Chromosome Painting Based on Bulked Oligonucleotides in Cotton.

Yuling Liu,Quanwei Lu,Fang Liu,Zhen Liu,Yangyang Wei,Xiuyuan Wang,Tao Zhang,Renhai Peng

doi:10.3389/fpls.2020.00802

Abstract

Chromosome painting is one of the key technologies in cytogenetic research, which can accurately identify chromosomes or chromosome regions. Oligonucleotide (oligo) probes designed based on genome sequences have both flexibility and specificity, which would be ideal probes for fluorescence in situ hybridization (FISH) analysis of genome structure. In this study, the bulked oligos of the two arms of chromosome seven of cotton were developed based on the genome sequence of Gossypium raimondii (DD, 2n = 2× = 26), and each arm contains 12,544 oligos. Chromosome seven was easily identified in both D genome and AD genome cotton species using the bulked chromosome-specific painting probes. Together with 45S ribosomal DNA (rDNA) probe, the chromosome-specific painting probe was also successfully used to correct the chromosomal localization of 45S rDNA in G. raimondii. The study reveals that bulked oligos specific to a chromosome is a useful tool for chromosome painting in cotton.

Highlights

Identification of an individual chromosome is the foundation of cytogenetic research
We developed oligo pools of G. raimondii chromosome seven on the basis of the released whole genomic sequence for chromosome identification as well as 45S ribosomal DNA (rDNA) location verification, which would provide evidence for the study of the evolutionary relationship between cotton species
Eleven cotton species were used in this study: four wild species of D genome cotton, Gossypium thurberi D1, Gossypium davidsonii D3−d, Gossypium klotzschianum D3−k, and G. raimondii D5 (DD, 2n = 2× = 26); two A genome cotton, Gossypium herbacium A1 and Gossypium arboretum A2 (AA, 2n = 2× = 26); and five allopolyploid lineage (AD) genome cotton, Gossypium hirsutum AD1, Gossypium barbadense AD2, Gossypium tomentosum AD3, Gossypium mustelinum AD4, Gossypium darwinii, and AD5 (AADD, 4n = 4× = 52)

Summary

Introduction

Identification of an individual chromosome is the foundation of cytogenetic research. Since 1980s, the development of fluorescence in situ hybridization (FISH) has led to the development of different chromosome identification techniques owing to its higher sensitivity and resolution. Chromosome painting is one of FISH techniques using chromosome-specific probes to detect specific chromosome regions or an entire chromosome (Pinkel et al, 1988). Chromosome painting using DNA probes prepared from flow-sorted or micro-dissected chromosomes was successfully used for chromosome identification, chromosome structure analysis, and comparative genomic analysis in human and other mammals with relatively small genomes (Breneman et al, 1993; Scherthan et al, 1994; Rens et al, 2006; Ferguson-Smith and Trifonov, 2007). The same type of probes could not work effectively in higher plants with large, complex genomes owing to the poor specificity of probes, which resulted from higher content of repetitive sequences (Fuchs et al, 1996; Lysak and Mandáková, 2013)

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Conclusion