[1]叶天文,李艳民,张 健,等.普通油茶染色体制片技术优化及核型分析[J].南京林业大学学报(自然科学版),2020,44(05):093-99.[doi:10.3969/j.issn.1000-2006.202005022]
 YE Tianwen,LI Yanmin,ZHANG Jian,et al.Optimization of chromosome mounting technique and karyotype analysis of Camellia oleifera[J].Journal of Nanjing Forestry University(Natural Science Edition),2020,44(05):093-99.[doi:10.3969/j.issn.1000-2006.202005022]
点击复制

普通油茶染色体制片技术优化及核型分析
分享到:

《南京林业大学学报(自然科学版)》[ISSN:1000-2006/CN:32-1161/S]

卷:
44
期数:
2020年05期
页码:
093-99
栏目:
研究论文
出版日期:
2020-09-23

文章信息/Info

Title:
Optimization of chromosome mounting technique and karyotype analysis of Camellia oleifera
文章编号:
1000-2006(2020)05-0093-07
作者:
叶天文李艳民张 健龚倩颖袁德义肖诗鑫*
(中南林业科技大学,经济林培育与保护教育部重点实验室,湖南 长沙 410004)
Author(s):
YE Tianwen LI Yanmin ZHANG Jian GONG Qianying YUAN Deyi XIAO Shixin*
(Key Laboratory of Non-Wood Forest Product of Ministry of Education, Central South University of Forestry and Technology, Changsha 410004,China)
关键词:
普通油茶 染色体制片 染色体 倍性 核型
Keywords:
Camellia oleifera Abel. chromosome mounting technique chromosome ploidy karyotype
分类号:
S794.4
DOI:
10.3969/j.issn.1000-2006.202005022
文献标志码:
A
摘要:
【目的】染色体是种内品种变异的遗传物质基础,明确普通油茶主栽品种染色体倍性及核型特征,可为育种和栽培中品种配置提供依据。【方法】以普通油茶3个主栽品种(‘华硕'、‘华鑫'、‘华金')的扦插苗和实生苗根尖为试材,对油茶染色体制片技术实验进行优化,对3个品种进行核型分析。【结果】①改良的去壁低渗火焰干燥法更适合普通油茶染色体制片,根尖以0.002 mol/L 8-羟基喹啉预处理5~6 h,1.75%纤维素酶和1.75%果胶酶酶解120 min,蒸馏水后低渗30 min,制片效果最佳。②3个品种扦插苗染色体数目均为2n=6x=90,为六倍体,而3个品种的实生苗染色体数目为90、87、85和75等。③对扦插苗根尖细胞中期分裂相进行核型分析可知,‘华硕'、‘华鑫'核型都为2A,‘华金'核型为2B,核型公式分别为2n=90=63m(3SAT)+27sm(‘华硕')、2n=90=58m(SAT)+32sm(‘华鑫')、2n=90=64m+26sm(SAT)(‘华金'),核型不对称系数分别是61.73%(‘华硕')、61.13%(‘华鑫')、61.44%(‘华金')。【结论】优化的去壁低渗火焰干燥法更适合于普通油茶染色体制片,‘华硕'、‘华鑫'、‘华金'均为六倍体,而其实生后代会发生倍性分离。本研究为进一步开展普通油茶的倍性与核型研究奠定了基础
Abstract:
【Objective】The Camellia oleifera Abel. has a wide distribution and a long cultivation history, but its ploidy has always been controversial, and there is a lack of research on ploidy and karyotype. The chromosome is the genetic basis of interspecies variation; the chromosomal ploidy and karyotype characteristics of the main varieties of C. oleifera are defined, providing a basis for the production and cultivation of new varieties. The chromosome mounting technique of C. oleifera is no longer suitable for current experimental requirements. This study aims to optimize the chromosome mounting technique of C. oleifera, compare and analyze the chromosome numbers of cuttings and seedlings, and the karyotype of some main varieties of C. oleifera.【Method】In this study, the root tips of cutting stecklings and seedlings of three main cultivars(‘Huashuo', ‘Huajin' and ‘Huaxin')were used as test materials. Firstly, the chromosome mounting technique was optimized by modifying key steps of the wall degradation hypotonic method, including pre-treatment, enzymatic hydrolysis and posterior hypotonicity. Secondly, to identify the material for a karyotype analysis, the modified wall degradation hypotonic method was used to unlock the chromosome number of the three main cultivars of C. oleifera. Finally, the karyotype of the three cultivars of C. oleifera was unlocked, and the karyotype formula and pivotal karyotype parameters were clarified.【Result】The following three points can be summarized from this study: ① The optimized chromosome mounting technique, which included pre-treatment of the tender root tip with a solution of 0.002 mol/L 8-hydroxyquinoline for 5-6 hours, hypotonicity for 60 min in 0.075 mol/L KCl solution, enzymolyzation with a mixture of 1.75% cellulase and 1.75% pectinase for 120 min at 20 ℃ in the dark, posterior hypotonicity in distilled water, and finally fixing with fresh Carnoy solution for more than 30 min, was found to be the best for chromosome preparation. The results showed that the modified wall degradation hypotonic method is the most suitable for preparation of C. oleifera chromosomes. ② The chromosome number of cuttings of three main cultivars was hexaploidy(2n=6x=90), and the chromosome number of seedlings of the main cultivars was 90, 87, 85, 75 and so on. As there is a tendency for chromosomal deletion in some seedlings, the stem tip or root tip of cutting stecklings should be used for a karyotype analysis of C. oleifera. ③ The cells at the mitotic metaphase stage of the root tip of cutting were used for a karyotype analysis; ‘Huashuo', ‘Huaxin' karyotypes are 2A, and ‘Huajin' karyotypes are 2B. The karyotype formulas are 2n=90=63m(3SAT)+27sm(‘Huashuo'), 2n=90=58m(SAT)+32sm(‘Huaxin')and 2n=90=64m+26sm(SAT)(‘Huajin'), and the karyotype asymmetry coefficient is 61.73%, 61.13% and 61.44%, respectively. The relative length of the chromosomes ranged from 4.81% to 8.64%, and the arm ratio ranged from 1.07 to 2.96.【Conclusion】There are many varieties of C. oleifera, and it is crucial to explore their genetic diversity, genetic relationships and the evolutionary trends among populations. Moreover, chromosome studies of C. oleifera can reflect the regular karyotype pattern of different cultivars. Through a comparative analysis of chromosome numbers of cuttings and seedlings of ‘Huashuo', ‘Huajin' and ‘Huaxin' by optimizing the chromosome mounting technique, it was found that the offspring of C. oleifera undergo polyploidy germplasm, reflecting the importance of choosing suitable test materials when studying C. oleifera karyotypes. The karyotype analysis of the three main cultivars further reflects the large karyotype difference between C. oleifera cultivars

参考文献/References:


[1] 庄瑞林.中国油茶[M].2版.北京: 中国林业出版社, 2008:65-108.ZHUANG R L. Camellia oleifera in China[M] 2nd ed. Beijing:China Forestry Publishing House,2008:65-108.
[2] 国家林业局国有林场和林木种苗工作总站. 中国油茶品种志[M]. 北京: 中国林业出版社, 2016: 27.State forestry admini-stration state-owned forest farm and tree seedling work master station. oil-tea camellia cultivars in China[M]. Beijing: China Forestry Publishing House, 2016: 27.
[3] 胡国珠,高伟,许怡欣,等.江西成林油茶叶片春梢末期营养诊断[J].南京林业大学学报(自然科学版),2018,42(4):193-197.HU G Z,GAO W,XU Y X,et al.Study on leaf nutrient of Camellia oleifera in Jiangxi Province[J].J Nanjing For Univ(Nat Sci Ed),2018,42(4):193-197.DOI: 10.3969/j.issn.1000-2006.201705003.
[4] 范晓明,袁德义,谭晓风,等.油茶花药离体培养影响因子研究[J].南京林业大学学报(自然科学版),2011,35(5):125-128.FAN X M,YUAN D Y,TAN X F,et al.Study of impact factor on the anther culture in vitro of Camellia oleifera[J].J Nanjing For Univ(Nat Sci Ed),2011,35(5):125-128.DOI: 10.3969/j.jssn.1000-2006.2011.05.028.
[5] 黎麦秋. 普通油茶、板栗染色体组型和Giemsa C-带的带型研究[J]. 林业科技通讯, 1981(4):9-12. LI M Q. Karyotypes and Giemsa C-banding analyses of Camellia oleifera and Castanea mollissima[J]. For Sci Tech, 1981(4):9-12. DOI: 10.13456/j.cnki.lykt.1981.04.008.
[6] 张文驹, 闵天禄. 山茶属的细胞地理学研究[J]. 云南植物研究, 1999(2):56-68. ZHANG W J, MIN T L. A cytogeological study of genus Camellia[J]. Acta Bot Yunnanica[J].1999(2):56-68. DOI: 10.3969/j.issn.2095-0845.1999.02.010.
[7] 李光涛,梁涛.山茶属植物的染色体数目和核型[J].广西植物,1990,10(2):127-137,191-194.LI G T,LIANG T.Chromosome numbers and karyotypes in the genus Camellia[J].Guihaia,1990,10(2):127-137,191-194.
[8] 顾志建,孙先凤.山茶属17个种的核形态学研究[J].云南植物研究,1997,19(2):159-170.GU Z J,SUN X F.A karyomorphological study of seventeen species of Chinese Camellia[J].Acta Bot Yunnanica,1997,19(2):159-170.
[9] 李懋学, 张学方. 植物染色体研究技术[M]. 哈尔滨: 东北林业大学出版社, 1991:31-48. LI M X, ZHANG X F. Plant chromosome technology research[M]. Harbin: N For Univ Pre, 1991:31-48.
[10] 陈瑞阳,宋文芹,李秀兰.植物染色体标本制备的去壁、低渗法及其在细胞遗传学中的意义[J].遗传学报,1982,9(2):151-159,173-174.CHEN R Y,SONG W Q,LI X L.Wall degradation hypotonic method of preparing chromosome samples in plant and its significance in the cytogenetics[J].Acta Genet Sin,1982,9(2):151-159,173-174.DOI: CNKI: SUN:YCXB.0.1982-02-012.
[11] 申建双,叶远俊,潘会堂,等.12份连翘种质资源的核型参数分析[J].植物遗传资源学报,2015,16(1):178-184.SHEN J S,YE Y J,PAN H T,et al.Karyotype analysis of twelve forsythia species and cultivars[J].J Plant Genet Resour,2015,16(1):178-184.DOI: 10.13430/j.cnki.jpgr.2015.01.028.
[12] 乔永刚,宋芸.利用EXCEL制作核型模式图[J].农业网络信息,2006(10):97-98.QIAO Y G,SONG Y.Making a nuclear pattern diagram with EXCEL[J].Agric Netw Inf,2006(10):97-98.DOI: 10.3969/j.issn.1672-6251.2006.10.036.
[13] 李懋学,陈瑞阳. 关于植物核型分析的标准化问题[J]. 武汉植物学研究,1985, 3(4):297-302. LI M X, CHEN R Y. The standardization about the karyotype analysis[J]. J Wuhan Bot Res,1985, 3(4):297-302.
[14] KUO S R, WANG T T, HUANG T C. Karyotype analysis of some formosan gymnosperms[J]. Taiwania,1972, 17(1):66-80. DOI: 10.6165/tai.1972.17.66.
[15] ARANO H. Cytological studies in subfamily Carduoideae(Compositae)of Japan[J]. Ⅸ Bot Mag,1963, 76:128-140. DOI: 10.15281/jplantres1887.77.54.
[16] STEBBINS G L. Chromosal evolution in higher plants[J]. London: Edward Arnold Ltd, 1971:87-89.
[17] 王霜, 王仲朗, 梁静, 等. 濒危植物杜鹃红山茶的细胞学研究[J]. 云南植物研究, 2007, 29(6):655-658. WANG S, WANG Z L, LIANG J, et al. Karyological study on the endangered species Camellia azalea(Theaceae)[J]. Acta Bot Yunnanica, 2007, 29(6): 655-658. DOI: CNKI:SUN:YOKE.0.2007-06-010.
[18] 贾文庆. 山茶花生殖生物学及倍性育种基础研究[D]. 北京: 中国林业科学研究院, 2015:32-33.JIA W Q. Fundamental study on reproductive biology and ploidy breeding of camellia flower[D]. Beijing: Chinese Academy of Forestry, 2015:32-33.
[19] KONDO K, TANIGUCHI K, TANAKA N, et al. A karyomorphological study of twelve species of Chinese camellia[J]. La Kromosomo II, 1991, 62:210-211. DOI: 10.2307/2007134.
[20] XIAO T, GU Z, XIA L, et al. A karyomorphological study of ten species of Chinese camellia[J]. La Kromosomo, 1991(2):2051-2085.
[21] 于小玉, 喻方圆, 刘建兵, 等. ISSR在油茶品种鉴别和遗传多样性分析中的应用[J]. 南京林业大学学报(自然科学版), 2013, 37(1): 061-66. YU X Y, YU F Y, LIU J B, et al. Identification and genetic diversity analysis of Camellia oleifera varieties using ISSR marker[J]. J Nanjing For Univ(Nat Sci Ed), 2013, 37(1): 061-66.DOI:10.3969/j.issn.1000-2006.2013.01.010.
[22] 李光涛. 山茶属植物核型研究进展[J]. 中国野生植物资源, 2001(5):9-14.LI G T. New Advance karyotype studies of genus Camellia[J]. Chin Wild Plant Reso, 2001(5):9-14.
[23] 王滑, 陈放, 段丽君, 等. 紫薇异倍体的杂交亲和性分析及子代倍性鉴定[J].湖南农业大学学报(自然科学版), 2016(3):291-295. WANG H, CHEN F, DUAN L J, et al. The crossability and progeny ploidyidentification of hybridization among the heteroploidincrape myrtle[J]. J Hunan Agr(Univ Nat Sci), 2016(3):291-295. DOI: 10.13331/j.cnki.jhau.2016.03.013.
[24] 王颖, 周攀, 王玉霞, 等. 苹果属皱叶型植株实生后代倍性鉴定研究. 北方园艺, 2008(11):43-46. WANG Y, ZHOU P, WANG Y X, et al. Chromosome ploidy identification of the seedling plants from the leaf-wrinkled and dwarf plants in Malus[J]. N Hortic, 2008(11):43-46. DOI: CNKI:SUN:BFYY.0.2008-11-013.
[25] 饶静云, 刘义飞, 黄宏文. 中华猕猴桃不同倍性间杂交后代倍性分离和遗传变异分析[J]. 园艺学报, 2012, 39(8):1447-1456.RAO J Y, LIU Y F, HUANG H W. Analysis of ploidy segregation and genetic variation of progenies of different interploidy crosses in Actinidia chinensis[J]. Acta Hortic Sinica, 2012, 39(8):1447-1456. DOI: CNKI: SUN: YYXB.0.2012-08-005.

相似文献/References:

[1]胡玉玲,姚小华*,任华东,等.普通油茶体胚再生体系研究[J].南京林业大学学报(自然科学版),2014,38(06):160.[doi:10.3969/j.issn.1000-2006.2014.06.030]
 HU Yuling,YAO Xiaohua*,REN Huadong,et al.Study on somatic embryogenesis regeneration system in Camellia oleifera[J].Journal of Nanjing Forestry University(Natural Science Edition),2014,38(05):160.[doi:10.3969/j.issn.1000-2006.2014.06.030]

备注/Memo

备注/Memo:
收稿日期:2020-03-09 修回日期:2020-04-13 基金项目:国家重点研发计划(2018YFD1000603); 国家自然科学基金项目(31500553); 中南林业科技大学创新基金项目(CZ2017B411)。 第一作者:叶天文(ytwdyx0401@163.com)。*通信作者:肖诗鑫(xsx1104@sina.com),讲师,ORCID(0000-0002-1958-8081)。
更新日期/Last Update: 1900-01-01