the field assays, especially in the case of the-0and+0h treatments in which the stigmas were washed immediately before or after hand-pollination. However, the-8,2,4,8,and12h treatments did not show significant differences from the control. On the other hand, the laboratory experiments showed that all washing treatments reduced the number of germinated pollen grains retained on the stigma, and immersion treatments at-30,-0,+0, or30min, and the additional ISPIS treatment showed significant differences from the control in the numbers of pollen grains retained on the stigmas. In conclusion, the results showed that the adhesion of pollen grains to the stigmas in loquat was rapid and strong, and the simulated rain was not able to wash-off pollen grains completely from the stigma surfaces, except in the-0and+0h treatments.7. Effects of temperature on stigma receptivity and pollen viabilityThe results in studying the effects of temperature on stigma receptivity and pollen viability showed that the blooming and pollen grain dispersion were delayed in the low temperature (5℃), the pollen viability could still reach a higher level (55%) up to the7d after cultivation, the ovule viability could exceed90%, and it was worth noting that the stigma receptivity was renewed and lasted a higher level with the rise of temperature. However, the blooming and pollen grain dispersion were promoted in the higher temperature (25-30℃), resulting in speeded pollen viability and stigma receptivity decrease. The pollen viability was only6.39%in the pollen grains cultivated at30℃at7DAA, simultaneously, the stigma receptivity decreased obviously in the flowers cultivated at30℃at3DAA, and there was no stigma receptivity at5DAA. These results showed that flowers at balloon stage might achieve pollination and fertilization after clearing up of weather after the flowers went through cold and wet weather, which was beneficial to the production in loquat. 8. Effects of temperature on pollen germinationThe difference in pollen germination of the flowers cultivated at different temperatures showed that the time of pollen germination was shortened gradually with the rise of temperature in some scope of temperature, and the rate of pollen germination was increased gradually. The rate of pollen germination was only10%at5℃, while the rate of pollen germination was improved obviously to more than75%at10,15and20℃. However, the rates of pollen germination were declined obviously to65%and45%respectively when the temperatures were raised to25and30℃.9. Effects of temperature on pollen tube growthThe effect of temperature on pollen tube growth was consistent with that on pollen germination. The speed of pollen tube growth was accelerated with the rise of temperature in some range of temperature. The rate of style or ovule traversed by pollen tubes in the flowers cultivated at10,15and20℃was significantly higher than those of flowers cultivated at5,25and30℃. And there were not styles or ovules traversed by pollen tubes in the flowers cultivated120HAP at5and30℃.10. Effects of temperature on ovule viabilityThe results of studying the effects of temperature on ovule viability showed that the rate of degenerated ovules was only about6%when the flowers were cultivated120HAP at5℃, while the rate of degenerated ovules was exceeded85%when the flowers were cultivated120HAP at25and30℃. The aforementioned results implied that the degeneration of ovules was accelerated gradually with the rise of temperature, while the low temperature was beneficial to maintaining the ovule viability, which indicated that the ovules might have the stronger ability of resisting the lower temperature.11. Establishment and optimization of AS-PCR reaction system for loquat’Dawuxing’,’Zaozhong6’and’ Longquan5’were used as materials. The concentrations of Mg2+, Taq polymerase, dNTPs, primer and template DNA were determined. Orthogonal design was used to optimized above components, and time and temperature of various procedures were also improved in AS-PCR in this study. The orthogonal design intuitive analysis method and DPS7.05software were adopted in the analysis of variance. The optimized PCR cocktail of25μL contained10×buffer2.5μL,2.0 mmol/LMg2+,1.5U Taq polymerase,0.5μmol/L primer,80ng template DNA, and0.4mmol/L dNTPs. The optimized reaction procedure was:94℃for1min, followed by35cycles at94℃for30s,54℃annealing for30s,72℃for1min, and terminated with a5min extension step at72℃.12. Identification and cloning novel S-RNases in loquatIn this investigation eight novel genes named S11, S12, S13, S15, S16, S18, S31and S41respectively were cloned from eighteen loquat cultivars using the optimization of AS-PCR reaction system for loquat. And they were deposited in GenBank under the accession numbers of KC131137, KC131138, KC131139, KC131135, KC131136, KC131134, KC131133and JX217035, respectively.13. Identification of S-genotypes of18cultivars in loquatPCR amplification from genomic DNAs of eighteen loquat cultivars was carried out using the aforementioned optimization of AS-PCR reaction system. Then the PCR products were extracted, cloned and sequenced. The amplified fragments were assigned to their respective S-RNases by Blast analysis. The S-genotypes of the eighteen tested self-incompatible cultivars were identified as follows:’Chuannong No.2’(S1-S41),’Huangmi’(S2-S5),’Zaozhong No.6’,’Youjian’ and ’Guifei’(S2-S6),’Longquan5’,’Xiangzhong11’,’Fangguang’,’Dawuxing’ and ’Guanyu’(S2-S41),’Zaohong’(S5-S6),’Dabaili’ and ’Wanzhong’ and ’Mogi’(S6-S13),’Lijian’(S6-S15),’Ruantiaobaisha’(S6-S31),’Chuannong1’(S11-S12) and ’Yoshi’(S16-S41).14. Alignment of the deduced amino acid sequences of S-RNasesThe deduced amino acid homology ranged from53.3to97.8%among these twelve S-RNases alleles in this study, and ranged from48.6to100%with S-RNases alleles from Pyrus and Malus. The deduced amino acid identities were100%between S13-RNase of Eriobotrya and S3-RNase of Pyrus, S2-RNase of Eriobotrya and S8-RNase of Pyrus,96%between S16-RNase of Eriobotrya and S1-RNase of Pyrus,93%between S18-RNase of Eriobotrya and S1-RNase of Malus, and S16-RNase of Eriobotrya and S6-RNase of Malus. The aforementioned results showed that the homology of S-RNases from different genera was higher than the S-RNases from same genus. Furthermore, the results of phylogenetic tree indicated that the thirty three S-RNases alleles from Eriobotrya, Pyrus and Malus were not assembled the specific group within genus, and were distributed randomly in the different locations of phylogenetic tree. Therefore, these results indicated that the S-RNases existence predated the speciation among Eriobotrya, Pyrus and Malus. |
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