Identification of chromosome and caryotype analysis in citrus by GISH method and in situ PCR method.

柑橘2n=18条染色体通过CMA染色的显带模式可分为A ~ E 5种类型，但很难识别同一类型的染色体。本研究采用CMA染色和GISH法对同型染色体进行鉴定，并对‘Tosa-Buntan’与‘Suisho-Buntan’杂交苗进行核型分析，明确‘Tosa-Buntan’1的染色体配对组。38个二倍体幼苗的染色体组成有13种差异。二倍体幼苗的核型分析表明，‘Tosa-Buntan’的染色体分离组为AB+CC+CC+CD+DE+EE+EE+EE+EE +EE或AB+CC+CC+DD+CE+EE+EE+EE+EE +EE+EE+EE。对三倍体和四倍体幼苗的核型分析表明，二倍体杂交产生的三倍体和四倍体幼苗既来自一个未还原的雄配子，也来自一个未还原的雄配子。以CMA（+）区域的相对大小为指标，c型染色体在谷川-邦坦和酸橙中可分为2组，d型染色体在吉田-本坎和酸橙中可分为3组，在木谷木舒中可分为2组。在GISH方法中，以蜜橘DNA为探针，根据信号的颜色和大小，将a ~ E型‘Tosa-Buntan’的9条染色体分为8组。3 .以‘Dancan’葡萄柚DNA为探针，根据信号的颜色和大小，将‘Suisho-Buntan’的a ~ E型9条染色体分为7组。利用几种探针DNA对‘Tosa-Buntan’染色体进行GISH分析，结果表明，‘Tosa-Buntan’与‘Suisho-Buntan’非常接近，与柑桔相对较近，与柑桔和橘子相对较远。‘Tosa-Buntan’和脐橙的GISH互反表明，这两个物种之间的同型染色体可能不是同源染色体。

Although citrus chromosomes, 2n=18, are classified to 5 types (A〜E) by the banding patterns of CMA staining, it is difficult to identify a chromosome in the same type. In this research, CMA staining and GISH method were performed for an identification of chromosome in the same type, and a caryotype analysis of hybrid seedlings from 'Tosa-Buntan' x 'Suisho-Buntan' pummelo was performed to clarify the chromosome paring set of 'Tosa-Buntan'1. Thirteen kinds of chromosome composition were manifested by 38 diploid seedlings from 'Tosa-Buntan' x 'Suisho-Buntan'. The caryotype analysis of the diploid seedlings indicated that the chromosome parting set of 'Tosa-Buntan' would by AB+CC+CC+CD+DE+EE+EE+EE+EE or AB+CC+CC+DD+CE+EE+EE+EE+EE. The caryotype analysis of the tri-and tetraploid seedlings showed that the triploid and tetraploid seedlings from diploid crossing also originated from an unreduced male gamete as well as from an unreduced male gamete.2. By using the relative size of CMA(+) regions as indices, type-C chromosomes were able to classify into two groups in 'Tanikawa-Buntan' and sour orange, and type-D chromosomes were able to classify into three groups in 'Yoshida-Ponkan' and sour orange, and into two groups in 'Mukaku-Kishu'.3. In GISH method, 9 chromosomes of 'Tosa-Buntan' in type-A to E were able to classify into 8 groups depended on coloration and size of the signals by using satsuma mandarin DNA as a probe. Nine chromosomes of 'Suisho-Buntan' in type-A to E were able to classify into 7 groups depended on coloration and size of the signals by using 'Dancan' grapefruit DNA as a probe.4. The GISH for 'Tosa-Buntan' chromosome using several kinds of probe DNA showed that 'Tosa-Buntan' is very closed to 'Suisho-Buntan', relative close to citron and relative far from mandarin and orange. The reciprocal GISH between 'Tosa-Buntan' and navel orange suggested that the same type chromosome between these species may not be homoeologous chromosome.