Study On Intraspecific Type Classification, Evaluation And Genetic Relationships Of Camellia Meiocarpa

Camellia meiocarpa is a kind of native oil plant species, whose cultivation area andannual output were only less than that of Camellia oleifera. While the genetic improvement,breeding and application of C. meiocarpa was just on its germinal stage. C. meiocarpa is across-pollination plant, which is rich in genetic variation. In order to genetic improve, theintraspecific type of C. meiocarpa was primarily divided based on previous work, and thegenetic relationships of four typical types that were studied on phenotypic, morphological,DNA and transcription level. The main conclusions were drawn as follows:1. The variation types of C. meiocarpa were classified according to the biologicalcharacteristics and population genetic characteristics of C. meiocarpa. The results showed thatthe phenotypic variation of C. meiocarpa plant type, fruit, leaves and flowers were very high.The variations of plant type (43.79%) were the highest among four phenotypic traits, thesecond was fruit traits (26.09%), and variations of leaf (16.84%) and flower (16.53%) were theleast. Based on the results the main principle for classifying the variation types of C.meiocarpa were fruit diameter, pericarp thickness and maturation stage. The intraspecific typeof C. meiocarpa was primarily divided into ten different types.2. Pollen morphology of Camellia meiocarpa and C. oleifera were analysted and thegenetic relationships of C. meiocarpa with other Camellia species were also studied. Underelectron microscope, the pollen morphology, polar view and equatorial view of different typesof C. meiocarpa were similar with Camellia pollen. The pollen gains were prolate orsubsphaeroidal, and its equatorial view was long ellipe, while the polar view was3-labedrounded with3narrow operculums. But there were some distinctive differences in pollen exineornament. The correlation analysis showed that the fruit weight and fresh seed ratio of C.meiocarpa was significant negatively related to pollen polar axis length (P) and positivelyrelated to the distance between colporates, respectively. Compared with the36species pollen morphology of Camellia, the pollen grain size of different types in C. meiocarpa were similarto that of Oleifera and Camellia, which indicated that C. meiocarpa had a close geneticrelationship with Oleifera and Camellia species.3. Morphological makers were used to evaluate the genetic relationships of someCamellia meiocarpa variation types and42Camellia species that seeds were used to beproduced oil. The results showed that the variation of phenotypic traits between46sampleswere very high, the coefficient of variation (CV) of39traits from leaf, flower and fruit wasfrom4.95%to159.41%, and the cv of fruit and seed weight reached up to159.41%and134.72%, respectively. The results showed that46samples were divided into2categories. C.meiocarpa cv.‘Longyan’,‘Yangshi’,‘Zhenzhu’and ‘Yichunbaipi’ were classificated togetherwith C. obtusifolia, then C. brevistyla, C. puniceiflora, C. confusa, C. oleifera, C. weiningensis,C. sasanqua, C. lanceoleosa, C. fluviatilis, C. yuhsienensis, C.grijsii, C. parafurfuracea, C.shensiensis were classificated together.4. AFLP makers were also used to evaluate the genetic relationships of four variationtypes of Camellia meiocarpa and42Camellia species. The results showed that642bands wereobtained using18primer combinations and95.69%of them were polymorphic loci. Thegenetic distance between ‘Yangshi’ and ‘Zhenzhu’ was maximum (0.9539), when that of C.Vietnamensis and C. boreali-yunnanica was minimum (0.5272). The results of clusteringanalysis indicated that46samples were divided into5groups. C. Vietnamensis and C. confusawere respectively clustered group Ⅰand Ⅱ, group Ⅲ were Section Furfuracea, group Ⅳwere Section Camellia and a subset of C.grijsii, C. shensiensis, C. yuhsienensis, which hadsimilar fruit traits. Group Ⅴ were C. meiocarpa, Section Oleifera and most of SectionParacamellia.5. A suitable cDNA-AFLP system for C. meiocarpa and C. oleifera was established afteroptimizing several key factors, including enzyme digestion system, pre-amplification andselective amplification. The results showed that total RNA of the tender leaf and kernel wasextracted by quick extraction kit of EASYspin Plus plant RNA and Trizol method. mRNA was synthesized into double stranded cDNA after it was isolated from total RNA, then it wasdigested completely by EcoRⅠand MseⅠat37℃for5hours. The digested product wasconnected with5U T4ligase at16℃for an overnight. Then reducible pre-amplification wasobtained when ligation product were diluted by10times for pre-amplification. And the optimalconditions for a20μl volume of selective amplification were obtained when the template ofpre-expanded product diluted by30times and with dNTP (10mM)1.5μL, primer (10μM)1.0μL, Taq enzyme1.25U. Moreover,61out of210primer pairs were successfully selected bythe improved method.6. In order to understand genetics bases of differentation of C. meiocarpa variation types,transcripts profile was analysted by cDNA-AFLP.3649transcript-derived fragments (TDFs)were obtained with138primer pairs, a3557TDFs appeared to be polymorphic loci (97.48%).Among them, the specific expression TDFs in C. meiocarpa cv.‘Longyan’ and C. oleifera upto4.27%of the total, the specific expression TDFs in C. meiocarpa cv.‘Longyan’ and‘Yichunbaipi’ was about4.53%, the specific expression TDFs in C. meiocarpa cv.‘Longyan’and ‘Yangshi’ were1.57%, and that of C. meiocarpa cv.‘Longyan’,‘Yangshi’ and‘Yichunbaipi’ were only1.88%, while the specific expression TDFs in C. meiocarpa cv.‘Longyan’,‘Yichunbaipi’ and C. oleifera occupied3.54%, which explained that C. meiocarpacv.‘Longyan’ may have closer relationship with C. meiocarpa cv.‘Yichunbaipi’ and C. oleifera,five materials were divided into3classese, cluster Ⅰ includes C. meiocarpa cv.‘Yangshi’ and‘Zhenzhu’, cluster Ⅱ includes ‘Longyan’ and ‘Yichunbaipi’, while C. oleifera separatecategorization. The result based on cDNA-AFLP was consistent with the AFLP taxa. A subsetof1176differential TDFs were sequenced, which789sequences could be obtained.716ofthem were annotated for their function with GeneBank database by BlastX/N, and functionalontology showed genes related to signal transduction, transcription factor, metabolism,transportation, cell defence mechanisms and protein sythesis.Theses results provided a theory basis for the further study of classificaton, geneticimprovement, resource protection and utilization of C. meiocarpa.