基于流式细胞仪鉴定番石榴倍性方法的建立及应用

doi:10.13560/j.cnki.biotech.bull.1985.2023-0844

摘要/Abstract

摘要：

【目的】利用流式细胞仪鉴定番石榴的染色体倍性，为番石榴倍性育种和杂交育种奠定基础。【方法】以番石榴为材料，比较了样品部位、离心处理和保存方式对倍性检测结果的影响。【结果】番石榴花瓣作为倍性检测材料时，收集到的细胞核DNA数目最多，CV值为1.96%，峰形效果最佳；在制备细胞核悬浮液时，过滤后不离心，直接染色后上机的测定效果最好；新鲜番石榴花瓣得到的细胞核DNA含量明显高于冷藏和其他冷冻处理组，且背景碎片少，主峰明显；-80℃冷冻处理对样本的破坏性最小，检测效果仅次于新鲜的番石榴花瓣。本研究建立的番石榴流式细胞术倍性分析的方法为：取番石榴花瓣0.50-1.00 cm²，加入1 mL的mGb裂解液中混合切碎，过滤后加入30 μL PI染色1 min即可上机检测。【结论】利用建立的流式细胞术对33份番石榴种质资源进行倍性鉴定，共检测出二倍体32份，六倍体1份。该方法执行简便、高效准确，为番石榴种质资源倍性鉴定提供了有效方法。

关键词: 番石榴, 花瓣, 倍性鉴定, 流式细胞术

Abstract:

【Objective】 The flow cytometry was used to identify the ploidy of Psidium guajava, which may lay the foundation for ploidy breeding and hybrid breeding of P. guajava. 【Method】 Using P. guajava as the material, the effects of sample parts, centrifugation treatment, and storage methods on ploidy detection results were compared. 【Result】 P. guajava petals had the highest number of collected nuclear DNA, with a CV value of 1.96% and the best peak shape. When preparing nuclear suspension, the best detection effect was achieved by directly staining and analyzing without centrifugation after filtration. The nuclear DNA content obtained from fresh P. guajava petals was significantly higher than that of refrigerated and other frozen treatment groups, with fewer background fragments and a clear main peak. The -80℃ freezing treatment caused the least damage to the samples, and the detection effect was second only to that of fresh P. guajava petals. The established flow cytometry method for ploidy analysis of P. guajava involved taking 0.50-1.00 cm² of pomegranate petals, mixing them with 1 mL of mGb lysis buffer, filtering, and adding 30 μL of PI staining for 1 min before analysis. 【Conclusion】 Using this established flow cytometry method, ploidy identification was performed on 33 P. guajava germplasm resources, resulting in the detection of 32 diploids and 1 hexaploid. This method is simple, efficient, and accurate, and may provide an effective approach for ploidy identification of P. guajava germplasm resources.

Key words: Psidium guajava, petal, ploidy identification, flow cytometry

邵雪花, 李桂兰, 肖维强, 赖多, 庄庆礼, 秦健. 基于流式细胞仪鉴定番石榴倍性方法的建立及应用[J]. 生物技术通报, 2024, 40(2): 48-54.

SHAO Xue-hua, LI Gui-lan, XIAO Wei-qiang, LAI Duo, ZHUANG Qing-li, QIN Jian. Establishment and Application of Ploidy Method for the Identification of Psidium guajava by Flow Cytometry[J]. Biotechnology Bulletin, 2024, 40(2): 48-54.

图/表 5

图1 番石榴不同组织流式图

Fig. 1 Flow chart of different tissues of guava

图2 番石榴不同离心方式的比较 L1：1 000 r/min离心5 min；L2：1 000 r/min离心10 min；L3：2 000 r/min离心5 min；L4：2 000 r/min离心10 min；CK：400目滤膜过滤后不离心

Fig. 2 Comparison of different centrifugation methods of guava L1: 1 000 r/min centrifuge for 5 min; L2: 1 000 r/min centrifuge for 10 min; L3: 2 000 r/min centrifuge for 5 min; L4: 2 000 r/min centrifuge for 10 min; CK: no centrifugation with filtration through 400 mesh membrane

图3 番石榴不同保存温度流式图

Fig. 3 Flow diagram of different storage temperatures of P. guajava

图4 不同倍性番石榴花瓣的相对DNA含量 A：‘珍珠’（二倍体）；B：‘紫果’（二倍体）；C：‘金斗香’（二倍体）；D：‘草莓’（六倍体）

Fig. 4 Relative DNA contents of P. guajava petals with different ploidy levels A: ‘Zhenzhu’（diploid）; B: ‘Ziguo’（diploid）; C: ‘Jindouxiang’（diploid）; D: ‘Strawberry’（hexaploid）

表1 以‘珍珠’为参照的33份番石榴种质资源倍性鉴定

Table 1 FCM identification of 33 P. guajava germplasms with ‘Zhenzhu’as reference

品种编号Sample No.	名称Name	倍性系数Coefficient of ploidy	倍性估算值Ploidy estimated value	倍性Ploidy
1	紫果Ziguo	0.49	1.96	2n
2	泰国2号Thailand 2	0.45	1.80	2n
3	泰国3号Thailand 3	0.48	1.92	2n
4	软香Ruanxiang	0.43	1.72	2n
5	木瓜Mugua	0.53	2.12	2n
6	红肉实生Hongrou shisheng	0.59	2.36	2n
7	红肉翁拔Hongrouwengba	0.51	2.04	2n
8	帝王Diwang	0.48	1.92	2n
9	草莓Strawberry	0.52	2.01	6n
10	西瓜红Xiguahong	0.44	1.76	2n
11	东莞红肉Dongguanhongrou	0.45	1.80	2n
12	佳硕Jiashuo	0.46	1.84	2n
13	水蜜Shuimi	0.49	1.96	2n
14	细叶小果Xiyexiaoguo	0.44	1.76	2n
15	细叶大果Xiyedaguo	0.43	1.72	2n
16	金斗香Jin douxiang	0.45	1.80	2n
17	白肉翁拔Bairouwengba	0.52	2.08	2n
18	翡翠Feicui	0.51	2.04	2n
19	青皮红肉Qingpihongrou	0.53	2.12	2n
20	金石宫Jinshigong	0.47	1.88	2n
21	珍珠Zhenzhu	0.50	2.00	2n
22	红心软果Hongxinruanguo	0.42	1.68	2n
23	粉红蜜Fenhongmi	0.51	2.04	2n
24	卵红Luanhong	0.50	2.00	2n
25	宫粉红Gongfenhong	0.58	2.32	2n
26	黄皮红肉Huangpihongrou	0.53	2.12	2n
27	B1	0.51	2.04	2n
28	B2	0.56	2.24	2n
29	B3	0.51	2.04	2n
30	B4	0.54	2.16	2n
31	B5	0.53	2.12	2n
32	B6	0.53	2.12	2n
33	穗红1号Suihong 1	0.49	1.96	2n

参考文献 23

[1]	Kumar M, Tomar M, Amarowicz R, et al. Guava(Psidium guaja-va L.)leaves: nutritional composition, phytochemical profile, and health-promoting bioactivities[J]. Foods, 2021, 10(4): 752. doi: 10.3390/foods10040752 URL
[2]	Zou XF, Liu HY. A review of meroterpenoids and of their bioactivity from guava(Psidium guajava L.)[J]. J Future Foods, 2023, 3(2): 142-154. doi: 10.1016/j.jfutfo.2022.12.005 URL
[3]	Jaiarj P, Khoohaswan P, Wongkrajang Y, et al. Anticough and antimicrobial activities of Psidium guajava Linn. leaf extract[J]. J Ethnopharmacol, 1999, 67(2): 203-212. pmid: 10619385
[4]	Gutiérrez RMP, Mitchell S, Solis RV. Psidium guajava: a review of its traditional uses, phytochemistry and pharmacology[J]. J Ethnopharmacol, 2008, 117(1): 1-27. doi: 10.1016/j.jep.2008.01.025 pmid: 18353572
[5]	Morais-Braga MFB, Carneiro JNP, Machado AJT, et al. Psidium guajava L., from ethnobiology to scientific evaluation: elucidating bioactivity against pathogenic microorganisms[J]. J Ethnopharmacol, 2016, 194: 1140-1152. doi: S0378-8741(16)31814-1 pmid: 27845266
[6]	Naseer S, Hussain S, Naeem N, et al. The phytochemistry and medicinal value of Psidium guajava(guava)[J]. Clin Phytosci, 2018, 4(1): 32. doi: 10.1186/s40816-018-0093-8
[7]	Amadike Ugbogu E, Emmanuel O, Ebubechi Uche M, et al. The ethnobotanical, phytochemistry and pharmacological activities of Psidium guajava L[J]. Arab J Chem, 2022, 15(5): 103759. doi: 10.1016/j.arabjc.2022.103759 URL
[8]	Liu HX, Wei SS, Shi LL, et al. Preparation, structural characterization, and bioactivities of polysaccharides from Psidium guajava: a review[J]. Food Chem, 2023, 411: 135423. doi: 10.1016/j.foodchem.2023.135423 URL
[9]	Kumar M, Kapoor S, Dhumal S, et al. Guava(Psidium guajava L.)seed: a low-volume, high-value byproduct for human health and the food industry[J]. Food Chem, 2022, 386: 132694. doi: 10.1016/j.foodchem.2022.132694 URL
[10]	邵雪花, 赖多, 肖维强, 等. 番石榴遗传多样性的ISSR分析及指纹图谱构建[J]. 中国农学通报, 2023, 39(21): 39-47. doi: 10.11924/j.issn.1000-6850.casb2022-0420
	Shao XH, Lai D, Xiao WQ, et al. Psidium guajava L.: genetic diversity analysis based on ISSR markers and construction of fingerprints[J]. Chin Agric Sci Bull, 2023, 39(21): 39-47.
[11]	陈宜木, 周群, 钟颖颖, 等. 利用流式细胞术鉴定68份三角梅基因组大小与染色体倍性[J]. 亚热带植物科学, 2022, 51(6): 417-423.
	Chen YM, Zhou Q, Zhong YY, et al. Genome size estimation and ploidy identification of 68 servings Bougainvillea by flow cytometry[J]. Subtrop Plant Sci, 2022, 51(6): 417-423.
[12]	Brown M, Wittwer C. Flow cytometry: principles and clinical applications in hematology[J]. Clin Chem, 2000, 46(8 Pt 2):1221-1229. pmid: 10926916
[13]	郑英转, 吕燕, 杨东旭, 等. 基于液氮研磨法的流式细胞术检测马铃薯倍性的研究[J]. 生物技术通报, 2021, 37(1): 282-288. doi: 10.13560/j.cnki.biotech.bull.1985.2020-0478
	Zheng YZ, Lü Y, Yang DX, et al. Study on the identification of potato ploidy using flow cytometry based on liquid nitrogen grinding method[J]. Biotechnol Bull, 2021, 37(1): 282-288.
[14]	陈林, 潘贞志, 戴毅, 等. 适合流式细胞仪分析的大豆细胞核解离液的筛选与应用[J]. 生物技术通报, 2020, 36(11): 230-237. doi: 10.13560/j.cnki.biotech.bull.1985.2020-0426
	Chen L, Pan ZZ, Dai Y, et al. Screening and application of the nuclear dissociation solutions of soybeans suitable for flow cytometry analysis[J]. Biotechnol Bull, 2020, 36(11): 230-237. doi: 10.13560/j.cnki.biotech.bull.1985.2020-0426
[15]	赵孟良, 任延靖, 田闵玉, 等. 基于流式细胞仪鉴定菊芋倍性方法的建立及应用[J]. 西北农林科技大学学报: 自然科学版, 2021, 49(3): 138-146.
	Zhao ML, Ren YJ, Tian MY, et al. Establishment and application of aploidy method for identification of Helianthus tuberosus L. by flow cytometry[J]. J Northwest A F Univ Nat Sci Ed, 2021, 49(3): 138-146.
[16]	王娅丽, 周利利, 王娜, 等. 利用流式细胞仪快速鉴定棉花倍性的方法比较[J]. 生物技术通报, 2022, 38(12): 144-148. doi: 10.13560/j.cnki.biotech.bull.1985.2022-0783
	Wang YL, Zhou LL, Wang N, et al. Comparison of methods for rapid determination of cotton ploidy by flow cytometry[J]. Biotechnol Bull, 2022, 38(12): 144-148.
[17]	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.
[18]	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 XM, Zhou XY, Gong N. 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.
[19]	熊文艳, 普冉, 刘云礼, 等. 基于流式细胞术对27种石斛的倍性鉴定和基因组大小分析[J]. 热带作物学报, 2022, 43(11): 2249-2257. doi: 10.3969/j.issn.1000-2561.2022.11.009
	Xiong WY, Pu R, Liu YL, et al. Estimation ploidy and genome size of 27 Dendrobium species by flow cytometry[J]. Chin J Trop Crops, 2022, 43(11): 2249-2257.
[20]	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 GF, Wang Y, Yan XQ, et al. Study on flow cytometer for detecting nuclear DNA contents in Dendrobium officinal[J]. Res Pract Chin Med, 2017, 31(1): 16-19.
[21]	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 QX, Ning JQ, et al. Establishment of Morus L. chromosome ploidy identification method using flow cytometry[J]. Sci Seric, 2017, 43(1): 8-17.
[22]	王利虎, 吕晔, 罗智, 等. 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, 2018, 26(3): 511-520.
	Wang LH, Lv Y, Luo Z, et al. Establishment and application of a method for chromosome ploidy identification and genome size estimation using flow cytometry in Ziziphus jujuba[J]. J Agric Biotechnol, 2018, 26(3): 511-520.
[23]	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]. Journal of Bioscience and Bioengineering, 2009, 107(4): 439-441. doi: 10.1016/j.jbiosc.2008.12.023 pmid: 19332305