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
宋子琪1,2(), 卞国良1, 林峰3, 胡凤荣4, 尚旭岚1,*(
)
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:
SONG Ziqi1,2(), BIAN Guoliang1, LIN Feng3, HU Fengrong4, SHANG Xulan1,*(
)
Received:
2023-02-14
Revised:
2023-03-23
Online:
2024-03-30
Published:
2024-04-08
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。
中图分类号:
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.
图4
流式细胞术鉴定青钱柳倍性方法的验证 a.二倍体待测样本与四倍体参照样本峰图 the the histogram of peak values with diploidy test sample and tetriploidy standard sample;b.二倍体参照样本与四倍体待测样本峰图the histogram of peak values with diploidy standard sample and tetriploidy test sample;c.初判二倍体待测样本与四倍体参照样本峰图 the histogram of peak values with diploidy test sample by preliminary judgement and tetriploidy standard sample;d.代表图c中的初判二倍体待测样本与二倍体参照样本峰图indicates the histogram of peak values with diploidy test sample by preliminary judgement and diploidy standard sample in c;e.二倍体参照样本与初判四倍体待测样本峰图 the histogram of peak values with diploidy standard sample and tetriploidy test sample by preliminary judgement;f.图e中的初判四倍体待测样本与四倍体参照样本峰图 the histogram of peak values with tetriploidy standard sample and tetriploid test sample by preliminary judgement in e; 均值mean.所对应峰的相对荧光强度值 the relative fluorescence intensity of the corresponding peak。 "
表2
流式细胞分析法对1 395份青钱柳种质资源倍性测定结果"
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 |
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[15] | 陈培, 周明明, 方升佐, 刘洋, 杨万霞, 尚旭岚. 光质对不同家系青钱柳叶酚类物质积累及抗氧化活性的影响[J]. 南京林业大学学报(自然科学版), 2020, 44(2): 17-25. |
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