Establishment and application of a flow cytometry method for chromosome ploidy identification of Cyclocarya paliurus

doi:10.12302/j.issn.1000-2006.202302014

JOURNAL OF NANJING FORESTRY UNIVERSITY ›› 2024, Vol. 48 ›› Issue (2): 61-68.doi: 10.12302/j.issn.1000-2006.202302014

Previous Articles Next Articles

Establishment and application of a flow cytometry method for chromosome ploidy identification of Cyclocarya paliurus

SONG Ziqi¹^,²(), BIAN Guoliang¹, LIN Feng³, HU Fengrong⁴, SHANG Xulan¹^,^*()

1. Co-Innovation Center for Sustainable Forestry in Southern China, College of Forestry and Grassland,Nanjing Forestry University, Nanjing 210037, China
2. Institute of Soil Science, Chinese Academy of Sciences, Nanjing 210018, China
3. Advanced Analysis and Testing Center, Nanjing Forestry University, Nanjing 210037, China
4. College of Landscape Architecture, Nanjing Forestry University, Nanjing 210037, China

Received:2023-02-14 Revised:2023-03-23 Online:2024-03-30 Published:2024-04-08

Abstract

Abstract:

【Objective】 To provide technical support and basic data for the germplasm identification and genetic breeding of Cyclocarya paliurus, this study determined the chromosome ploidy using flow cytometry. 【Method】 C. paliurus leaves were used as study materials to compare the ploidy detection effects of different nuclei isolation buffers, centrifugation treatments, and leaf preservation methods. The ploidy identification method for C. paliurus using flow cytometry was as follows: 0.50-1.00 cm² leaves of the reference sample and the test sample were mixed and chopped with 1 mL of mGb buffer. After filtration, 20 μL PI was added for staining for 1 min. The ploidy of 1 395 C. paliurus germplasm resources was determined by the established method. 【Result】 The optimal choice for isolating nuclei and achieving a clear peak in the resulting nucleus suspension was the use of Modified Gitschier buffer (mGb). Nuclear suspensions could be directly stained after filtration without centrifugation treatment. The optimal detection effect was obtained for leaves preserved at 4 ℃, and the most suitable storage time was 7 d. Drying leaves with silica gel yielded superior test results compared to freezing them, and the most suitable storage time for silica gel drying was 150 d. The coefficients of variation of 100 test samples ranged from 2.13% to 5.04%. If the estimated value of ploidy was 1.80-2.40, the sample was identified as diploid. If the estimated value of ploidy was 3.60-4.20, the sample was identified as tetraploid. When the estimated value of ploidy was 3.00 ± 0.40, the reference sample with the same ploidy as the initial judgment was used for the second detection. Using this method for the ploidy identification of 1 395 germplasm resources 104 diploids and 1 291 tetraploids were detected. 【Conclusion】 Samples with an abnormal estimated value of ploidy could be identified quickly by the second detection with the same ploidy standard. The method is simple, efficient, and accurate, and provides an effective method for ploidy identification of C. paliurus germplasm.

Key words: Cyclocarya paliurus, ploidy identification, flow cytometry, nuclei isolation buffer, internal standard method

CLC Number:

S722

SONG Ziqi, BIAN Guoliang, LIN Feng, HU Fengrong, SHANG Xulan. Establishment and application of a flow cytometry method for chromosome ploidy identification of Cyclocarya paliurus[J]. JOURNAL OF NANJING FORESTRY UNIVERSITY, 2024, 48(2): 61-68.

Figures/Tables 7

Fig. 1

Fig. 2

Fig. 3

Table 1

Fig. 4

Fig. 5

Table 2

References 40

[1]	方升佐. 青钱柳产业发展历程及资源培育研究进展[J]. 南京林业大学学报(自然科学版), 2022, 46(6): 115-126.
	FANG S Z. A review on the development history and the resource silviculture of Cyclocarya paliurus industry[J]. J Nanjing For Univ (Nat Sci Ed), 2022, 46(6): 115-126. DOI: 10.12302/j.issn.1000-2006.202206019.
[2]	洪俊溪. 青钱柳人工林材性试验研究[J]. 福建林学院学报, 1997, 17(3): 214-217.
	HONG J X. Experimental study on wood properties of Cyclocarya paliurus artificial forest[J]. J Fujian Coll For, 1997, 17(3): 214-217.
[3]	ZHOU M M, LIN Y, FANG S Z, et al. Phytochemical content and antioxidant activity in aqueous extracts of Cyclocarya paliurus leaves collected from different populations[J]. Peer J, 2019, 7: e6492. DOI: 10.7717/peerj.6492.
[4]	YANG H M, YIN Z Q, ZHAO M G, et al. Pentacyclic triterpenoids from Cyclocarya paliurus and their antioxidant activities in FFA-induced HepG2 steatosis cells[J]. Phytochemistry, 2018, 151: 119-127. DOI: 10.1016/j.phytochem.2018.03.010.
[5]	FU X X, ZHOU X D, DENG B, et al. Seasonal and genotypic variation of water-soluble polysaccharide content in leaves of Cyclocarya paliurus[J]. South For, 2015, 77(3): 231-236. DOI: 10.2989/20702620.2015.1010698.
[6]	QU Y Q, SHANG X L, ZENG Z Y, et al. Whole-genome duplication reshaped adaptive evolution in a relict plant species, Cyclocarya paliurus[J]. Genomics Proteomics Bioinformatics, 2023:S1672-229(23)00033.DOI: 10.1016/j.gpb.2023.02.001.
[7]	SOLTIS P S, SOLTIS D E. The role of hybridization in plant speciation[J]. Annu Rev Plant Biol, 2009, 60(1): 561-588. DOI: 10.1146/annurev.arplant.043008.092039.
[8]	PIKAARD C S. Genomic change and gene silencing in polyploids[J]. Trends Genet, 2001, 17(12): 675-677. DOI: 10.1016/s0168-9525(01)02545-8.
[9]	何法慧, 左倩倩, 于景金, 等. 35份狗牙根种质材料指纹图谱构建及染色体倍性鉴定[J]. 南京农业大学学报, 2023, 46(1):42-54.
	HE F H, ZUO Q Q, YU J J, et al. Fingerprint construction and chromosome ploidy identification of 35 germplasms in bermudagrass[J]. J Nanjing Agric Univ, 2023, 46(1):42-54.DOI: 10.7685/jnau.202201035.
[10]	赵帅琪, 张伟伟, 牛俊芳, 等. 森林草莓和栽培草莓在果实发育和成熟过程中细胞壁变化的比较[J]. 植物生理学报, 2021, 57(12): 2323-2336.
	ZHAO S Q, ZHANG W W, NIU J F, et al. Comparison of cell wall changes of Fragaria vesca and Fragaria × ananassa during fruit development and ripening[J]. Plant Physiol J, 2021, 57(12): 2323-2336. DOI: 10.13592/j.cnki.ppj.2021.0053.
[11]	康向阳. 杜仲良种选育研究现状及展望[J]. 北京林业大学学报, 2017, 39(3): 1-6.
	KANG X Y. Status and prospect of improved variety selection in Eucommia ulmoides[J]. J Beijing For Univ, 2017, 39(3): 1-6. DOI: 10.13332/j.1000-1522.20160377.
[12]	李秀兰, 陈力. 三倍体丹参的培育及其可持续利用研究[J]. 中草药, 2012, 43(2): 375-379.
	LI X L, CHEN L. Breeding for triploids of Salvia miltiorrhiza and its sustainable utilization[J]. Chin Tradit Herb Drugs, 2012, 43(2): 375-379. DOI: 10.7501/j.issn.0253-2670.
[13]	DAS S K, SABHAPONDIT S, AHMED G, et al. Biochemical evaluation of triploid progenies of diploid×tetraploid breeding populations of Camellia for genotypes rich in catechin and caffeine[J]. Biochem Genet, 2013, 51: (5/6). DOI: 10.1007/s10528-013-9569-x.
[14]	XU C G, TANG T X, CHEN R, et al. A comparative study of bioactive secondary metabolite production in diploid and tetraploid Echinacea purpurea (L.) Moench[J]. Plant Cell Tiss Organ Cult, 2014, 116(3): 323-332. DOI: 10.1007/s11240-013-0406-z.
[15]	陶抵辉, 李小红, 王利群, 等. 植物染色体倍性鉴定方法研究进展[J]. 生命科学研究, 2009, 13(5): 453-458.
	TAO D H, LI X H, WANG L Q, et al. Progresses on determination of cell chromosome ploidy level of plants[J]. Life Sci Res, 2009, 13(5): 453-458. DOI: 10.16605/j.cnki.1007-7847.2009.05.011.
[16]	SCHWARZACHER T, WANG, M L, LEITCH A R, et al. Flow cytometric analysis of the chromosomes and stability of a wheat cell-culture line[J]. Theor Appl Genet, 1997, 94(1): 91-97. DOI: 10.1007/s001220050386.
[17]	SLIWINSKA E. Flow cytometry:a modern method for exploring genome size and nuclear DNA synthesis in horticultural and medicinal plant species[J]. Folia Hortic, 2018, 30(1): 103-128. DOI: 10.2478/fhort-2018-0011.
[18]	金亮, 徐伟韦, 李小白, 等. DNA流式细胞术在植物遗传及育种中的应用[J]. 中国细胞生物学学报, 2016, 38(2): 225-234.
	JIN L, XU W W, LI X B, et al. Application of DNA flow cytometry to plant genetics and breeding[J]. Chin J Cell Biol, 2016, 38 (2): 225-234. DOI: 10.11844/cjcb.2016.02.0308.
[19]	宫雅昕, 岳涵, 向宇, 等. GABA代谢负调控叶片细胞内复制发生的机制研究[J]. 植物生理学报, 2020, 56(2): 235-246.
	GONG Y X, YUE H, XIANG Y, et al. Mechanism study of negative regulation of GABA metabolism on endoreplication in Arabidopsis thaliana leaf development[J]. Plant Physiol J, 2020, 56(2): 235-246. DOI: 10.13592/j.cnki.ppj.2019.0452.
[20]	GALBRAITH D W. Simultaneous flow cytometric quantification of plant nuclear DNA contents over the full range of described angiosperm 2C values[J]. Cytometry A, 2009, 75(8): 692-698. DOI: 10.1002/cyto.a.20760.
[21]	田新民, 周香艳, 弓娜. 流式细胞术在植物学研究中的应用检测植物核DNA含量和倍性水平[J]. 中国农学通报, 2011, 27(9): 21-27.
	TIAN X M, ZHOU X Y, GONG N, et al. Applications of flow cytometry in plant research-analysis of nuclear DNA content and ploidy level in plant cells[J]. Chin Agric Sci Bull, 2011, 27(9): 21-27.
[22]	韩杰, 沈海萍, 储冬生, 等. 4个薄壳山核桃品种核型分析[J]. 分子植物育种, 2018, 16(17): 5704-5711.
	HAN J, SHEN H P, CHU D S, et al. Karyotype analysis of four pecan cultivars[J]. Mol Plant Breed, 2018, 16(17): 5704-5711. DOI: 10.13271/j.mpb.016.005704.
[23]	任伟超, 徐姣, 樊锐锋, 等. 应用流式细胞术对柳属染色体倍性与基因组大小测定[J]. 东北林业大学学报, 2021, 49(4): 56-61.
	REN W C, XU J, FAN R F, et al. Chromosome ploidy and genome size determination of Salix using flow cytometry[J]. J Northeast For Univ, 2021, 49(4):56-61. DOI: 10.13759/j.cnki.dlxb.2021.04.010.
[24]	PELLICER J, LEITCH I J. The application of flow cytometry for estimating genome size and ploidy level in plants[M]//Molecular Plant Taxonomy. Totowa, NJ: Humana Press, 2014: 279-307. DOI: 10.1007/978-1-62703-767-9_14.
[25]	DPOOLEŽEL J, BINAROVÁ P, LCRETTI S. Analysis of nuclear DNA content in plant cells by flow cytometry[J]. Biol Plant, 1989, 31(2): 113-120. DOI: 10.1007/BF02907241.
[26]	XU J, WEI X P, LIU J S, et al. Genome sizes of four important medicinal species in Kadsura by flow cytometry[J]. Chin Herb Med, 2021, 13(3): 416-420. DOI: 10.1016/j.chmed.2021.05.002.
[27]	SONG P, WANG X F, CAI M, et al. Research on identification of polyploids by flow cytometry in Lagerstroemia indica and Lagerstroemia subcostata[J]. Acta Hortic, 2012 (935): 207-212. DOI: 10.17660/actahortic.2012.935.29.
[28]	LOUREIRO J, RODRIGUEZ E, DOLEZEL J, et al. Flow cytometric and microscopic analysis of the effect of tannic acid on plant nuclei and estimation of DNA content[J]. Ann Bot-London, 2006, 98(3): 515-527. DOI: 10.1093/aob/mcl140.
[29]	张桂芳, 王艳, 闫小巧, 等. 流式细胞仪检测铁皮石斛核DNA初探[J]. 现代中药研究与实践, 2017, 31(1): 16-19.
	ZHANG G F, WANG Y, YAN X Q, et al. Study on flow cytometer for detecting nuclear DNA contents in Dendrobium officinal[J]. Res Pract Chin Med, 2017, 31(1): 16-19. DOI: 10.13728/j.1673-6427.2017.01.005.
[30]	于红梅, 王静, 赵密珍, 等. 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, 2012, 43(10): 1530-1533.
	YU H M, WANG J, ZHAO M Z, et al. Optimization of strawberry ploidy identification method using flow cytometry[J]. J South Agric, 2012, 43(10): 1530-1533. DOI: 10.3969/j.issn.2095-1191.2012.10.1530.
[31]	杨静, 宋勤霞, 宁军权, 等. 利用流式细胞术鉴定桑树染色体倍性的方法[J]. 蚕业科学, 2017, 43(1): 8-17.
	YANG J, SONG Q X, NING J Q, et al. Establishment of Morus L. chromosome ploidy identification method using flow cytometry[J]. Sci Seric, 2017, 43(1): 8-17. DOI: 10.13441/j.cnki.cykx.2017.01.002.
[32]	何婷, 郭桂梅, 陆瑞菊, 等. 两份大麦材料小孢子诱导愈伤及再生植株的倍性研究[J]. 植物生理学报, 2021, 57(8): 1708-1714.
	HE T, GUO G M, LU R J, et al. Study on the ploidy of microspore-derived calli and regenerated plants of two barley materials[J]. Plant Physiol J, 2021, 57(8): 1708-1714. DOI: 10.13592/j.cnki.ppj.2021.0100.
[33]	REUTEMANN A V, HONFI A I, KARUNARATHNE P, et al. Variation of residual sexuality rates along reproductive development in apomictic tetraploids of Paspalum[J]. Plants, 2022, 11(13): 1639. DOI: 10.3390/plants11131639.
[34]	PLASCHIL S, ABEL S, KLOCKE E. The variability of nuclear DNA content of different Pelargonium species estimated by flow cytometry[J]. PLoS One, 2022, 17(4): e0267496. DOI: 10.1371/journal.pone.0267496.
[35]	VIRUEL J, CONEJERO M, HIDALGO O, et al. A target capture-based method to estimate ploidy from herbarium specimens[J]. Front Plant Sci, 2019, 10: 937. DOI: 10.3389/fpls.2019.00937.
[36]	TOMASZEWSKA P, PELLNY T K, HERNÁNDEZ L M, et al. Flow cytometry-based determination of ploidy from dried leaf specimens in genomically complex collections of the tropical forage grass Urochloas L.[J]. Genes, 2021, 12(7): 957. DOI: 10.3390/genes12070957.
[37]	李雯雯, 刘立强, 帕米尔·艾尼, 等. 利用流式细胞术鉴定新疆野杏染色体倍性和DNA含量[J]. 农业生物技术学报, 2019, 27(3): 542-550.
	LI W W, LIU L Q, AINI P M E, et al. Identification of chromosomal ploidy and DNA content in Xinjiang Armeniaca vulgaris by flow cytometry[J]. J Agric Biotechnol, 2019, 27(3):542-550. DOI: 10.3969/j.issn.1674-7968.2019.03.019.
[38]	CASTRO S, LOUREIRO J, RODRIGUEZ E, et al. Evaluation of polysomaty and estimation of genome size in Polygala vayredae and P. calcarea using flow cytometry[J]. Plant Sci, 2007, 172(6): 1131-1137. DOI: 10.1016/j.plantsci.2007.03.002.
[39]	GEORGIEV V, WEBER J, BLEY T, et al. Improved procedure for nucleus extraction for DNA measurements by flow cytometry of red beet (Beta vulgaris L.) hairy roots[J]. J Biosci Bioeng, 2009, 107(4): 439-441. DOI: 10.1016/j.jbiosc.2008.12.023.
[40]	吕顺, 任毅, 王芳, 等. 利用流式细胞术快速鉴定169份香蕉种质资源的染色体倍性[J]. 果树学报, 2018, 35(6): 668-684.
	LÜ S, REN Y, WANG F, et al. Ploidy identification of 169 Musa germplasms by flow cytometry[J]. J Fruit Sci, 2018, 35(6): 668-684. DOI: 10.13925/j.cnki.gsxb.20170419.

Related Articles 15

[1]	WANG Ji, FANG Shengzuo. Effects of different anti-browning agents on enzyme activity and growth in callus of Cyclocarya paliurus [J]. JOURNAL OF NANJING FORESTRY UNIVERSITY, 2023, 47(6): 167-174.
[2]	HUANG Ziliang, XU Ziheng, SUN Caowen. Study on seasonal dynamics of seed rain and characteristics of soil seed banks in Cyclocarya paliurus [J]. JOURNAL OF NANJING FORESTRY UNIVERSITY, 2023, 47(2): 18-26.
[3]	MA Qiuyue, WANG Yuxiao, LI Qianzhong, LI Shushun, WEN Jing, ZHU Lu, YAN Kunyuan, DU Yiming, XIE Zhijun, LI Shuxian, OUYANG Fangqun, LU Chengdai. Estimation of genome sizes of six Acer species by flow cytometry and K-mer analysis [J]. JOURNAL OF NANJING FORESTRY UNIVERSITY, 2023, 47(1): 163-170.
[4]	FANG Shengzuo. A review on the development history and the resource silviculture of Cyclocarya paliurus industry [J]. JOURNAL OF NANJING FORESTRY UNIVERSITY, 2022, 46(6): 115-126.
[5]	XU Zhanhong, ZHU Ying, JIN Huiying, SUN Caowen, FANG Shengzuo. Variations in the contents of leaf pigments and polyphenols and photosynthesis traits in Cyclocarya paliurus with different leaf colors [J]. JOURNAL OF NANJING FORESTRY UNIVERSITY, 2022, 46(2): 103-110.
[6]	SUN Caowen, ZHONG Wenwen, FU Xiangxiang, SHANG Xulan, FANG Shengzuo. A study on growth and aboveground biomass production of juvenile Cyclocarya paliurus plantations [J]. JOURNAL OF NANJING FORESTRY UNIVERSITY, 2022, 46(1): 138-144.
[7]	DUAN Yifan, LI Lan, YANG Xinxin, WANG Xianrong, ZHANG Min, ZHANG Cheng, CHAI Zihan. Study on ploidy and genome sizes of Osmanthus fragrans and its related species [J]. JOURNAL OF NANJING FORESTRY UNIVERSITY, 2021, 45(5): 47-52.
[8]	LI Na, ZHU Peilin, FENG Cai, WEN Minxue, FANG Shengzuo, SHANG Xulan. Variations in physiological characteristics of rootstock-scion and its relationship to graft compatibility during the grafting union process of Cyclocarya paliurus [J]. JOURNAL OF NANJING FORESTRY UNIVERSITY, 2021, 45(1): 13-20.
[9]	ZHOU Yongsheng, XU Ziheng, YUAN Fayin, SHANG Xulan, SUN Caoweng, FANG Shengzuo. Comparisons of community characteristics among three natural forests of Cyclocarya paliurus in the subtropical region of China [J]. JOURNAL OF NANJING FORESTRY UNIVERSITY, 2021, 45(1): 29-35.
[10]	CHEN Pei, ZHOU Mingming, FANG Shengzuo, LIU Yang, YANG Wanxia, SHANG Xulan. Effects of light quality on accumulation of phenolic compounds and antioxidant activities in Cyclocarya paliurus (Batal.) Iljinskaja leaves from different families [J]. JOURNAL OF NANJING FORESTRY UNIVERSITY, 2020, 44(2): 17-25.
[11]	LIN Yuan, CHEN Pei, ZHOU Mingming, SHANG Xulan, FANG Shengzuo. Key bioactive substances and their antioxidant activities in Cyclocarya paliurus (Batal.) Iljinskaja leaves collected from natural populations [J]. JOURNAL OF NANJING FORESTRY UNIVERSITY, 2020, 44(2): 10-16.
[12]	KOU Xiaolin, XIE Nan, WU Caie, FAN Gongjian, FU Xiangxiang. Isolation and identification of endophytic fungi from Cyclocarya paliurus (Batal.) Iljinskaja [J]. JOURNAL OF NANJING FORESTRY UNIVERSITY, 2020, 44(2): 26-34.
[13]	YUE Xiliang, QIN Jian, FU Xiangxiang, SHANG Xulan, FANG Shengzuo. Effects of nitrogen fertilization on secondary metabolite accumulation and antioxidant capacity of Cycolcurya paliurus (Batal.) Iljinskaja leaves [J]. JOURNAL OF NANJING FORESTRY UNIVERSITY, 2020, 44(2): 35-42.
[14]	LI Weiqiang, DAI Xiaogang, LI Xiaoping, LI Shuxian. Assessment of ploidy levels in the Populus deltoides Marsh. germplasm [J]. JOURNAL OF NANJING FORESTRY UNIVERSITY, 2019, 43(5): 51-58.
[15]	MA Qiuyue, LI Shushun, MA Xiang, WEN Jing, ZHU Lu, TANG Ling, LI Rui, GUO Wei, LI Qianzhong. Estimation of genome size of two Acer Linn. by flow cytometry [J]. JOURNAL OF NANJING FORESTRY UNIVERSITY, 2018, 42(05): 201-205.

Metrics

Comments

www.nldxb.njfu.edu.cn

Edited ＆ Published by Editorial Department of Nanjing Forestry University(Natural Sciences Edition)
Address： No.159 Longpan Road,Nanjing 210037 Jiangsu,P.R.China
E-mail ：xuebao@njfu.edu.cn , xuebao@njfu.com.cn
Telephone +86-25-85428247,85427076
Distributed by China International Book Trading Corporation P.O. Box 399, Beijing ,P.R. China

倍性 ploidy	倍性系数 coefficientof ploidy	倍性估算值 ploidy estimated	样本数量/份 number of samples	变异系数/% CV
2	0.45~0.60	1.80~2.40	34	2.13~4.53
	0.65~0.70	2.60~2.80	3	3.23~4.66
4	1.80~2.10	3.60~4.20	58	2.43~5.04
	1.50~1.70	3.00~3.40	5	4.46

来源 resource	数量 number of samples	倍性 ploidy	占比/% proportion	变异系数/% CV
种质资源库 germplasm bank	1 087	2	4.05	2.45~4.22
		4	95.95	2.05~4.86
安徽省 Anhui Province	75	2	38.67	3.02~5.54
		4	61.33	2.76~5.21
湖北省 Hubei Province	73	2	42.46	4.32~6.08
		4	57.54	2.98~5.41
四川省 Sichuan Province	32	4	100.00	3.42~5.03
江西省 Jiangxi Province	80	4	100.00	3.10~5.41
广西壮族自治区 Guangxi Zhuang Autonomous Region	48	4	100.00	2.68~4.97

Establishment and application of a flow cytometry method for chromosome ploidy identification of Cyclocarya paliurus

RichHTML

PDF

Knowledge

Abstract

Cite this article

share this article

Figures/Tables 7

References 40

Related Articles 15

Recommended Articles

Metrics

Comments

Author

Reader

Reviewer