Karyotype And Banding Patterns Of The Amphioxus Branchiostoma Belcheri Tsingtauense (Cephalochordata)

Amphioxus or lancelet, a cephalochordate, is the extant invertebrate chordate considered to be the closest relationship to the vertebrates. It has been more than 100 years since the first description of the chromosomes of the amphioxus Branchiostoma lanceolatum by Stricht in 1895. However, only three species of the cephalochordate including B. lanceolatum, B. floridae, and B. belcheri, have so far been studied for their chromosome numbers, and the karyotypic analysis and banding study of amphioxus chromosomes remains largely untouched. Knowledge of the chromosomal karyotype and banding in amphioxus could be of particular use for the extensive study of comparative genome and cytogenetics. In this paper, the karyotype and banding pattern of amphioxus B. belcheri tsingtauense were reported for the first time. 1. Chromosome number (2n) and fundamental number (FN)The chromosome number and karyotype of amphioxus Branchiostoma belcheri tsingtauense were studied using embryonic cells of the animal. The counting of 150 well-spread samples revealed that the number of the chromosomes ranged from 18 to 38, but the modal number of the chromosome was 36, which was in proportion to 66%. This showed that the diploid chromosome number (2n) of amphioxus B. belcheri tsingtauense was 36.The chromosomes were extremely small, ranging from 1.4 to 3.7 um in length. The first pair of the 36 chromosomes of B. belcheri tsingtauense was subtelocentric and the other 17 pairs telocentric. Neither metacentric nor submetacentric chromosomes nor satellites were observed. Therefore, the karyotype of theamphioxus is 2n=36, 2st+34t, FN=36. This is the first report on the karyotype of the cephalochordata. It is likely that telocentric chromosomes with minute sizes may be ancestral types of the vertebrate chromosomes.2. G-bandingThe GTG banding (G-banding) was carried out by the standard trypsin method with slight modification, which works well for protochordate because a good number of reproducible G-bands are consistently obtained from the embryonic cells of late blastulae and early gastrulae of amphioxus B. belcheri tsingtauense. The trypsin treatment for G-banding is an important factor in obtaining well-banded chromosomes, while the slide aging is not. After G-banding, 65.5% of the chromosome surface is positively stained and the total number of G-banded is 149. There are 77 positive, 65 negative and 7 variable bands among the 149 G-bands.3. R-bandingThe RHG banding (R-banding) technique used was the method of Dutrillaux and Lejeune (1971) slightly modified. The total number of R-bands found in the chromosomes is 100, which is approximately 1/4 to 1/5 of that of the vertebrate metaphase chromosome R-bands. They consist of 57 positive, 33 negative and 10 variable bands. As the number of R-bands is associated with that of genes, and most genes that have been mapped to mammalian chromosomes with sufficient precision are usually distributed in R-bands rather than in G-bands. This is apparently in agreement with the hypothesis of two rounds of extensive gene duplication leading to the origin of the vertebrates. In addition, there exists 72.2% of the chromosome surface positively stained with R-banding.4. Complementarity of G- and R- banding patternIt is generally accepted that chromosome banding patterns appear closely related to differences in DNA base composition, and R-bands correspond to GC-rich DNA and G-bands to AT-rich DNA. Bands positively stained after G-banding are commonly negatively stained after R-banding and vice versa.The complementarity of G- and R-band patterns was evaluated by comparingthe features of each positive and negative chromosomal G- and R-band. It was found that the complementarity is only illustrated to some extent by the parts of some chromosomes. The G- and R-band patterns are found to be only about 38% complementary. The reason for this is unknown at the present. Possibly, CG-rich and AT-rich regions in amphioxus DNA are not as "rich" as those in the vertebrate DNA, that is, a...