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

柑桔染色体2n=18，根据CMA染色带型可分为5种类型（A、E、E），但同一类型的染色体很难鉴别。本研究采用CMA染色和GISH法对同一类型的染色体进行鉴定，并对'土佐文旦' × '水照文旦'柚杂种苗进行核型分析，以明确'土佐文旦' 1号的染色体配对组。38株土佐文旦×水昌文旦的二倍体植株表现出13种染色体组成。二倍体苗的核型分析表明，土佐文旦的染色体分型为AB+CC+CC+CD+DE+EE+ EE + EE +EE或AB+CC+CC+DD+CE+EE+EE+EE+ EE;三倍体和四倍体苗的核型分析表明，二倍体杂交的三倍体和四倍体苗既起源于未减数的雄配子，也起源于未减数的雄配子.以CMA（+）区域的相对大小为指标，C型染色体在谷川文丹和酸橙子中可分为两大类，D型染色体在吉田庞柑和酸橙子中可分为三大类，而在Mukaku-Kishu中可分为两大类.以温州蜜柑DNA为探针，采用GISH方法，根据信号的颜色和大小，将A ~ E型的土佐文旦9条染色体分为8类。以“丹参”葡萄柚DNA为探针，根据信号的颜色和大小，将“水照文旦”的9条A ~ E型染色体分为7类.用几种探针DNA对土佐文旦染色体进行GISH分析，结果表明，土佐文旦与水昌文旦的亲缘关系很近，与香橼的亲缘关系较近，与普通柑橘和橙子的亲缘关系较远。“土佐文旦”与脐橙橙子之间的GISH互作表明，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.