CMA fluorescence banding of somatic metaphase chromosomes in Citrus ichangensis

Abstract:【Objective】Citrus ichangensis Swingle is a very important breeding germplasm of Citrus with high resistances and somatic chromosome association which is a specific phenomenon comparing with other plants. Chromosome research is an important method for studying plant origin and development, gene function and chromosome structure. Here we studied the somatic chromosome fluorescence banding stained by chromomycin A3 in eight different types of C. ichangensis Swingle which would contribute to the research of relationship between structure and composition of chromosomes, origin and evolution of Citrus. This paper explored the chromosome characteristics and the somatic chromosome association in C. ichangensis Swingle as well.【Methods】Fluorescence banding of metaphase chromosomes was analyzed using CMA staining. First, the preparation of somatic metaphase chromosome samples was conducted by wall degradation hypotonic method according to Chen Ruiyang. The procedures were as follows: the tender leaves and stem tips were put into 0.002 mol·L^-1 8-hydroxyquinoline solution for 2.5 h, and then immersed into mix solution of methylalcohol and acetic acid(3∶1 in volume) for over 2 h, then the samples were digested with 3% cellulase and pectinase solution mix for 2.5 h and washed with deionized water andinserting mix solution of methylalcohol and acetic acid(3:1 in volume) in the tubes carefully. The samples were spread on the glass sides and stained with 5% Giemsa solution. The second, CMA staining followed the methods of M. Guerra and YANG Xiaolin.【Results】Eight accessions had different CMA(+) bands. According to pattern characteristics and the results of previous studies, CMA banding pattern were divided into four different types: C-type: the ends of the arms possessed CMA(+) fluorescence bands; D-type: the end of the long arm or short arm possessed CMA(+) fluorescence band; E-type: no fluorescence band; Bsttype: the end of the long arm, centromeric region and satellite chromosomes possessed CMA(+) fluorescence band. The accessions were 8 different strains of C. ichangensis Swingle including Sanye, No. 4, No. 1,2586, 6-3, Guizhou, Yuandaguangpi and Huaihua. CMA(+) banding patterns were as follows: Sanye: 2n=2x=18=3C+11D+2E+2F, No. 4: 2n=2x=18=4C+12D+2F, No. 1: 2n=2x=18=2C+13D+1E+2F, 2568: 2n=2x=18=3C+13D+2F, Guizhou: 2n=2x=18=2C+12D+2E+2F, Yuandaguangpi: 2n=2x=18=5C+8D+3E+2F,Huaihua: 2n=2x=18=6C+8D+2E+2F. Among them, No. 4, and 2586 showed different CMA(+) fluorescence bands, which were defined as C, D, and Bst types in their 18 chromosomes. The other six accessions showed different CMA(+) fluorescence bands from those of No. 4, and 2586, with no any CMA(+) fluorescence band on 1 to 5 chromosomes respectively. Different amount of bands in our 8 accessions reflected the differences of their chromosome structures. However, the two same Bst type bands on a same pair of homologous chromosomes with satellites were detected in every accession. Compared with previously published results, we concluded that somatic chromosome association was related to the regions with abundant hetero chromatin. Meanwhile, we found that there were some polymorphic bands among every pair of homologous chromosomes except the 9thchromosomes with satellites in the eight materials detected.【Conclusion】Polymorphism and divergence were found in the CMA(+) banding patterns in eight strains of Citrus and this suggested that the chromosome structures and compositions of the eight strains were different, and some of the homologous chromosomes showed CMA(+) heterozygosity. CMA fluorescence banding would be an effective indicator to be associated with genetic classification of C. ichangensis Swingle. Therefore, the technique of CMA staining would be helpful for analysis of chromosome characteristics, chromosome identification and systematic classification in C. ichangensis Swingle and other Citrus plants.