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

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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.

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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

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

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