Abstract：The wild apricot (Armeniaca vulgaris) distributed in Yili Valley of Xinjiang, China, is a relic community of the Tertiary warm temperate broad-leaved forest. It has a vast distribution area, large seedling population and abundant genetic diversity. In this study, different materials (young leaves and old leaves) of wild apricot were treated by different preservation methods (fresh, refrigerated, liquid nitrogen preservation). Using Medicago sativa as external standard, the cell ploidy and DNA content of wild apricot were determined by screening and optimizing 10 common dissociation solutions through flow cytometry (FCM). The results showed that the optimum mixture of MgSO4 buffer (MgSO4) and Lysis buffer (LB01) was used to detect the fresh tender leaves, and its coefficient of variation (CV) was the lowest, and the sample rate was 150 events/μL. Aging leaves could not produce a clear main peak. The CV value of samples increased with the increase of cold storage time. The CV value of young leaves within 4 d cold storage was less than 5%. The CV value of the cell nuclear suspension prepared by the old leaves and liquid nitrogen leaves was higher, showing a peak of poor resolution and more background fragments. The results showed that 20 populations of 2 populations of A. vulgaris were diploid and the content of DNA was 288~321 Mb/C. Fresh young leaves or tender leaves in cold storage within 4 days are ideal materials for FCM. The optimized mixture of MgSO4-LB01 was the best dissociation solution for apricot. FCM could be used to detect ploidy and DNA content of apricot plants quickly and accurately. The results of this study provide technical support for ploidy breeding and omics research of apricot plants. The ploidy and DNA content of wild apricot in Xinjiang are preliminarily discussed, which provide important information for the future study of molecular genetics and cytogenetics.
[1] 富新年. 2012. 黄花苜蓿种质倍性鉴定及遗传多样性研究[D]. 硕士学位论文, 甘肃农业大学, 导师: 师尚礼, pp. 1-2. (Fu X N.2012. Study on ploidy level and genetic diversity of Medicago falcata L.[D]. Thesis for M. S., Gansu Agricultural University, Suppervisor: Shi S L, pp. 1-2.) [2] 何天明. 2006. 中国普通杏(Prunus armeniaca)种质资源遗传多样性及紫杏(Prunus dasycarpa)起源研究[D]. 博士学位论文, 山东农业大学, 导师: 陈学森, pp. 1-2. (He T M.2006. Study on genetic diversity of Chinese common apricot (Prunus armeniaca) germplasm and origin of purple apricot (Prunus dasycarpa) [D]. Thesis for Ph. D., Shandong Agricultural University, Suppervisor: Chen X S, pp. 70.) [3] 李贇, 石荫坪, 束怀瑞, 等. 1998. 利用流式细胞光度术鉴定苹果倍性的研究[J]. 西北植物学报, 18(4): 26-30. (Li Y, Shi Y P, Shu H R, et al.1998. Use of flow cytometry for identification of ploidy in apple[J]. Acta Botanica Boreali-Occidentalia Sinica, 18(4): 26-30.) [4] 林峰, 周翔宇, 徐莉, 等. 2017. 几种鼠尾草属植物基因组C值测定[J]. 农业生物技术学报, 25(10): 1622-1628. (Lin F, Zhou X Y, Xu L, et al.2017. Estimation of genomic C value in several species of Salvia[J]. Journal of Agricultural Biotechnology, 25(10): 1622-1628.) [5] 林盛华, 张加延,方成泉.1999. 杏属野生种和栽培品种染色体研究[C]//中国园艺学会. 中国园艺学会成立70周年纪念优秀论文选编, 北京: 中国科学技术出版社, pp.121-125. (Lin S H, Zhang J Y, Fang C Q.1999. Chromosome study of wild species and cultivars of apricot[C]// Chinese Horticultural Society. Selected papers for the 70th anniversary of the establishment of the Chinese Horticultural Society,Beijing: Science and technology of China press, pp. 121-125.) [6] 刘凤霞, 李京一, 王志刚, 等. 2018. 适合流式细胞仪分析的梨叶片细胞核提取缓冲液筛选[J]. 农业生物技术学报, 26(6): 1034-1042. (Liu F X, Li J Y, Wang Z G, et al.2018. The screening of the optimum buffer to produce pear (Pyrus) leaf nuclear suspensions for flow cytometry analysis[J]. Journal of Agricultural Biotechnology, 26(6): 1034-1042.) [7] 田新民, 周香艳, 弓娜. 2011. 流式细胞术在植物学研究中的应用[J]. 中国农学通报, 27(9):21-27. (Tian X M, Zhou X Y, Gong Na.2011.Applications of Flow Cytometry in Plant Research[J]. Chinese Agricultural Science Bulletin, 27(9): 21-27. ) [8] 汪艳, 肖媛, 刘伟, 等. 2015. 流式细胞仪检测高等植物细胞核DNA含量的方法[J]. 植物科学学报, 33(1):126-131. (Wang Y, Xiao Y, Liu W, et al.2015. Operation skills of flow cytometer for detecting nuclear DNA contents in higher plant cells[J]. Plant Science Journal, 33(1):126-131.) [9] 王利虎, 吕晔, 罗智, 等. 2018. 流式细胞术估测枣染色体倍性和基因组大小方法的建立及应用[J]. 农业生物技术学报, 26(3): 511-520. (Wang L H, Lv Y, Luo Z, et al.2018. Establishment and application of a method for chromosome ploidy identification and genome size estimation using flow cytometry in Ziziphus jujuba[J]. Journal of Agricultural Biotechnology, 26(3): 511-520.) [10] 王晓庆, 骆军, 施春晖, 等. 2014. 一种利用流式细胞术鉴定梨倍性的方法[J]. 中国农学通报, 30(31): 46-51. (Wang X Q, Luo J, Shi C H, et al.2014. A method of ploidy identification in pear using flow cytometry[J]. Chinese Agricultural Science Bulletin, 30(31): 46-51.) [11] 魏高明, 方炎明. 2015. 栎属植物流式细胞术实验体系的建立与优化[J]. 南京林业大学学报(自然科学版), 39(1): 167-172. (Wei G M, Fang Y M.2015. Establishment and optimization of an experiment system for flow cytometry in Quercus[J]. Journal of Nanjing Forestry University (Natural Sciences Edition), 39(1): 167-172.) [12] 于红梅, 王静, 赵密珍, 等. 2012. 利用流式细胞仪检测草莓倍性方法的优化[J]. 南方农业学报, 43(10): 1530-1533. (Yu H M, Wang J, Zhao M Z, et al.2012. Optimization of strawberry ploidy identification method using flow cytometry[J]. Journal of Southern Agriculture, 43(10): 1530-1533.) [13] 苑兆和. 2007. 杏属植物种质资源分子系统学研究[D]. 博士学位论文, 山东农业大学, 导师: 陈学森, pp. 70. (Yuan Z H.2007. Studies on molecular systematics of apricot genus (Armeniaca Mill.) Germplasm[D]. Thesis for Ph.D., Shandong Agricultural University, Suppervisor: Chen X S, pp. 70.) [14] Arumuganathan K, Earle E D.1991. Estimation of nuclear DNA content of plants by flow cytometry[J]. Plant Molecular Biology Reporter, 9(3): 229-241. [15] Baird W V, Estager A S, Wells J K.1994. Estimating nuclear DNA content in peach and related diploid species using laser fow cytometry and DNA hybridization[J]. Journal of the American Society for Horticulturalence, 119(6): 1312-1316. [16] Ben T H, Walker D, Ben M S, et al.2015. DNA variation and polymorphism in Tunisian plum species (Prunus spp): contribution of flow cytometry and molecular markers[J]. Genetics & Molecular Research Gmr, 14(4): 18034. [17] Cires E, Cuesta C, Peredo E L, et al.2009. Genome size variation and morphological differentiation within Ranunculus parnassifolius group (Ranunculaceae) from calcareous screes in the Northwest of Spain[J]. Plant Systematics & Evolution, 281(1-4): 193-208. [18] Galbraith D W, Harkins K R, Maddox J M, et al.1983. Rapid flow cytometric analysis of the cell cycle in intact pant tissues[J]. Science, 220: 1049-1051. [19] Hopping M E.1993. Preparation and preservation of nuclei from plant tissues for quantitative DNA analysis by flow cytometry[J]. New Zealand Journal of Botany, 31(4): 391-401. [20] Loureiro J, Rodriguez E, Doležel J, et al.2007. Two new nuclear isolation buffers for plant DNA flow cytometry: a test with 37 species[J]. Annals of Botany, 100(4): 875-888. [21] Marie D. Brown S C.1993. A cytometric exercise in plant DNA histograms with 2C values for 70 species[J]. Biology of the Cell, 78:41-51. [22] Michaelson M J.1991. Comparison of Plant DNA contents determined by feulgenmicrospectrophotometry and laser flow cytometry[J]. American Journal of Botany, 78(2): 183-188. [23] Otto F.1990. DAPI staining of fixed cells for high-resolution flow cytometry of nuclear DNA[J]. Methods in Cell Biology, 33(33): 105. [24] Pfosser M, Amon A, Lelley T, et al.1995. Evaluation of sensitivity of flow cytometry in detecting aneuploidy in wheat using disomic and ditelosomic wheat-rye addition lines[J]. Cytometry Part B Clinical Cytometry, 21(4): 387-393. [25] Postman J, Bassil N, Bell R.2015. Ploidy of USDA world pear germplasm collection determined byflow cytometry[J]. Acta Horticulturae, (1094): 75-81. [26] Puite K J, Broeke W R R T.1983. DNA staining of fixed and non-fixed plant protoplasts for flow cytometry with hoechst 33342[J]. Plant Science Letters, 32(1): 79-88. [27] Sakhanokho H F, Rinehart T A, Stringer S J, et al.2018. Variation in nuclear DNA content and chromosome numbers in blueberry[J]. Scientia Horticulturae, 233: 108-113. [28] Suda J, Trávnícek P.2006. Estimation of Relative Nuclear DNA Content in Dehydrated Plant Tissues by Flow Cytometry[M]// Robinson J P. Current protocols in cytometry. pp: 30. [29] Yan J, Zhang J, Sun K, et al.2016. Ploidy level and DNA content of erianthusarundinaceus as determined by flow cytometry and the association with biological characteristics[J]. PLoS One, 11(3): e0151948.
