| Garcinia paucinervis is mainly distributed in Limestone Mountains in Western Guangxi and Southeastern Yunnan Provinces,China.G.paucinervis has economic,ecological,ornamental and medicinal value.Because of the wealth of its wood,G.paucinervis was overharvested and it became a rare and endangered species.Expanding its application in urban landscaping will be a more realistic and feasible way to protect it.And it could be developed as a spring leaf tree and a landscape tree in South and parts of Southwest China.However,the resource has been few cultivated artificially,and its genetic characteristics and reproduction mode have not been studied systematically.Moreover,the slow germination of seeds seriously impedes its rapid propagation and large-scale cultivation.In this study,based on the investigation of the habitats and resource status of G paucinervis,the conditions of introduction and cultivation were predicted.The genetic diversity and population genetic differentiation were discussed in terms of population genetics,and populations with prior conservation units and great development values could be found out.The morphology,germination,storage characteristics,dormancy type and molecular mechanisms of the seeds were also studied to find the efficient reproduction mode of G paucinervis.The study will provide a scientific basis for studying the molecular mechanism of dormancy of G.paucinervis seeds and even the recalcitrant seeds with dormancy characteristics,so as to lay a foundation for the resource conservation and development,genetic improvement,breeding,cultivation and landscape use.The results are as follows:(1)The results of investigation for 14 populations in 13 counties at maturity showed that G.paucinervis was distributed in north tropics and south subtropics with an average annual temperature of 20?22?,and prefers loose,moist neutral or slightly alkaline lime soil.G paucinervis could coexist with many vegetative plants of tropical and subtropical species.Populations distributed dispersedly.Most of intra-population individual numbers were below 100.The trees with diameter at breast height over 50 cm and fruiting individuals were rare.It was difficult for G.paucinervis to gain the advantage in the competition of resources.Most of the populations' habitats were affected by human activities.The age structures of the populations were unbalanced except for the population in Nonggang National Nature Reserve.The young seedlings were few under the woods in habitats.The weak natural regeneration ability of G.paucinervis existed.It is inferred that G paucinervis could be introduced in warm and rainy areas,and the requirements for soil fertility and light were not strict.(2)Nineteen polymorphic microsatellite markers were used to investigate genetic diversity and population structure of the 11 extant natural populations(a total of 360 individuals)of G.paucinervis in Guangxi and Yunnan Provinces.Our results revealed a low to moderate genetic diversity in G.paucinervis remnants(the expected heterozygosity was 0.468,Shannon's diversity index was 0.885,the allelic richness was 3.334).The highest genetic diversity was found in Hekou,Long'an and Malipo populations,and the lowest in Xincheng population.The global inbreeding coefficient(FIS=0.051)showed a significant deviation from Hardy-Weinberg equilibrium,implying that the risk of inbreeding depression was probably due to severe habitat fragmentation and decreasing population sizes.Significant bottlenecks were detected in Chongzuo and Hekou populations.There was relatively high genetic differentiation(FST=0.174)and relatively limited gene flow(Nm=1.187)across G.paucinervis populations.There were 28.127%variance among populations and 71.873%variance within populations,respectively.The genetic distance was not related to the geographical distance,suggesting a limitation of gene flow.The Malipo population from Yunnan Province with the largest number of private alleles probably existed the most developmental values.And the population could be classified as an independent cluster separated from other populations,which should be considered as a prior conservation unit and a prior selection for genetic improvement material.It is recommended to use the seeds of the Hekou,Long'an and Malipo populations showing higher genetic diversity as the sources for landscape.(3)The basic characteristics of G.paucinervis seeds were observed.And the effects of various ecological factors and biological characteristics of the seed on seed germination were studied.The results show that there was evident morphological differentiation among the fruits and seeds of three populations.The seeds contained ergastic substances,such as fat,protein and starch.The germination processes of G.paucinervis seeds were slow and irregular.The seeds were preserved after seed germination,and adventitious roots eventually replaced the primary roots and then became taproots,gradually.Both endocarp and seeds without coats delayed seed germination.The seeds could germinate at 25?35?,but the germination was inhibited at 35?.The seeds did not germinate at below 18? and above 37?.The germination process could be obviously accelerated at 32?.The G.paucinervis seed was the intermediate light seed,the germination speed was the fastest under 3000 lx,12h·d-1 condition.Sand and peat soil were the most suitable germination substrates.The seed germination was slow when seeds were sowed in limestone soil and seeds could not germinate under 20%moisture content.In the same burial depth,compared with limestone soil as substrate,the germination energy(GE)and the germination percentage(GP)of seeds sowed in sand was higher.With the increase of burial depth,both GE and GP decreased.There were significant differences in GP among the seeds of three populations.Seed weight had no significant effect on seed germination.Thus,the temperature range for the G.paucinervis seed germination is narrow and seed germination has a higher requirement for soil permeability and moisture.Its slow and irregular germination characteristics severely limit its cultivation and popularization.(4)The G paucinervis seeds are sensitive to water loss,and seed storage restricts its seedling breeding.Its desiccation tolerance and storage method were studied accordingly.The results show that the initial moisture content of G.paucinervis seeds was 45.29%.The seeds almost lost their moisture when placed after 35 d at room temperature.When the dehydration rate was less than 18%,GP and the water absorption rate after rewatering were not significantly different from those of fresh seeds.Furthermore,when the dehydration rate exceeded 18%,GP and the water absorption rate after rewatering were significantly decreased.GP was 0 at dehydration rate 42%.The critical moisture content of seeds was 27.29%,and the lethal moisture content of 50%was 12.72%.The relative conductivity,the contents of soluble sugar and proline rose gradually with the deepening of seed dehydration.The content of malondialdehyde(MDA)changed little when the dehydration rate was less than 24%,and increased significantly when exceeding 24%.Both activities of superoxide dismutase(SOD)and peroxidase(POD)showed a trend of fluctuation and peaked at dehydration rate 18%.Seeds with desiccation storage for one month at room temperature and seeds with storage in damp sand at-1? and-20? for one month all could not germinate.GP of seeds with water immersion for 1 month dropped significantly.The germination processes of seeds with storage in damp sand for one,three or six months at 4? were delayed,but there was no significant effect on GP.It indicated that when the dehydration rate of G.paucinervis seeds was less than 18%,seeds could regulate resistance to maintain normal metabolisms of cells.The seeds could endure a certain degree of dehydration and low temperature.When the dehydration rate exceeded 18%,the metabolism of seeds was unbalanced,and then deteriorated until death.The seeds belong to minimally recalcitrant seeds.Storage in damp sand at 4?(moisture content 7.5%)is a good method for short-term storage of G.paucinervis seeds.(5)Based on the germination processes of G.paucinervis seeds were slow and irregular,the studies have been undertaken on the reasons and types of seed dormancy.Also,the methods of releasing dormancy of seeds were studied based on soaking seeds with different exogenous substances.The results showed that the seed coat of G.paucinervis was mainly composed of parenchymatous layer without palisade layer,and the cuticle of abaxial surface was thinner.The seed coat had less inhibition to seed water absorption.With the extent of the elimination of seed coats and endosperm at the side of micropyle,the germination processes of seeds extended gradually,and its germination percentage was even reduced.The treatment of cutting off at the side of hilum had little effect on seed germination.There were probably endogenous inhibitors hindering germination of G.paucinervis seeds and seedling growth inside endocarp,seed coats,endosperm and embryos.The embryo rate of fresh seeds reached 86.12%,and no significant change was found after cold stratification.A better way to break seed dormancy was soaking seeds with 0.36 mmol·L-16-BA for 24 h.The best way for GP combined with the growth of seedlings was soaking seeds with 0.22 mmol·L-1 6-BA for 24 h.During the germination process of seeds,the content of ABA(abscisic acid)decreased.With seed germination,the ratios of GA(gibberellin)to ABA and IAA(indole-3-acetic acid)to ABA increased significantly.The ratios of endogenous accelerants to inhibitors were gradually partial to acceleration.However,the content of ethylene decreased significantly from sowing to germination.Thus,G.paucinervis seeds belong to physiological dormancy caused by existence of endogenous inhibitors and lack of germination accelerants.It belongs to minimally recalcitrant seeds with physiological dormancy characteristics.(6)In order to study the molecular mechanism of the dormancy of G paucinervis seeds,the transcriptome sequencing were carried out on the fresh mature seeds(OS),the ungerminated seeds sown for 60 days(UG)and the germinated seeds sown for 60 days(GS).It was found that the transcriptome levels of embryos and endosperm in three groups of samples were significantly different.Of the 120040 unigenes obtained,46808 and 56224 were annotated in GO and KEGG databases,respectively.The screened differentially expressed genes(DEGs)were analyzed through results of pathway enrichments.During the process of seed dormancy release,carbohydrate metabolism,fat metabolism,protein synthesis and catabolism,signal transduction,hormone action,cell division and metabolism were more active.During the process of seed dormancy release,the 40 DEGs related to hormones such as ABA,GA,auxin and ethylene were screened(7 genes related to hormone synthesis,decomposition or derivation,3 receptors,17 genes related to signal transduction,12 transcription factors and 1 other gene).After sowing,the variation trend of relative expression of the four endogenous hormone-related genes(CYP707A2 GA3ox2 GAI and ZEP)corresponded to the decrease of ABA content and the increase of GA content during dormancy release of seeds.In terms of CYP707A2 and GA3ox2,the significant difference of relative expression between at 25? and 32?corresponded to the rapid germination of seeds at 32? at the same time points.Combined with the related gene expression of ABA,GA and ethylene in transcriptome sequencing results,it was speculated that the decreased ratios of ABA-GA in embryo and endosperm probably played a key role in the breaking of G.paucinervis seed dormancy.Ethylene was probably involved in the regulation of seed dormancy. |
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