Study for citrus chromosome mapping

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For making physical map of citrus chromosomes , chromosome preparation method in which vegetative tissue of mother plant use as a material and chromosomes are relatively long and useful for karyotype analysis , and identification of chromosomes should be confirmed.1. In chromosome preparation method, using young leaves of 3-5mm, 120-180min treatment of 0.3% cellurase +0.2% pectolyase solution at 37℃ and dropping suspended cells to slide-glass were useful for relatively long chromosome prepared.2 . Citrus chromosomes were classtfied to 5 types based on CMA banding patterns . That is , CMA(+) regions in terminal of both arms and a proximal (type-A) , in terminal of one arm and a proximal (type-B) , in terminal of both arms (type-C), in terminal of one arm (type-D) and no CMA(+) region (type-E) on chromosome.3. Citrus chromosome compositions depended on CMA banding pattern were 2B+8D+8E in fingered citron, 1C+8D+9E in koji, 1A+1C+8D+8E in unshiu, 2B+2C+7D+7E in 'Washington' navel orange, 2A+1B+1C+6D+8D in 'Duncan' grapefruit, 1A+1B+5C+2D+9E in 'Tosa-Buntan' , 3A+3C+3D+9E in 'Suisho-Buntan' , 2A+1B+3C+3D+9E in ' Ban-okan ' , 1A+2B+2C+4D+8E+1A or B in 'Banpeiyu ' and 4B+8D+6E in triforiate orange.4. For 'Nankan N0.20' unshiu chromosomes, genomic in situ hybridization (GISH) was performed using Dig labeled 'Nankan N0.20 ' DNA and Biotin labeled 'Tosa-Buntan ' DNA, and chromosomes were stained with anti-Dig rhodamine and avidin FITC. Eight type-D chromosomes of ' Nankan N0.20 ' were classified to I chromosome with large yellow signal, I with small yellow signal, 3 with large orange signal, I with small orange signal and 2 with small red signal. Furthermore in 'Nankan N0.20 ' chromosomes, 8 type-E chromosomes were classified to 2 long, 2 medium and 4 short chromosomes by Giemsa staining.