| Cornus wilsoniana(Wanger.),belonging to the genus Cornus(Cornaceae),is an important woody oil plant resource in China with great prospect of industrialization development.Lack of systematic and profound study on the national wide germplasm resources collection and the evaluation of genetic diversity,fruit development and fatty acid accumulation in molecular level,due to its wild or semi-artificial cultivation status,has seriously affected the process of breeding and industrialization of Cornus wilsoniana.In this study,516 plus trees were collected from 60 counties around 8provinces/municipality/autonomous regions,and two germplasm resource banks were established.Using these germplasms,the phenotype variations on fruit and oil traits,the photosynthetic physiology of plus tree clones,the gene expression involved in oil synthesis at different stages of fruit development,the development of EST-SSR and the population genetic diversity were studied.On this basis,the core germplasm construction of Cornus wilsoniana and molecular identification were carried out.,which provided theoretical basis for evaluation of germplasm resources and genetic breeding of Cornus wilsoniana in China.The main conclusions are as follows:(1)Two germplasm resource banks containing 516 plus trees from distribution area of Cornus wilsoniana were established.After investigation and selection,516 plus trees were collected from 8provinces/municipalities/autonomous regions,including 171 from Hunan,108 from Chongqing,60 from Guangdong,50 from Guangxi,46 from Hubei,42 from Jiangxi,24 from Fujian and 15 from Guizhou.Two germplasm resource banks of Cornus wilsoniana were established in the forest farm of Hunan Academy of forestry and Longshan county of Hunan Province respectively.(2)Significant differences in leaf morphology and photosynthetic physiology among the plus tree clones of Cornus wilsoniana were found.The analysis of leaf morphological indexes based on 20 plus tree clones from Jiangxi and Guangdong provinces showed that there were differences on leaf length,width,length/width ratio and leaf area among plus trees,and the variation of leaf area was the most significant.The fitting results of photosynthesis-light response curves showed significant differences on the maximum net photosynthetic rate,apparent quantum efficiency and dark respiration rate of leaves among plus trees.The average maximum net photosynthetic rate was 10.61 ?mol·m-2s-1 with CV at10.60 %;the average apparent quantum efficiency was 0.034 ?mol·mol-1 with CV at 18.00 %,and the average respiration rate was 1.32?mol·m-2s-1 with CV at 21.50 %.(3)The population variation characteristics of fruit traits of Cornus wilsoniana were analyzed.The thousand grain weight(TGW)of fresh fruits,diameter of fresh fruits,TGW of seeds,seed diameter,and oil content of fruits of plus trees showed highly significant differences among populations.The average TGW of fresh fruits was 143.89 g with coefficient of variation(CV)at 22.24%;the average diameter of fresh fruits was 0.65 cm with CV at 11.76%;the TGW of seeds was 71.77 g with CV at 37.16%;the average diameter of seeds was 0.48 cm with CV at 11.38%;the average oil content of fruits was 27.76%with CV at 30.43%.(4)The fatty acid contents in fruits of plus trees among different provenances were significantly different.The variations of several fatty acid contents,such as linoleic acid,oleic acid and arachidic acid,in fruits of the plus trees among different provenances were significant.The average contents of linoleic acid,oleic acid and arachidic acid were 38.93%,33.55% and 1.32%,respectively.The content of linolenic acid in fruits of the plus trees among different provenances was various,with CV at 73.09%;while the variation of palmitic acid contents in fruits of the plus trees from different provenances was relatively limited,with CV at 13.76 %.(5)Gene expression patterns involved in oil synthesis during fruit development of Cornus wilsoniana were analyzed.Based on RNA-seq,the transcriptome analysis was carried out on fruits at different developmental stages,i.e.the early stage of fruit embryo development(T1),the early stage of fruit oil formation(T2),the peak period of fruit growth(T3),and the mature stage of fruit(T4).It was found that there was no significant difference in the expression of genes related to oil synthesis between T1 and T2 which were the stages for fruit growth and development.The DEGs related to oil synthesis,such as ACAA,KAS II,nd AAD,were all up-regulated during the period from T2 to T3,which was the initial stage for oil synthesis.Genes involved in the fat synthesis,elongation and desaturation pathway,such as MAT,KAR,HAD,EAR,KAS II,KCS,FATA,FAD2,ACSL,PP,PDAT1,MAGL and FAD2,were significantly up-regulated during the period from T3 to T4,which was the oil accumulation stage.(6)Core germplasms of Cornus wilsoniana were screened based on EST-SSR markers.Two hundred pairs of EST-SSR primers were developed based on transcriptome data of fruits and 29 valid pairs with high polymorphism and good stability were screened through 2 round tests.A total of 362 polymorphic loci were amplified by the 29 pairs of EST-SSR primers in 224 plus trees of Cornus wilsoniana.The average allelic variation was up to 12.48.In the polymorphic loci,133 unique loci were detected,accounting for 36.74 %.The provenance with the largest number of unique polymorphic loci was Hunan.After analysis,the average genetic heterozygosity of the plus tree populations was 0.5395,showing a relatively high level of population genetic diversity;the AMOVA analysis explained that the genetic variation among populations accounted for 12 %.According to sampling strategies based on the classification of population genetic structure of germplasm resources,the clustering of genetic relationship,and the number and proportion of specific SSR polymorphic sites,a core germplasm population SW3 of Cornus wilsoniana was constructed at a sampling rate of 30%,including 9 from Guangdong,3 from Guangxi,4 from Guizhou,5 from Hubei,25 from Hunan,4 from Jiangxi and 13 from Chongqing.The diversity parameters of the SW3 were almost consistent with the original plus tree population.(7)Molecular fingerprints and a two-dimensional code identification system of 63 core germplasms of Cornus wilsoniana were constructed.Sixty-three core germplasms could be distinguished based on 29 EST-SSR locus.Furthermore,fingerprint for each core germplasm was generated.Two-dimensional code for each core germplasm was also formed based on its fingerprint in QR-code software.The results will provide useful information for collection,management,protection and utilization of Cornus wilsoniana. |
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