Oligo-FISH painting reveals the structures and pairings of the pachytene chromosome 1 in Citrus maxima

Abstract:【Objective】The cytogenetic study is not only an important tool for the structural analysis of the chromosomes in Citrus species, but also a direct tool for the pairing investigation of homologous or homeologous chromosomes during meiosis. Then, the precise identification of a specific chromosome, especially a single full-length extended chromosome in meiotic prophase Ⅰ, plays a crucial role in realizing the above studies. However, it has been a tough challenge for plant species, let alone most of the non- model plants. Recently, bioinformatically chosen oligonucleotides have been synthesized and labeled as chromosome painting probe libraries in identifying specific portions or entire chromosomes through fluorescence in situ hybridization (FISH). This study aimed to explore and compare the cytogenetic characteristics of a specific citrus chromosome at meiotic pachytene and mitotic stages by chromosome painting with synthesized oligonucleotide probes.【Methods】The representative Citrus species C. maxima (Burm.) Merr.‘Shatian pummelo’(2n = 2x = 18), cultivated in the experimental base of Horticulture Institute, Sichuan Academy of Agricultural Sciences, Chengdu, China, was used for chromosome painting studies. Firstly, the former bioinformatically chosen and synthesized oligonucleotide library for Citrus chromosome 1-specific FISH painting was labeled as a probe with digoxin. Secondly, the vigorous root tips containing interphase nuclei, prometaphase, and metaphase chromosomes were used for the preparation of mitotic slides. The immature inflorescence was adopted to harvest meiotic slides with pachytene chromosomes. Thirdly, both mitotic and meiotic slides were applied for chromosome painting by oligo-FISH with Citrus chromosome 1-specific oligonucleotide probe. The digoxin-labeled probe was detected by anti- digoxigenin FITC conjugated antibody (Roche Diagnostics, USA). The chromosomes were counterstained with an antifade solution containing 4’, 6-diamidino-2-phenylindole (DAPI, Vector Laboratories, USA). Finally, the FISH images were captured with a CCD camera (ORCA Flash 4.0, Hamamatsu, Japan) attached to a Zeiss Axio Scope A1 fluorescence microscope. The brightness and contrast of images were adjusted with Adobe Photoshop 5.0. The lengths of the metaphase and pachytene chromosomes were determined with the Image J software, through which the bent pachytene chromosomes were straightened using the“Straighten”plugin.【Results】To enable uniform coverage of oligonucleotides on the chromosome 1, a total of 27 392 single-copy oligos of 45 bases had been screened from the assembled genome sequence, with a distribution density of 0.85 oligo · kb^-1 . After oligo- FISH, a pair of homologous chromosome 1 were clearly identified at mitotic metaphase through green signals derived from the probe. However, the distribution of the FISH signals on the chromosome 1 was not uniform, showing very weak signals in some parts of the chromosome, indicating a much lower density of single-copy oligonucleotides in the corresponding regions. In the mitotic interphase nuclei, chromosome painting signals showed that the two homologous chromosomes located in separated regions, were still being discrete chromatin with extremely low condensation. Both homologous chromosomes at prometaphase were observed much more condensed than at interphase, but they were less condensed compared with those at metaphase according to the FISH signals. The chromosomal segments with no or weak signals were more clearly visible than their counterparts in the metaphase chromosomes. As for pachytene chromosome 1, on which a small segment in the centromeric and adjacent pericentromeric region was observed absent of signal, dense FISH signals were covered from its end to end, exhibiting clearly full pairing of two homologous chromosomes 1 from end to end in all 15 examined cells. Thus, the chromosome painting oligo probe exhibited robust capacity in accurately and effectively tracing specific pachytene chromosome and its pairing of homologous chromosomes. Enormous differences in the chromatin structures between the pachytene and metaphase stage of chromosome 1 were observed. With the help of oligo-FISH signals and centromere positions that were lightly stained by DAPI, the means of the full length, long arm length, and short arm length of pachytene chromosome 1 were measured, which were (27.48 ± 1.89) µm, (18.67 ± 1.29) µm, and (8.82 ± 0.80) µm. The mean full length, long arm length, short arm length, and arm ratio of pachytene chromosome 1 were 13.93 times, 16.54 times, 10.44 times, and 1.54 times that of metaphase, respectively, indicating that the long arms at pachytene hold higher extension rate than the full-length chromosome, let alone the short arms. As a result, a much higher resolution of the FISH signals was obtained on pachytene chromosome 1. The oligo probe was screened from the assembled genomic sequence of 32.08 Mb in the pummelo chromosome 1, on which covered by the FISH signals, accounting for 91.42% of the full length of pachytene chromosome 1. According to the hypothesis that the DNA content was evenly distributed on the full-length pachytene chromosome, the signal absent centromeric and adjacent pericentromeric region, which accounted for 8.58% of the full length of pachytene chromosome 1, was estimated to be about 3.01 Mb after calculation. Accordingly, the total length of chromosome 1 might be calculated as 32.08 Mb plus 3.01 Mb equals 35.09 Mb, suggesting that the genomic sequence assembly in the signal absent centromeric and adjacent pericentromeric heterochromatin regions might not have been achieved completely.【Conclusion】These results demonstrated the evident differences in chromo-somal structures and their corresponding resolutions of oligo-FISH signals between meiotic pachytene and mitotic chromosomes through Citrus chromosome- specific painting probes. This pioneering work on detailed tracing and pairing examinations of Citrus specific full-length homologous chromosomes at meiotic pachytene phase would provide a powerful tool kit for studies on Citrus pachytene chromosomes, especially the pairing and inheritance of homologous chromosomes in different polyploid species.【Objective】The cytogenetic study is not only an important tool for the structural analysis of the chromosomes in Citrus species, but also a direct tool for the pairing investigation of homologous or homeologous chromosomes during meiosis. Then, the precise identification of a specific chromosome, especially a single full-length extended chromosome in meiotic prophase Ⅰ, plays a crucial role in realizing the above studies. However, it has been a tough challenge for plant species, let alone most of the non- model plants. Recently, bioinformatically chosen oligonucleotides have been synthesized and labeled as chromosome painting probe libraries in identifying specific portions or entire chromosomes through fluorescence in situ hybridization (FISH). This study aimed to explore and compare the cytogenetic characteristics of a specific citrus chromosome at meiotic pachytene and mitotic stages by chromosome painting with synthesized oligonucleotide probes.【Methods】The representative Citrus species C. maxima (Burm.) Merr.‘Shatian pummelo’(2n = 2x = 18), cultivated in the experimental base of Horticulture Institute, Sichuan Academy of Agricultural Sciences, Chengdu, China, was used for chromosome painting studies. Firstly, the former bioinformatically chosen and synthesized oligonucleotide library for Citrus chromosome 1-specific FISH painting was labeled as a probe with digoxin. Secondly, the vigorous root tips containing interphase nuclei, prometaphase, and metaphase chromosomes were