| Schima superba is the main constructive species in humid subtropical evergreen broadleaf wood in east China.The plant species is not only used as a biological firebreak,but it is also considered a valuable timber tree,with fine timber quality and strong resistance.We have constructed more than 100 ha of S.superba clones seed orchard,since developed S.superba breeding work at 2001.But the reproductive biology characteristics and genetic management of S.superba seed orchard are lacking in systematic research,that inhibitted the management control and fine variety production of seed orchard.In this thesis,the research object is located in S.superba clonal seed orhcard which has entered normally blossom.Further study the floral syndrome,pollination mechanism,and breeding system,in order to reveal the reproductive biology characteristics and provide practical guidance and scientifically basis for seed production,resource utilization and crossbreeding of S.superba.Simultaneously,the synchronization,genetic mating system,and pollination regularity are also analyzed to provide important theories for genetic management in higher quality and yield of the clonal seed orhcard of S.superba.The results are as follows:1.The floral syndrome,pollen viability,and stigma receptivity were studied by location observations.Results showed,S.superba bloomed from May to July.The life span of single flower was approximately 4-5 days.The white flowers were arranged on a single racemose containing an average of 7 flowers.The stigma receptivity started from bloom and remained highly receptive for the entirety flowers’ lifetime.The viability of the pollen showed a sharp decreasing trend which decreased rapidly from 83.78% to 5.45%.On the day of anthesis,the stigma rapid elongation,until the stigma slightly beyond anther.As a result,there is a spatial isolation of anther canister and stigma with in the same floral.The outcrossing index(OCI)value was 4 and the pollen ovule ratio(P/O)value of S.superba was 6686.67,and controlled pollination test showed that there was no phenomenon of apomixis,and S.superba was supposed to self-incompatibility plant and pollinators depended.2.S.superba is a kind of xenogamous plants and must depend on the pollinators for its production.Apis cerana,Protaetia brevitarsis,and Popillia mutans were the major pollinators.The peak types of foraging activities were from 10:00 to 11:00 in A.cerana and P.mutans,and there was no peak type of foraging activities in P.brevitarsis.The body of three pollinators were able to carry pollens.The carried quantity of pollen grains of P.brevitarsis was significantly higher than P.mutans(P<0.001)and A.cerana(P<0.001),and the carried quantity of pollen grains of P.mutans was significantly higher than A.cerana(P<0.001).There were different carried amount of pollen at different parts.The main parts of carried pollen were thoracic and abdominal of all three pollinators,meanwhile,thoracic and abdominal were the most frequent and strength part which touched the anther and stigma.So,the pollination methods of all three pollinators were abdomen touching pollination.The removed and deposited pollen grains and pollinator efficiency of A.cerana were all less than P.brevitarsis and P.mutans,but not significant(P>0.05).However,A.cerana was dominant in visitation frequency,which had 29 and 8.3 times of P.brevitarsis and P.mutans,respectively.The duration of each visit of A.cerana was lower than that of P.brevitarsis and P.mutans.Therefore,we suggested that A.cerana was the most efficient pollinator for S.superba.3.Further comparative studied the compatibility between cross and self-pollination of S.superba.The results showed that both the pollen of self and cross-pollination could normally germinate on the stigma,and the self and cross-pollination pollen tube could successfully go into the base of style.The cross-pollination pollen tube could successfully go into the ovary and embryo sac,while the self-pollination pollen tube could not go into the ovary and embryo sac.Studies showed that S.superba was late-acting self-incompatibility(LSI)plants.The activity of enzyme(SOD,POD and CAT)in ovaries were all increased after self and cross-pollination.There were no significant difference of enzymatic activity between self and cross-pollination,expect the activity of POD in selfing ovaries at 48 h was significant higher than crossing ovaries.There was the biggest difference of content of amino acid between self and cross-pollination at 48 h.The content of arginine(Arg)in self-pollination ovaries was significant higher than crosspollination(P<0.001),indicated that the change of Arg may be correlated with the late-acting self-incompatibility of S.superba.The auxin(IAA)and zeatin(ZT)increased in initial stage and then declined after self and cross-pollination.The content of IAA and ZT in cross-pollination ovaries were significant higher than self-pollination at 48 h(P<0.05),indicated that high level of IAA and ZT in the ovaries were benefitted to pollination fertilization.4.The results of differential proteomics showed that there were 82 different protein spots in ovaries after 48 h of pollination between self-pollination and cross-pollination.58 proteins were identified homologous to known proteins by MASCOT analysis,including 15 up-expressed and 43 down-expressed in self-pollination ovaries compared with that in cross-pollination ovaries.Actin and proteasome were higher expressed in cross-pollination ovaries compared with that in self-pollination ovaries.But peroxidase,protein disulfide isomerase and lectin had a lower expressed level in cross-pollination ovaries than self-pollination ovaries.The transcriptome information was obtained from S.superba ovaries RNA after 48 h self-and cross-pollination by RNA-seq.509 differentially expressed genes were identified that compared with selfed and crossed ovaries.The expressed level of signal transduction related components(such as Ca2+ signaling,protein kinase and protein phosphatases),defense-associated(cytochrome P450 and MLP31),and amino acid metabolism(oxaloacetate decarboxylase)genes in self-pollination ovaries were significantly higher than cross-pollination ovaries.The expressed level of promotes growth hormone metabolism genes in self-pollination ovaries were significantly lower than cross-pollination ovaries.Selfed ovaries also specifically or preferentially express two unique transcription factor genes(MYB and bHLH)that could potentially be involved in the novel mechanisms of LSI.The expression of these unigenes indicated by qRT-PCR agreed well with transcriptome analysis.The present study indicated that the late-acting self-incompatibility of S.superba was a process that active inhibited the growth of self-pollination pollen tube in the ovaries,along with a series of signal transduction and defense response.5.Based on collected data of florescence period and flower number of 19 clones in a seed orchard of S.superba for two consecutive years,the flowering phenology and synchronization of each clone were estimated.The correlation analysis showed that there were a significant positive correlation between the number of flowers and flowering duration and fruit set.The analysis of variance showed that there was a significant variance of flowering synchronization(C)value between every clones during the year(P<0.01).The C value ranged from 0.552 to 0.857 and from 0.406 to 0.808 in 2015 and 2016,respectively.The coefficient of variation of C value were ranged from 12.016% to 46.476% and 15.375%~51.202%,respectively,during the year.The average index of flowering synchronization was 0.758 and 0.713 in 2015 and 2016,and there was no significant variance of C value between years(P>0.05).The coefficient of correlation between 2015 and 2016 was 0.229,indicated that there was correlation of C value between years.The C value between years was stability in the seed orhcard.We could screen and optimize the clones in seed orchard based on the flowering phenology and synchronization,and combined with hand-pollination to decrease the difference of flowering synchronization between clones,that would benefit to increase the yield and quality of seed in S.superba seed orhcard.6.Simultaneously,a total of 44 parents and 328 open-pollination progenies from 11 clone individuals in the initial stage of S.superba seed orchard were identified by 13 polymorphic SSR loci.The progeny population had all alleles detected in maternal population.The level of genetic diversity of progeny population was lower than parent population,but there is no significant differences.Observed heterozygosity was higher than parent population,but there was no significant difference.The results indicated that the genetic uniformity and the ratio of observed heterozygosity per tree accounted for an increase than their parents,but the difference was small.There were an obvious heterozygosis excess in progeny population(F=-0.143).Results from the multilocus mating system analysis indicated that the multilocus outcrossing rate in the seed orchard was 1.000,and there was no significant inbreeding between parents.A small number of biparental inbreeding was detected in this seed orchard.We found a small number of effective pollen donors.The D-value between multilocus correlation of paternity and singlelocus correlation of paternity is greater than zero,but not significant,indicating that only few part of the pollen donor is close relative relationship.The difference of correlation of paternity between the 11 families was from 0.210 to 0.762.And the number of valid pollen donor was 1.3-4.8,showed that the degree of correlation was inconsistent in each paternal,the highest was 31 clones,and the lowest was 48 clones.On the whole,there was a higher outcrossing rate,and a lower biparental inbreeding in this seed orchard.The progeny of the generation clonal seed orchard still had abundant genetic diversity,gene exchange among clones was relatively sufficient,and the progenies could keep the high levels of genetic diversity of parents.7.The patterns of effective pollen dispersal were explored in an experimental population of the clonal seed orchard of S.superba.13 microsatellite markers were highly polymorphic for the parent and progeny populations,with an average polymorphic information content(PIC)was 0.725.The average observed and expected heterozygosity(Ho and He)were 0.679 and 0.714,respectively.A total of 203 full-sib families were identified and were nested among the 11 studied seed donors under 95% confidence level.Every mother tree with 18 male parents produced progenies.The self-crossing rate reached 1.5% in open-pollination condition,and outcrossing was primary mating mode of the clonal seed orchard of S.superba.The male reproductive success rate from 0.49% to 7.77%,with an average of 2.44%.The mating success rate was more than 80% within 60 m,the maximum dispersal distance reached 120 m.There were some pollen contamination(7.01%)from outside the seed orchard.Overall,the pollen dispersal was uniform in the seed orchard,and the isolation of seed orchard must be far more than 60 m from the source of contamination,the best more than 120 m,to avoid high pollen contamination that caused by long-distance pollen movement. |
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