Developing repetitive oligo probes from the whole genome sequencing data of peanut
Genome sequences of Tifrunner and A. ipaensis were analyzed using the Tandem Repeats Finder (TRF) [40], resulting in 4,595 and 894 repetitive sequences, respectively. The lengths of these sequences varied between 4 bp and 723 bp while copy numbers varied between 50 and 29,162. After CD-HIT [41] elimination, a total of 80 and 35 TRs belonging to Tifrunner and A. ipaensis, respectively, were selected for further development of the oligos.
A total of 249 oligos were designed using Oligo 7 [42] and mapped in silico based upon the reference sequences of Tifrunner using B2DSC [32]. The designed oligos were first labeled via a random primer labeling method [28] and hybridized with the chromosomes of peanut. We observed by microscopy that 114 of the designed oligos produced clear signals in different positions of the chromosomes of Tifrunner (Fig. 1, Fig. 2, Table S1). The 114 oligo probes were categorized into 28 types based upon the pattern and position distribution observed by FISH(Table S2); oligos with the same position and overlapping signals in the karyotype were classified as the same type. For each of the 28 types, a single oligo was selected and further modified with 6-carboxyfluorescein (FAM) or 6-carboxytetramethylrhodamine (TAMRA). Figure 1 shows the results of oligo Ipa-1463 after in silico mapping and FISH. 1,260 copies were observed in chromosome plots mapped in the region 63–77 Mbp region of chromosome B9 (Fig. 1a). The FISH analysis confirmed that the signals of oligo Ipa-1463 were only present in one pair of chromosomes at a similar region. Thus, we conclude that this chromosome corresponded with chromosome B9 in the reference sequence (Fig. 1b).
Development of a genome map-based karyotype of Tifrunner
Based on the unique patterns and sequence composition of the 28 oligo probes, two new oligo probe cocktails, Multiplex #3 and Multiplex #4, were developed using eight synthesized oligos. Multiplex #3 included FAM-modified TIF-439, TIF-185-1, TIF-134-3, and TIF-165-3. Multiplex #4 included TAMRA-modified Ipa-1162, Ipa-1137, DP-1, and DP-5. Both DP-1 and DP-5 were derived from a previous study by Du et al. [17]. Following the sequential FISH/GISH with Multiplex #3 and Multiplex #4, total genomic DNAs of A. duranensis and A. ipaensis, and 45S and 5S rDNA assays, a robust karyotype of Tifrunner was established (Fig. 3a–e).
Comparing the distributions of the eight oligos in the karyotype and chromosome plots, we found that most signal sites and intensities in the actual chromosomes corresponded well with their respective positions and copy numbers in the reference sequence (Fig. S1). Additionally, a genome map-based karyotype of Tifrunner was established. Each of the actual chromosomes in the karyotype were renumbered as A1~A10 and B1~B10, according to their pseudo-molecule number in the genome map of Tifrunner [36]. We found short arms of some chromosomes in the karyotype, such as A1 and B7, were not positioned on the short arm (upper arm) in the genome map. In order to facilitate understanding, the long arm (L) and short arm (S) in this study were assigned according to the upper arm and lower arm of the genome map. Based upon this karyotype, nine significant non-correspondent signals were observed across seven chromosomes (Fig. 3e–f). For example, the oligos Ipa-1162 and Ipa-1137 evidently had distribution sites on chromosomes A1 and B1 in the chromosome plots, but no signal was observed upon probe hybridization to the actual chromosomes. In contrast, oligos TIF-439, TIF-185-1, TIF-134-3, and TIF-165-3 produced strong signals in the centromeric regions of chromosomes B2 and B10, but were not in silico mapped in the chromosome plots (Fig. 3d–f, Fig. S1).
To validate the genome map-based karyotype, two chromosome-specific single-copy sequence oligo libraries, L1A-1 and L3A-1, from the short arm of chromosomes A1 and A3 in the genome map, were used for sequential FISH/GISH analysis. The libraries were combined with the Multiplex #3, Multiplex #4, and total genomic DNAs of A. duranensis and A. ipaensis as the probes used for the subsequent analysis (Fig. S2). The specific signals of the two chromosome-specific oligo libraries were clearly observed on the two expected chromosomes, indicating considerable correspondence between the actual chromosomes of the karyotype and the genome maps.
Among the A subgenome chromosomes of this karyotype, chromosomes A1 and A8 both contained intense green signals in the centromeric regions, while A1 also had strong red signals in the terminal region of its long arm. Chromosomes A6, A7, and A10 had 45S or 5S rDNA sites. Chromosomes A2, A3, and A4 had green signals in the terminal regions of the short arms, while A3 and A4 had green signals in the centromeric regions. Chromosome A5 had red signals within the short arm, while A9 had red signals at the subtelomeric region of the long arm. Among the B subgenome, chromosomes B6, B7, and B8 showed signals of the probes for 45S or 5S rDNA. Chromosome B9 and B3 had red and green signals in the centromeric regions respectively. The other chromosomes had green signals either in the centromeric or telomeric regions with varying intensities (Fig. 3e). Based on the unique patterns observed, all chromosomes could be clearly differentiated in the karyotype.
Chromosome allocation of the oligo probe
To map TRs in the genome map-based karyotype of Tifrunner, the 28 oligos representative of each type were analyzed by both FISH and in silico mapping (Fig. 4). The 28 oligos produced more signals on the B subgenome chromosomes than in those of the A subgenome (Fig. 4). Among the 28 oligos, six (TIF-165-3, TIF-439, TIF-556, TIF-198-1, TIF-384-3, and TIF-185-1) produced signals in the interstitial or terminal regions of the chromosomes. Four oligos (TIF-198-2, TIF-416-3, TIF-497, and TIF-342-2) had signals exclusively on the B subgenome, indicating that these oligos are specific for the B subgenome. Two oligos (Ipa-1137 and Ipa-1162) had signals only at the secondary constrictions, which fully overlapped with the signals of 45S rDNA, following sequential FISH. Oligo Ipa-1463 exclusively showed signals on one pair of chromosomes, which indicated that it is chromosome-specific. The other 15 oligos had signals located within the pericentric regions (Fig. S3).
Among the 28 oligos physically mapped via FISH, the distributions of 22 oligos were the same or similar to those in the in silico mapping results(Fig. 4, Fig. S4). However, six oligos (TIF-89-3, TIF-155-5, TIF-198-1, TIF-359-3, TIF-76-1, and Ipa-1757) showed significant differences between the two maps. For example, TIF-89-3 and TIF-155-5 were in silico mapped to two pairs of chromosomes with a high number of copies. However, eight pairs of chromosomes evidently showed FISH signals. Similarly, TIF-198-1 was in silico mapped to only one pair of chromosomes, but produced signals on 16 pairs of chromosomes. In contrast, obvious sites of TIF-76-1 were in silico mapped to 13 pairs of chromosomes but produced FISH signals on five pairs of chromosomes. This may indicate that TRs were not unambiguously assembled within the genome map of Tifrunner.
Identification of chromosomal variations of Silihong (SLH) induced by radiation exposure
To check chromosomal variations in peanut, sequential FISH and GISH were conducted on Chinese variety SLH and 70 radiation-induced M1 SLH plants with Multiplex #3 and Multiplex #4, total genomic DNAs of A. duranensis and A. ipaensis, and 45S and 5S rDNA assays (Fig. 5). The fourteen M1 plants used within this study showed chromosomal variations. For example, in plant 161-1a one reciprocal translocation was evident, which likely occurred between chromosomes 1A and 3B. The segment of one chromosome translocation from the A subgenome had extensive green signals spanning the entire arm and red signals at the terminal end of the chromosome. This pattern was similar to that of the signals on the centromere and the long arm (L) of 1A. The signal distribution pattern of another segment was like the pattern of 3B from the end of short arm (S) to centromere, which indicates that they were T 3BL-1AS·1AL and T 1AS·3BL-3BS. Additional FISH was performed using the single-copy oligo library probes L1A-1 and L3A-1 which exclusively hybridize to the upper arms of 1A and 3A. The results of this experiment confirmed the identity of the two translocated chromosomes (Fig. 5f–h).
A total of 13 other plants were identified to have 17 translocations, one deletion, and eight monosomic chromosomes (Fig. S5). Among the identified chromosomal variations, eight translocations were observed between homoeologous chromosomes, and nine translocations were observed between non-homologous chromosomes. Chromosomes 1, 3, and 5 showed a greater number of translocations (Fig. 6).