Physical mapping of repetitive oligonucleotides facilitates the establishment of a genome map-based karyotype to identify chromosomal variations in peanut

Abstract

Background Chromosomal variants play important roles in crop breeding and genetic research. The development of single-stranded oligonucleotide (oligo) probes simplifies the process of fluorescence in situ hybridization (FISH) and facilitates chromosomal identification in many species. Genome sequencing provides rich resources for the development of oligo probes. However, little progress has been made in peanut due to the lack of efficient chromosomal markers. Until now, the identification of chromosomal variants in peanut has remained a challenge. Results A total of 114 new oligo probes were developed based on the genome-wide tandem repeats (TRs) identified from the reference sequences of the peanut variety Tifrunner (AABB, 2n = 4x = 40) and the diploid species Arachis ipaensis (BB, 2n = 2x = 20). These oligo probes were classified into 28 types based on their positions and overlapping signals in chromosomes. For each type, a representative oligo was selected and modified with green fluorescein 6-carboxyfluorescein (FAM) or red fluorescein 6-carboxytetramethylrhodamine (TAMRA). Two cocktails, Multiplex #3 and Multiplex #4, were developed by pooling the fluorophore conjugated probes. Multiplex #3 included FAM-modified oligo TIF-439, oligo TIF-185-1, oligo TIF-134-3 and oligo TIF-165. Multiplex #4 included TAMRA-modified oligo Ipa-1162, oligo Ipa-1137, oligo DP-1 and oligo DP-5. Each cocktail enabled the establishment of a genome map-based karyotype after sequential FISH/genomic in situ hybridization (GISH) and in silico mapping. Furthermore, we identified 14 chromosomal variants of the peanut induced by radiation exposure. A total of 28 representative probes were further chromosomally mapped onto the new karyotype. Among the probes, eight were mapped in the secondary constrictions, intercalary and terminal regions; four were B genome-specific; one was chromosome-specific; and the remaining 15 were extensively mapped in the pericentric regions of the chromosomes. Conclusions The development of new oligo probes provides an effective set of tools which can be used to distinguish the various chromosomes of the peanut. Physical mapping by FISH reveals the genomic organization of repetitive oligos in peanut chromosomes. A genome map-based karyotype was established and used for the identification of chromosome variations in peanut following comparisons with their reference sequence positions.

Oligo-FISH Can Identify Chromosomes and Distinguish Hippophaë rhamnoides L. Taxa

“…campanulatus [ 30 ], G. biloba and P. densiflora [ 52 ], H. rhamnoides [ 17 ], R. wichurana [ 53 ], Passiflora species [ 62 ], Cestrum species [ 56 ], and V. foetida [ 57 ]. However, 5S rDNA has also showed high diversity in other plants, including A. hypogaea [ 20 ], Fragaria L. species [ 63 ], Crocus sativus L., Crocus vernus (L.) Hill [ 64 , 65 ], and P. concolor [ 33 ].…”

“…These oligonucleotide sequences can then be readily produced and tagged with fluorescent markers for use as oligo-probes in FISH [ 18 ]. Species identification based on such oligo-probes has been reported in an increasing number of plant species, such as Avena L. species [ 19 ], Arachis hypogaea L. [ 20 ], Saccharum spontaneum L. [ 21 ], Citrus L. species [ 22 ], Citrus sinensis (L.) Osbeck × Poncirus trifoliata (L.) Raf., CC [ 23 ], Populus L. species [ 24 ], Strobus Opiz species [ 25 ], and Pinus L. species [ 26 ]. However, information regarding H. rhamnoides is limited.…”

Multi-Omics Profiling Identifies Candidate Genes Controlling Seed Size in Peanut

“…We found that the ratio was relatively stable at 0 along the whole chromosome ( Figure 2 A,B), indicating no large segment deletions (more than 1 Mb), duplications, or translocations in the mutants. In order to validate the WGS observations, we designed two cocktails, Multiplex #3 and Multiplex #4 [ 21 ]. Multiplex #3 included FAM-modified oligo TIF-439, oligo TIF-185-1, oligo TIF-134-3, and oligo TIF-165.…”

Developing Oligo Probes for Chromosomes Identification in Hemp (Cannabis sativa L.)

“…Oligos are being developed for easy application, which allows researchers to obtain results by fast oligo-FISH or ND-FISH in a short time. Oligos have been developed and successfully used for cereals [ 40 , 41 , 42 , 43 , 51 , 52 ] and other crops [ 53 , 54 , 55 ]. In this article, we report the development of oligo-labeled probes, based on CS-1 [ 56 ] and CS-237 [ 31 ] repeats for rapid oligo-FISH.…”