Industrial Crops Research Institute, Henan Academy of Agricultural Sciences/Key Laboratory of Oil Crops in Huang-Huai-Hai Plains, Ministry of Agriculture/Henan Provincial Key Laboratory for Oil Crops Improvement, Zhengzhou, 450002, Henan, China.
State Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing, 210095, China.
BMC Plant Biol. 2018 Oct 17;18(1):240. doi: 10.1186/s12870-018-1468-1.
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.
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 F hybrids, and one radiation-induced M 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.
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.
落花生属包含 80 个种，这些种携带许多有益基因，可用于落花生（Arachis hypogaea L. 2n = 4x = 40，基因组 AABB）的遗传改良。染色体工程是一种强大的技术，可以通过该技术将这些基因转移并应用于栽培落花生中。然而，其小染色体和不足的细胞遗传学标记使得染色体的鉴定以及与基因组进化相关的研究变得非常困难。开发有效的细胞遗传学标记或探针对于栽培落花生的染色体工程和基因组鉴别都是非常必要的。
基于从重复序列中衍生的 8 个单链寡核苷酸（SSON），开发了一种用于区分花生基因组、染色体和染色体畸变的简单高效的寡核苷酸多重探针。然后通过荧光原位杂交（FISH）使用寡核苷酸多重、45S 和 5S rDNA 以及以 A. duranensis（2n = 2x = 20，AA）和 A. ipaënsis（2n = 2x = 20，BB）的总基因组 DNA 作为探针的基因组原位杂交（GISH），开发了 16 种 Arachis 种、两个种间 F1 杂种和一个辐射诱导的 M 植物的高分辨率核型。在建立的核型中，可以清楚地识别基因组、染色体和畸变。所有 8 个栽培品种的核型相似，而 8 个野生种则表现出各种染色体变异。此外，基于 A. duranensis 染色体 6A 的单拷贝序列开发了染色体特异性 SSON 文库。与重复 SSON 和 rDNA FISH 结合使用，单拷贝 SSON 文库用于鉴定 A. duranensis 核型中相应的 A3 染色体。
用于 FISH 和 GISH 的重复和单拷贝 SSON 探针的开发为染色体的分化和结构染色体重排的鉴定提供了有用的工具。它促进了 Arachis 种高分辨率核型的发展和染色体变异的检测。据我们所知，本研究中提出的方法首次证明了在花生中，测序染色体区域与细胞遗传学鉴定的染色体之间存在相关性。
