基于FCM测定的香蕉种质倍性分析

Ploidy investigation of bananas(Musa spp.) by flow cytometry

【目的】基于流式细胞术(FCM)分析结果,从细胞学层面上准确揭示不同基因组类型香蕉种质的倍性差异,为香蕉遗传多样性研究及选择育种提供基础支撑。【方法】以二倍体(AA)‘陵水野生蕉’(Musa AA acuminate‘Lingshui Yesheng Jiao’)为内参,利用FCM技术,对11份香蕉种质资源进行了倍性鉴定分析。【结果】依据(FCM)结果分析,参与分析样品的倍性可分为4大类:第I类有‘红河矮’蕉(M.AAA Cavendish‘Honghe Ai’)和‘陵水野生蕉’,属于2 N;第II类有‘始兴BB’(M.balbisiana‘Shixing BB’)、‘东莞中把大蕉’(M.ABB Cavendish‘Dongguan Zhongba Dajiao’)、‘Pisang Ceylan’和‘FHIA-18’4个种质,属于3 N;第III类有‘Cachaco’‘FHIA-17’‘FHIA-03’和‘TMB×5259-1’4个种质,均属于4 N;第IV类‘红香蕉’(‘Red banana’),在FCM分析中,此种质出现了2个峰值,第一个峰值为52.79,第二个峰值为103.24,测定时2个峰同时出现,且重复性好,可能是二倍体与四倍体混倍形成。【结论】较之传统方法,FCM准确揭示了DNA含量与倍性的关系,能更好地反映香蕉种质资源之间的遗传特征。

[Objective] Banana（Musa spp.）, origined from the southeast Asia, is the forth food crop in the world, which are important staple food and income-generating crops, especially to the people in the tropical and subtropical regions Therefore, global banana production plays an important role in dealing with the problems derived from the global warming and the population expansion of the world. However, there are some problems exsiting in banana genetic improvement due to uncertainty of ploidy of varieties or breeds. The accurate determination of banana ploidy level is important for proper genetic manipulation and is a prerequisite for interspecific hybridization. So far, various methods, including conventional breeding, genetic transformation, morphological classification, cytological study, biochemical means and molecular markers, have been used to genetically manipulate bananas so as to improve its production. Chromosome counting is tedious and laborious to determine the ploidy of bananas. Flow cytometry（FCM） was employed to detect the ploidy of bananas in this study. 11 banana varieties, named Musa AAA Cavendish ＇Honghe Ai＇, M. AA acuminate ＇Lingshui YeshengJiao＇, M. balbisiana ＇Shixing BB＇, ＇Red banana＇, M. ABB Cavendish ＇ Dongguan Zhongba Dajiao＇, ＇ Pisang Ceylan＇ （AAB）, ＇ FHIA- 18＇ （AAAB）, ＇ Cachaco＇ （ABB）, ＇ FHIA- 17＇ （AAAA）, ＇ FHIA-03＇ （AABB） and ＇ TMB ×5259-1＇ （ABBB） were chosen as materials. [ Methods ] Approximately 0.5 cm^2 of fresh leaves were chopped with a sharp razor blade in a plastic Petri dish containing 2 mL HRA buffer for each sample. The suspension of nuclei was filtered through a 30 μm nylon mesh to remove large cellular materials. After that, the nuclear DNA of the sample was stained by adding 1 600 μL of 0.02 g·L^-1 propidium iodide. The sample was mixed gently and incubated briefly on ice before mixing again. A known diploid wild banana germplasm resource, M. AA acuminate ＇Lingshui YeshengJiao＇, was used to test the standardisation of flow cytometry by using a Partec CyFlow Space Flow Cytometer with a 405 nm laser. At least 5 000-10 000 nuclei were analyzed for each sample. Three leaf samples were tested from each of four plants of each accession. [Results ] The FCM was successfully used to analyze the ploidy of 11 banana germplasm resources in present study,. The ploidy of 11 banana clones were divided into four groups. The first group included M. AAA Cavendish ＇Honghe Ai＇ and M. AA acuminate. ＇Lingshui YeshengJiao＇, identified as 2 N. The second group contained M. BB balbisiana ＇Shixing BB＇, M. ABB Cavendish ＇Dongguan Zhongba Dajiao＇, ＇Pisang Ceylan＇ and ＇FHIA-18＇, identified as 3 N. The third group consisted of ＇ Cachaco＇ ＇FHIA-17＇ ＇FHIA-03＇ and ＇TMB×5259- 1 ＇, identified as 4 N. The fourth group only included ＇ Red banana＇ , which showed two peak values in the analysis atlas, 52.79 and 103.24, with good repeatability. Our study had some different results from the pi：evous research. M. AAA Cavendish ＇Honghe Ai＇ previously was classified as triploid, M. BB balbisiana. ＇ Shixing BB＇ as diploid, ＇FHIA-18＇ as tetraploid, and ＇Cachaco＇ as triploid, they were found to be diploid, triploid, triploid and triploid, respectively ,in our study. [ Conclusions ] The ploidy of 11 banana varieties were identified by means of the FCM method and were divide into four groups. ＇Red banana＇ might be a mixoploid derived from two diploid and tetraploid, and this needs further investigation to confirm.