Abstract:

Objective: The lack of chromosome physical markers seriously hinders the development of cytogenetics research in the genus Carya. In this study, we used the whole genome sequencing data to develop some oligonucleotide type chromosomal physical markers and established a development method of oligonucleotide probes for Carya cathayensis, in order to provide reference for the related research of other species in Carya. Method: The repetitive sequences in the genome of C. cathayensis were analyzed by the Tandem Repeat Finder software and filtered out according to the principle of Period size greater than 4, Copy number more than 100, and Period size * Copy number more than 3 000. About 60 oligonucleotide probes were designed and synthesized, and then these probes were further analyzed and screened by the technology of fluorescence in situ hybridization in C. cathayensis, C. hunanensis, C. tonkinensis, C. dabieshanensis, C. kweichowensis, C. illinoinensis and C. sinensis. Result: 1) The oligonucleotide probes of sht-2, sht-3, sht-4, sht-5, sht-5S and sht-45S were able to produce fluorescence signals in C. cathayensis. The probes of sht-2, sht-3, sht-4 and sht-5 had the same distribution patterns on the chromosome, and there were two pairs of signals with different fluorescence intensities. The sht-45S produced one pair of signals on the C. cathayensis chromosome, while sht-5S only had one signal site on the single chromosome of C. cathayensis. These probes were all located in the near centromere region of the chromosome. 2) Among the two pairs of signal sites of sht-5, the stronger pair was completely overlapped with the distribution of the signal sites of sht-45S on the chromosome of C. cathayensis. 3) The distribution positions of 45S rDNA and sht-45S on the chromosomes of C. cathayensis were completely overlapped, and the positions of 5S rDNA and sht-5S were also completely overlapped. 4) The oligonucleotide probe of sht-5 was able to produce fluorescence signals in C. hunanensis, C. tonkinensis, C. dabieshanensis and C. kweichowensis, and the distribution patterns were similar with C. cathayensis. However, there were no signals produced by sht-5 in C. illinoinensis and C. sinensis. 5) The oligonucleotide probes of sht-5S and sht-45S were able to produce fluorescence signals in C. hunanensis, C. tonkinensis, C. dabieshanensis, C. kweichowensis and C. illinoinensis, but no signals in C. sinensis. 6) The probe of sht-45S only had one pair signal sites on the chromosome of C. hunanensis, C. dabieshanensis and C. kweichowensis, and their distribution patterns were similar. The sht-45S produced two pairs of signals sites on the chromosome of C. illinoinensis, which located in the near centromere region of two pairs of homologous chromosomes. 7) The probe of sht-45S only had one pair signal sites on the chromosome of C. tonkinensis, C. kweichowensis, C. dabieshanensis and C. illinoinensis, and the signal sites were located in the near centromere region of homologous chromosomes and had the similar distribution patterns. However, there were two different distribution patterns in C. hunanensis. The first distribution pattern only had one single fluorescence signal site, and the second pattern had two different intensities of signal sites, which all located in the near centromere region of chromosomes. Conclusion: 1) The oligonucleotide probes of sht-2/3/4/5, sht-5S and sht-45S can be used as physical markers for C. cathayensis, C. hunanensis, C. tonkinensis, C. dabieshanensis and C. kweichowensis. 2 ) The oligonucleotide probes of sht-5S and sht-45S can replace the plasmid probes of 45S rDNA and 5S rDNA to analyze the chromosomes of C. cathayensis, C. hunanensis, C. tonkinensis, C. dabieshanensis, C. illinoinensis and C. kweichowensis.

Key words: Carya, repetitive sequence, oligonucleotide probe, chromosome, Oligo-FISH.