普通油茶染色体制片技术优化及核型分析

【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。

Optimization of chromosome mounting technique and karyotype analysis of Camellia oleifera

【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.