流式细胞仪鉴定青钱柳倍性方法的建立及其应用

doi:10.12302/j.issn.1000-2006.202302014

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南京林业大学学报（自然科学版） ›› 2024, Vol. 48 ›› Issue (2): 61-68.doi: 10.12302/j.issn.1000-2006.202302014

流式细胞仪鉴定青钱柳倍性方法的建立及其应用

宋子琪¹^,²(), 卞国良¹, 林峰³, 胡凤荣⁴, 尚旭岚¹^,^*()

1.南京林业大学南方现代林业协同创新中心,南京林业大学林草学院,江苏南京 210037
2.中国科学院南京土壤研究所,江苏南京 210018
3.南京林业大学现代分析测试中心,江苏南京 210037
4.南京林业大学风景园林学院,江苏南京 210037

收稿日期:2023-02-14 修回日期:2023-03-23 出版日期:2024-03-30 发布日期:2024-04-08
通讯作者: *尚旭岚(shangxulan@njfu.edu.cn),副教授。
作者简介:宋子琪(2632583724@qq.com)。
基金资助:
国家自然科学基金项目(31971642);国家自然科学基金项目(32271959);江苏省重点研发计划(现代农业)重点项目(BE2019388)

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

摘要/Abstract

摘要：

【目的】利用流式细胞仪检测青钱柳(Cyclocarya paliurus)的染色体倍性,为青钱柳种质鉴定及遗传育种研究提供技术支持和基础数据。【方法】以青钱柳叶片为材料,比较了裂解液种类、离心处理和叶片保存方式对倍性检测效果的影响。建立的青钱柳流式细胞分析倍性的方法为:取参照样本和待测样本叶片各0.50~1.00 cm²,加入1 mL的mGb裂解液中快速切碎,过滤后加入20 μL的碘化丙啶(PI)染色1 min即可上机检测。利用建立的方法对1 395份青钱柳种质资源的倍性进行检测。【结果】 mGb裂解液的裂解效果最好,峰图质量最优。细胞核悬浮液过滤后可无需离心而直接染色并进行测定。4 ℃冷藏叶片的测定效果最佳,保存时间以7 d为宜。硅胶干燥样本测定效果优于冷冻,硅胶干燥保存时间以150 d为宜。100份试验样本的DNA含量峰值的变异系数(CV)值为2.13%~5.04%,倍性估算值为1.80~2.40的样本判断为二倍体,倍性估算值为3.60~4.20的样本判断为四倍体。倍性估算值为3.00±0.40时,需采用与初判倍性相同的参照样本进行第2次标定。对1 395份青钱柳种质资源进行倍性鉴定,共检测出二倍体104份,四倍体1 291份。【结论】bbbb对于倍性估算值异常的样本,采用二次标定法可快速确定其倍性。该方法操作简单,高效准确,为青钱柳种质倍性鉴定提供了有效方法。

关键词: 青钱柳, 倍性鉴定, 流式细胞术, 细胞核裂解液, 内标法

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

中图分类号:

S722

宋子琪,卞国良,林峰,等. 流式细胞仪鉴定青钱柳倍性方法的建立及其应用[J]. 南京林业大学学报（自然科学版）, 2024, 48(2): 61-68.

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 (Natural Science Edition), 2024, 48(2): 61-68.DOI: 10.12302/j.issn.1000-2006.202302014.

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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

流式细胞仪鉴定青钱柳倍性方法的建立及其应用

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

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