Cloning And Location Of Centromeric And Telomeric Repeat Sequences Of Morus Notabilis

Repeat sequence is one of the important components in the plant genomes. It is important to maintain chromosome structure in space, regulate the expression of gene and genetic recombinations. The cluster of tandem repeat DNA often has a specificity of genus, species or even the chromosome. The genome of M. notabilis is about 330 Mb with 128 Mb repeats sequences. In this research, we firstly investigated the centromeric tandem repetitive sequences(CTR) and telomere associated sequence(TAS) of M. notabilis. Karyotype analysis of fluorescence in situ hybridization technique(FISH) was used in the present study. The main research results are as follows.Using bioinformatic methods, repetitive sequences in the mulberry genome arranged from high to low complexity were examined and four DNA sequences were screened out with highest copy number. These sequences were selected as the candidate centromeric tandem repeats of M. notabilis and named as CCTR964, CCTR723, CCTR327, and CCTR251. Compared with CTRs in other species, they have sequence similarity between 1% and 28%, suggesting their species specificities. Then they were used as probes and FISH was performed with the metaphase chromosomes of M. Notabilis. The results indicated that:(1) The hybridization signals of CCTR964 were on the centromeres of the chromosome 1, 2, 4, 6, and the pericentromeric region of the short arm in chromosome 3.(2) CCTR723 signals were located on the pericentromeric region of the short arm in chromosome 2 and the centromeres of the chromosome 5.(3) CCTR327 signals distributed on the centromeres of No. 3, 4, 5 chromosomes. In chromosome 3, 4, the signal was weak and in chromosome 5 the signals was strong. In addition, the signals of CCTR723 scattered in the centromeres of No. 1 and 2 chromosome.(4) CCTR251 signals were spread on almost all the centromeres of M. notabilis chromosomes except chromosome 6. Among them, the signals distributed on the chromosome 2 and 3 were obvious. Therefore, it is concluded that these four sequences were the centromeric tandem repeat sequences of M. notabilis.Using Arabidopsis telomeric repeat sequences(TTTAGGG)4 and(CCCTAAA)3 as primers, one DNA sequence of 540 bp was obtained from the former amplified products and two sequences(403bp and 420bp) were obtained from the latter. These sequences were named as TAS1, TAS2, and TAS3, respectively. Sequence analysis showed that they were all riched in AT, which was consistent with the characteristics of satellite sequences. In these TAS fragments, there are some mutant of Arabidopsis TAS units, it also contains telomeric repeat sequence of other creatures, such as silkworm(CCTAA) and tomato(TTTGGG). The homology analysis showed that the sequences similarities of TAS1, TAS2, TAS3 and other species of telomere associated sequences were very low. The results of FISH study showed that:(1) TAS1 signals were distributed at the end of the short arm of the chromosome 2, both ends of chromosomes 4, 5 and one end of chromosome 6.(2) TAS1 signals were distributed at both ends of the chromosomes 1, 6, the short arm of the chromosome 2, the long arm of the chromosome 3, and one end of chromosomes 4, 5. No signal was detected in chromosome 7.(3) The signals of TAS3 were distributed on all the seven pairs of chromosomes, not on the end of all chromosomes, and the intensity of the signal was weak. When using the strict elution condition, the signals of TAS3 sequence were not be detected. Therefore, it was speculated that TAS3 had low copy number on the chromosomes of M. Notabilis.Combined with the hybridization signals of CCTR964, CCTR723, CCTR251, TAS1 and TAS2 probes, the chromosome idiogram of M. Notabilis was established, which provided a new basis for the karyotype analysis of M. Notabilis and reference for the accurate identification of the chromosomes in M. Notabilis.