利用流式细胞术快速鉴定169份香蕉种质资源的染色体倍性
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摘要
【目的】利用流式细胞术快速测定169份香蕉种质资源的倍性,以期为香蕉遗传进化以及杂交育种研究提供理论依据。【方法】研究以‘小果野蕉’(AA)为内参,分析香牙蕉(Musa AAA Cavendish)、大蕉(Musa Dajiao)、粉蕉(Musa ABB Pisang Awak)、棱指蕉(Musa ABB Bluggoe)、龙牙蕉(AAB)、贡蕉(AA)、野生蕉等不同类型香蕉种质资源的染色体倍性。【结果】野生蕉、贡蕉均为二倍体,香牙蕉、棱指蕉、龙牙蕉均为三倍体,大蕉和粉蕉多数为三倍体,‘畦头大蕉’‘景洪野大蕉’‘粉杂1号’为四倍体。本研究中的香蕉杂交组合‘高州中把大蕉’ב那邦野蕉’、‘高州中把大蕉’ב广宁野生蕉’、‘高州中把大蕉’ב阿宽蕉’、‘华农中把大蕉’ב阿宽蕉’的后代均为四倍体,而‘广粉1号’ב那邦野蕉’为三倍体。‘海南红蕉’和‘飞亚-25’为三倍体。【结论】香蕉基因组和倍性十分复杂,根据形态特征鉴定香蕉染色体倍性,其结果可靠性很低。以嫩叶为材料,采用FCM技术可以快速、准确鉴定香蕉资源的倍性。
【Objective】Banana(Musa spp.) is one of the most important fruits in the world and one of the important economic crops in southern China. Banana germplasms are rich and diverse. Its chromosomal ploidy is quite complicated due to the long evolution. The identification of chromosomal ploidy is the basis for studying the genetic evolution, classification and breeding of banana. The identification of chromosomal ploidy by chromosome squashing is difficult because the banana chromosome witch are small, and the satellite DNA is easy to mix with the small chromosome. Flow cytometry(FCM) is now the prevailing method for identification of chromosomal ploidy in plants, because of the ease of sample preparation and the high sample throughput. In this study, the chromosomal ploidy levels of 169 Musa germplasms were identificated by flow cytometry in order to provide theoretical basis for the study of genetic evolution and hybridization of banana.【Methods】The leaves unopened of 169 Musa germplasms were selected with ice boxes from Banana Germplasm Resource Garden of Dongguan Banana and Vegetable Institute and Banana and Litchi Germplasm Resource Garden in Guangzhou of Na-tional Fruit tree Germplasm. 20-30 mg fresh leaves contained an equal number of sample and standard sample were chopped with a sharp razor blade in a Petri dish containing 1.5 m L OTTO buffer Ⅰ which was precooled. The mixture was placed on ice 5-10 min and filtered with a 22 μm strainer to remove large cellular materials and the remaining 50 μL sample was mixed again after discarding the upper liquid. Then, 200 μL OTTO buffer Ⅱ was added to the mixture. The cell suspension was stained by Propidium Iodide and RNase 5-10 min. In the end, 200 μL cell suspension was used for the analysis of cell flow analyzer. Xiaoguo Yejiao, a known diploid wild banana(M. acuminata) was used as the internal standard, and we analyzed the relative genome size of musa germplasms containing cavendish, pisang awak, pisang mas, bluggoe, wild banana etc. by Beckman Cell Lab Quanta SC, and the chromosomal ploidy levels were then estimated.【Results】According to the analysis, thirty-seven different types of wild bananas from Yunnan, Guangxi, Guangdong, Jiangxi, Hunan and Shaanxi were all diploid. The existing triploidy and tetraploid bananas were originated from intra-and interspecific hybridization between two wild diploid species: Musa acuminata Colla. and M. balbisiana Colla, so the genomes and ploidy of these bananas were very complex. In this study, pisang mas are all diploid. All of the germplasms belonged to cavendish, longyajiao, bluggoe, and most of germplasms derived from dajiao and pisang awak were triploid except for three varieties:‘Qitou Dajiao'‘Yunnan Jinghong Yedajiao'‘Fenza No.1'. They were tetraploid due to the calculation values of ploidy levels were 3.84, 3.59, 3.43.‘Hainan Hongjiao'was triploid. The plant of‘FHIA-25'whose genotype and ploidy were unknown in the Banana Germplasm System(Musa Germplasm Information System) is very thick, and it was found to be triploid according to the results of repeated detection and analysis. Similarly,‘Indonesia Dafenjiao'which was very strong, was found to be triploid. In contrast,‘FX2'which was thin, and‘Jinghong Yedajiao'which was thin and short, were tetraploid. The individuals derived from‘Gaozhou Zhongba Dajiao'בNabang Yejiao',‘Gaozhou Zhongba Dajiao'בGangning Yeshengjiao',‘Gaozhou Zhongba Dajiao'בAkuanjiao',‘Huanong Zhongba Dajiao'בAkuanjiao'(The hybrid offsprings of triploid Dajiao and Akuanjiao) were all tetraploid, while those derived from‘Guangfen No.1'בNabang Yejiao'were triploid in this study. The point of the average DNA relative content of the diploid was AA>M. basjoo>M. itinerans>AA(cultivar)>BB wild. The point of the average DNA relative content of the the triploid was AAA>AAB>ABB.【Conclusion】After a long period of evolution, the genome and the ploidy of bananas, especially cultivars are complex. The reliability of identification of banana chromosomal ploidy according to the size of the plant and other morphological characteristics, especially some allopolyploid bananas, is very low. FCM is suitable for quickly identification of the ploidy of bananas because of its simplexity, high sensitivity, resolution and accuracy.
【Objective】Banana(Musa spp.) is one of the most important fruits in the world and one of the important economic crops in southern China. Banana germplasms are rich and diverse. Its chromosomal ploidy is quite complicated due to the long evolution. The identification of chromosomal ploidy is the basis for studying the genetic evolution, classification and breeding of banana. The identification of chromosomal ploidy by chromosome squashing is difficult because the banana chromosome witch are small, and the satellite DNA is easy to mix with the small chromosome. Flow cytometry(FCM) is now the prevailing method for identification of chromosomal ploidy in plants, because of the ease of sample preparation and the high sample throughput. In this study, the chromosomal ploidy levels of 169 Musa germplasms were identificated by flow cytometry in order to provide theoretical basis for the study of genetic evolution and hybridization of banana.【Methods】The leaves unopened of 169 Musa germplasms were selected with ice boxes from Banana Germplasm Resource Garden of Dongguan Banana and Vegetable Institute and Banana and Litchi Germplasm Resource Garden in Guangzhou of Na-tional Fruit tree Germplasm. 20-30 mg fresh leaves contained an equal number of sample and standard sample were chopped with a sharp razor blade in a Petri dish containing 1.5 m L OTTO buffer Ⅰ which was precooled. The mixture was placed on ice 5-10 min and filtered with a 22 μm strainer to remove large cellular materials and the remaining 50 μL sample was mixed again after discarding the upper liquid. Then, 200 μL OTTO buffer Ⅱ was added to the mixture. The cell suspension was stained by Propidium Iodide and RNase 5-10 min. In the end, 200 μL cell suspension was used for the analysis of cell flow analyzer. Xiaoguo Yejiao, a known diploid wild banana(M. acuminata) was used as the internal standard, and we analyzed the relative genome size of musa germplasms containing cavendish, pisang awak, pisang mas, bluggoe, wild banana etc. by Beckman Cell Lab Quanta SC, and the chromosomal ploidy levels were then estimated.【Results】According to the analysis, thirty-seven different types of wild bananas from Yunnan, Guangxi, Guangdong, Jiangxi, Hunan and Shaanxi were all diploid. The existing triploidy and tetraploid bananas were originated from intra-and interspecific hybridization between two wild diploid species: Musa acuminata Colla. and M. balbisiana Colla, so the genomes and ploidy of these bananas were very complex. In this study, pisang mas are all diploid. All of the germplasms belonged to cavendish, longyajiao, bluggoe, and most of germplasms derived from dajiao and pisang awak were triploid except for three varieties:‘Qitou Dajiao'‘Yunnan Jinghong Yedajiao'‘Fenza No.1'. They were tetraploid due to the calculation values of ploidy levels were 3.84, 3.59, 3.43.‘Hainan Hongjiao'was triploid. The plant of‘FHIA-25'whose genotype and ploidy were unknown in the Banana Germplasm System(Musa Germplasm Information System) is very thick, and it was found to be triploid according to the results of repeated detection and analysis. Similarly,‘Indonesia Dafenjiao'which was very strong, was found to be triploid. In contrast,‘FX2'which was thin, and‘Jinghong Yedajiao'which was thin and short, were tetraploid. The individuals derived from‘Gaozhou Zhongba Dajiao'בNabang Yejiao',‘Gaozhou Zhongba Dajiao'בGangning Yeshengjiao',‘Gaozhou Zhongba Dajiao'בAkuanjiao',‘Huanong Zhongba Dajiao'בAkuanjiao'(The hybrid offsprings of triploid Dajiao and Akuanjiao) were all tetraploid, while those derived from‘Guangfen No.1'בNabang Yejiao'were triploid in this study. The point of the average DNA relative content of the diploid was AA>M. basjoo>M. itinerans>AA(cultivar)>BB wild. The point of the average DNA relative content of the the triploid was AAA>AAB>ABB.【Conclusion】After a long period of evolution, the genome and the ploidy of bananas, especially cultivars are complex. The reliability of identification of banana chromosomal ploidy according to the size of the plant and other morphological characteristics, especially some allopolyploid bananas, is very low. FCM is suitable for quickly identification of the ploidy of bananas because of its simplexity, high sensitivity, resolution and accuracy.
引文
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