High-resolution chromosome painting with repetitive and single-copy oligonucleotides in Arachis species identifies structural rearrangements and genome differentiation

Abstract

Background: Arachis contains 80 species that carry many beneficial genes that can be utilized in the genetic improvement of peanut (Arachis hypogaea L. 2n = 4x = 40, genome AABB). Chromosome engineering is a powerful technique by which these genes can be transferred and utilized in cultivated peanut. However, their small chromosomes and insufficient cytological markers have made chromosome identification and studies relating to genome evolution quite difficult. The development of efficient cytological markers or probes is very necessary for both chromosome engineering and genome discrimination in cultivated peanut.

Results: A simple and efficient oligonucleotide multiplex probe to distinguish genomes, chromosomes, and chromosomal aberrations of peanut was developed based on eight single-stranded oligonucleotides (SSONs) derived from repetitive sequences. High-resolution karyotypes of 16 Arachis species, two interspecific F₁ hybrids, and one radiation-induced M₁ plant were then developed by fluorescence in situ hybridization (FISH) using oligonucleotide multiplex, 45S and 5S rDNAs, and genomic in situ hybridization (GISH) using total genomic DNA of A. duranensis (2n = 2x = 20, AA) and A. ipaënsis (2n = 2x = 20, BB) as probes. Genomes, chromosomes, and aberrations were clearly identifiable in the established karyotypes. All eight cultivars had similar karyotypes, whereas the eight wild species exhibited various chromosomal variations. In addition, a chromosome-specific SSON library was developed based on the single-copy sequence of chromosome 6A of A. duranensis. In combination with repetitive SSONs and rDNA FISH, the single-copy SSON library was applied to identify the corresponding A3 chromosome in the A. duranensis karyotype.

Conclusions: The development of repetitive and single-copy SSON probes for FISH and GISH provides useful tools for the differentiation of chromosomes and identification of structural chromosomal rearrangement. It facilitates the development of high-resolution karyotypes and detection of chromosomal variations in Arachis species. To our knowledge, the methodology presented in this study demonstrates for the first time the correlation between a sequenced chromosome region and a cytologically identified chromosome in peanut.

Keywords: Arachis species; Chromosome painting; Genomic evolution; High-resolution karyotype; Oligonucleotide multiplex.

Fig. 1

FISH mapping of DP-1 (red), DP-2 (green), DP-3 (green), DP-4 (red), DP-5 (green), DP-6 (red), DP-7 (red), and DP-8 (green) by sequential FISH using 45S rDNA (red), 5S rDNA (green), and A. duranensis (green) and A. ipaënsis (red) total genomic DNA as probes in Arachis hypogea cv. SLH, A. duranensis, and A. ipaënsis. Blue color represents chromosomes counterstained with DAPI

Fig. 2

Karyotypes of eight peanut varieties. Lines labeled as SSON and 45S/5S refer to signals from Multiplex #1 and rDNAs, respectively. Blue color represents chromosomes counterstained with DAPI. In SSON, red represents signals of DP-1, DP-3, DP-4, and DP-6; and green represents signals of DP-2, DP-5, and DP-7. In 45S/5S, red represents signals of 45S rDNA and green represents signals of 5S rDNA

Fig. 3

Karyotypes of nine Arachis species. Columns labeled as SSON and 45S/5S refer to signals from Multiplex #1 and rDNAs, respectively. Probe color same as Fig. 2

Fig. 4

Sequential FISH of two hybrids F₁ w1510 and w1612 using Multiplex #1 (a and c), and 5S and 45S rDNA (b and d) plasmid clones as probes. a–b: w1510 derived from SLH (A. hypogaea) and A. batizocoi; c–d: w1612 derived from N734 (A. hypogaea) and A. stenophylla. Probe color as Fig. 2

Fig. 5

Chromosome aberrations detected in radiation-induced M₁ plant 162–30 of peanut cultivar SLH after sequential FISH/GISH. a–f Results of sequential FISH/GISH in SLH (a–c) and M1 plant 162–30 (d–f) using Multiplex #2 (a and d), A. duranensis genomic DNA (b and e; green), A. ipaënsis genomic DNA (b and e; red), 5S rDNA (c and f; green), and 45S rDNA (c and f; red). g Karyotypes of SLH and 162–30. h Translocated chromosomes in 162–30. Multiplex #2 contains four SSONs, including TAMRA-DP-8, FAM-DP-2, FAM-DP-5, and FAM-DP-7

Fig. 6

Oligopainting using single-copy oligonucleotide library 6A-1 combined with sequential FISH using DP-5, and plasmid clones 45S rDNA and 5S rDNA as probes in A. duranensis. Blue color represents chromosomes counterstained with DAPI; a and c shows oligopainting using single-copy SSON library 6A-1 (green); b and d, sequential FISH using repetitive SSON DP-5 (green), 45S rDNA (red), and 5S rDNA (white inverted from the original green); c From left to right: chromosome with signals of library 6A-1 (green); karyotype of A3, probe signals are same as (b) and (d); idiograms of the chromosome displaying signals from library 6A-1 and the karyotype of A3; and sequence map of A6 showing 0–8.5 Mb regions corresponding to library 6A-1. Yellow arrows in panels (a–d) indicate chromosomes with signals from library 6A-1