Enhancing Hybridization Signal of Sugarcane Chromosome Oligonucleotide Probe via Multiple Fluorescence Labeled Primers

doi:10.13560/j.cnki.biotech.bull.1985.2022-1085

Abstract

Abstract:

Fluorescence in situ hybridization（FISH）is one of the most important methods in plant molecular cytogenetics. In recent years, low copy oligonucleotide probes based on reference genome design have been widely used in FISH. However, the resolution of oligo-FISH is limited due to the large number of repeats distributed in plant genomes. The sugarcane（Sacchanum spp.）chromosome specific oligo probes were amplified by using fluorescent PCR primers containing multiple fluorophore groups, and the sugarcane fluorescence in situ hybridization system was further optimized to improve the efficiency of sugarcane oligo probes in recognizing chromosomes of related species. By developing a new method of multi-fluorescent labeled oligo probes in sugarcane and optimizing the sugarcane fluorescence hybridization system, the minimum resolution of fluorescence signal was effectively broadened，the signal-to-noise ratio（SNR）increased, and sugarcane oligo probes were used to successfully identify sorghum chromosomes 1-10. The new method of multi-fluorescent labeled primers enhanced the signal of oligo probe and the optimization of FISH system provide a reference for improving oligo-FISH of identifying chromosomes and capturing weak fluorescence signals in other species in the future.

Key words: sugarcane, fluorescence in situ hybridization, oligonucleotide probes, chromosome identification, oligo-FISH

LI Xin-yi, JIANG Chun-xiu, XUE Li, JIANG Hong-tao, YAO Wei, DENG Zu-hu, ZHANG Mu-qing, YU Fan. Enhancing Hybridization Signal of Sugarcane Chromosome Oligonucleotide Probe via Multiple Fluorescence Labeled Primers[J]. Biotechnology Bulletin, 2023, 39(5): 103-111.

Figures/Tables 8

Table 1 Amplification sequences of oligo probe fluoresc-ence labeled primers

引物名称 Primer name	引物序列 Primer sequence
T7-Cy3	/iCy3dT/ACGACTCACTATAGGGAGA
D9-Cy3	/iCy3dA/GTGTGTACGGACTAATAAT
T7-FAM	/iFAMdT/ACGACTCACTATAGGGAGA
D9-FAM	/iFAMdA/GTGTGTACGGACTAATAAT
T7-Cy3-3	/iCy3dT/ACGAC/iCy3dT/CAC/iCy3dT/ATAGGGAGA
D9-Cy3-3	/iCy3dA/G/iCy3dT/G/iCy3dT/GTACGGACTAATAAT
T7-FAM-3	/iFAMdT/ACGAC/iFAMdT/CAC/iFAMdT/ATAGGGAGA
D9-FAM-3	/iFAMdA/G/iFAMdT/G/iFAMdT/GTACGGACTAATAAT

Fig. 1 Amplification electrophoretogram of multiple fluo-rescence labeled primers

Fig. 2 Comparison of flow before and after optimization of FISH Blue area: Pre-optimization hybridization step; red area: post-optimization hybridization step; yellow marker: improvement step; dashed part: pre-hybridization step area

Fig. 3 Signal capture of Cy3 and FAM with different exposure gradients based on hybridization system optimization a: Imaging of the FAM probe at different exposure duration in the unoptimized hybridization system; b: imaging of the FAM probe at different exposure duration in the optimized hybridization system; c: imaging of the Cy3 probe at different exposure duration in the unoptimized hybridization system; d: imaging of the Cy3 probe at different exposure duration in the optimized hybridization system; e: comparison of fluorescence density before and after optimization system, the red background is marked with Cy3, the green background is marked with FAM.IntDen: Intensity Image. bar=10 μm

Fig. 4 Signal capture of Cy3 and FAM with different exposure gradients based on different fluorophore group probes a: Imaging of FAM probe at different exposure duration under single fluorescence modification; b: imaging of FAM probe at different exposure duration under three fluorescence modifications; c: imaging of Cy3 probe at different exposure duration under unoptimized hybridization system; d: imaging of Cy3 probe at different exposure duration under three fluorescence modifications; e: comparison graph of fluorescence density under different number of fluorescence modifications, red background for Cy3 marker, green background is FAM marker; IntDen is gray scale value; scale bar is 10 μm

Table 2 Number statistics of sorghum genome aligned by S. officinarum oligos probes

oligo探针 oligo probes	高粱染色体 Sorghum chromosome
oligo探针 oligo probes	1	2	3	4	5	6	7	8	9	10
Chr1	18 400	63	120	79	51	84	68	46	55	66
Chr2	73	12 522	55	65	29	55	30	80	34	42
Chr3	45	46	15 771	61	54	47	41	30	125	41
Chr4	28	47	48	11 448	46	42	29	28	18	65
Chr5	27	34	26	107	3 680	29	16	53	25	44
Chr6	85	20	70	93	48	7 959	32	39	41	47
Chr7	75	81	54	33	40	36	7 760	30	39	95
Chr8	29	34	21	65	143	25	25	5 218	24	40
Chr9	28	39	43	15	25	33	15	26	8 592	57
Chr10	86	112	47	28	46	21	33	60	46	9 573

Fig. 5 S. officinarum Oligos sequences distribute on the chromosome of sorghum a: The hot map of Oligo probe distribution. b: The statistics of oligo probe density

Fig. 6 Oligo probe located on sorghum chromosome a: Probe Chr1 and Chr2 located on sorghum chromosome; b: probe Chr3 and Chr4 located on sorghum chromosome; c: probe Chr5 and Chr6 located on sorghum chromosome; d: probe Chr7 and Chr8 located on sorghum chromosome; e: probe Chr9 and Chr10 located on sorghum chromosome; Cy3 is red, FAM is green; bar=10 μm

References 30

[1]	Garsmeur O, Droc G, Antonise R, et al. A mosaic monoploid reference sequence for the highly complex genome of sugarcane[J]. Nat Commun, 2018, 9(1): 2638. doi: 10.1038/s41467-018-05051-5 pmid: 29980662
[2]	Speicher MR, Gwyn Ballard S, Ward DC. Karyotyping human chromosomes by combinatorial multi-fluor FISH[J]. Nat Genet, 1996, 12(4): 368-375. pmid: 8630489
[3]	Xin HY, Zhang T, Wu YF, et al. An extraordinarily stable karyotype of the woody Populus species revealed by chromosome painting[J]. Plant J, 2020, 101(2): 253-264. doi: 10.1111/tpj.v101.2 URL
[4]	Jiang JM. Fluorescence in situ hybridization in plants: recent developments and future applications[J]. Chromosome Res, 2019, 27(3): 153-165. doi: 10.1007/s10577-019-09607-z
[5]	Jiang JM, Gill BS. Current status and the future of fluorescence in situ hybridization(FISH)in plant genome research[J]. Genome, 2006, 49(9): 1057-1068. doi: 10.1139/g06-076 URL
[6]	Liu XY, Sun S, Wu Y, et al. Dual-color oligo-FISH can reveal chromosomal variations and evolution in Oryza species[J]. Plant J, 2020, 101(1): 112-121. doi: 10.1111/tpj.v101.1 URL
[7]	刘玉玲, 刘震, 李兆国, 等. 棉花oligo-FISH技术建立及其初步应用[J]. 棉花学报, 2017, 29(3): 213-221.
	Liu YL, Liu Z, Li ZG, et al. Construction and primary application of oligos fluorescence in situ hybridization technology in cotton[J]. Cotton Sci, 2017, 29(3): 213-221.
[8]	Song XY, Song RR, Zhou JW, et al. Development and application of oligonucleotide-based chromosome painting for chromosome 4D of Triticum aestivum L[J]. Chromosome Res, 2020, 28(2): 171-182. doi: 10.1007/s10577-020-09627-0
[9]	Li GR, Zhang T, Yu ZH, et al. An efficient oligo-FISH painting system for revealing chromosome rearrangements and polyploidization in Triticeae[J]. Plant J, 2021, 105(4): 978-993. doi: 10.1111/tpj.v105.4 URL
[10]	do Vale Martins L, Yu F, Zhao HN, et al. Meiotic crossovers characterized by haplotype-specific chromosome painting in maize[J]. Nat Commun, 2019, 10(1): 4604. doi: 10.1038/s41467-019-12646-z pmid: 31601818
[11]	Albert PS, Zhang T, Semrau K, et al. Whole-chromosome paints in maize reveal rearrangements, nuclear domains, and chromosomal relationships[J]. Proc Natl Acad Sci USA, 2019, 116(5): 1679-1685. doi: 10.1073/pnas.1813957116 pmid: 30655344
[12]	Braz GT, Yu F, Zhao HN, et al. Preferential meiotic chromosome pairing among homologous chromosomes with cryptic sequence variation in tetraploid maize[J]. New Phytol, 2021, 229(6): 3294-3302. doi: 10.1111/nph.17098 pmid: 33222183
[13]	Yu F, Zhao XW, Chai J, et al. Chromosome-specific painting unveils chromosomal fusions and distinct allopolyploid species in the Saccharum complex[J]. New Phytol, 2022, 233(4): 1953-1965. doi: 10.1111/nph.v233.4 URL
[14]	Meng Z, Zhang ZL, Yan TY, et al. Comprehensively characterizing the cytological features of Saccharum spontaneum by the development of a complete set of chromosome-specific oligo probes[J]. Front Plant Sci, 2018, 9: 1624. doi: 10.3389/fpls.2018.01624 URL
[15]	Han YH, Zhang T, Thammapichai P, et al. Chromosome-specific painting in Cucumis species using bulked oligonucleotides[J]. Genetics, 2015, 200(3): 771-779. doi: 10.1534/genetics.115.177642 URL
[16]	He L, Zhao HN, He J, et al. Extraordinarily conserved chromosomal synteny of Citrus species revealed by chromosome-specific painting[J]. Plant J, 2020, 103(6): 2225-2235. doi: 10.1111/tpj.v103.6 URL
[17]	Šimoníková D, Němečková A, Karafiátová M, et al. Chromosome painting facilitates anchoring reference genome sequence to chromosomes in situ and integrated karyotyping in banana(Musa spp.)[J]. Front Plant Sci, 2019, 10: 1503. doi: 10.3389/fpls.2019.01503 pmid: 31824534
[18]	Yu F, Chai J, Li XT, et al. Chromosomal characterization of Tripidium arundinaceum revealed by oligo-FISH[J]. Int J Mol Sci, 2021, 22(16): 8539. doi: 10.3390/ijms22168539 URL
[19]	Rauscher JT, Doyle JJ, Brown AHD. Multiple origins and nrDNA internal transcribed spacer homeologue evolution in the Glycine tomentella(Leguminosae)allopolyploid complex[J]. Genetics, 2004, 166(2): 987-998. pmid: 15020482
[20]	McCarthy EM, Liu JD, Gao LZ, et al. Long terminal repeat retrotransposons of Oryza sativa[J]. Genome Biol, 2002, 3(10): RESEARCH0053.
[21]	Schnable PS, Ware D, Fulton RS, et al. The B73 maize genome: complexity, diversity, and dynamics[J]. Science, 2009, 326(5956): 1112-1115. doi: 10.1126/science.1178534 pmid: 19965430
[22]	Zhang JS, Zhang XT, Tang HB, et al. Allele-defined genome of the autopolyploid sugarcane Saccharum spontaneum L[J]. Nat Genet, 2018, 50(11): 1565-1573. doi: 10.1038/s41588-018-0237-2
[23]	Braz GT, Yu F, do Vale Martins L, et al. Fluorescent in situ hybridization using oligonucleotide-based probes[J]. Methods Mol Biol, 2020, 2148: 71-83.
[24]	Bi YF, Zhao QZ, Yan WK, et al. Flexible chromosome painting based on multiplex PCR of oligonucleotides and its application for comparative chromosome analyses in Cucumis[J]. Plant J, 2020, 102(1): 178-186. doi: 10.1111/tpj.v102.1 URL
[25]	Zhang T, Liu GQ, Zhao HN, et al. Chorus2: design of genome-scale oligonucleotide-based probes for fluorescence in situ hybridization[J]. Plant Biotechnol J, 2021, 19(10): 1967-1978. doi: 10.1111/pbi.13610 pmid: 33960617
[26]	Braz GT, He L, Zhao HN, et al. Comparative oligo-FISH mapping: an efficient and powerful methodology to reveal karyotypic and chromosomal evolution[J]. Genetics, 2018, 208(2): 513-523. doi: 10.1534/genetics.117.300344 pmid: 29242292
[27]	Meng Z, Han JL, Lin YJ, et al. Characterization of a Saccharum spontaneum with a basic chromosome number of x=10 provides new insights on genome evolution in genus Saccharum[J]. Theor Appl Genet, 2020, 133(1): 187-199. doi: 10.1007/s00122-019-03450-w
[28]	Piperidis N, D'Hont A. Sugarcane genome architecture decrypted with chromosome-specific oligo probes[J]. Plant J, 2020, 103(6): 2039-2051. doi: 10.1111/tpj.v103.6 URL
[29]	Hou LL, Xu M, Zhang T, et al. Chromosome painting and its applications in cultivated and wild rice[J]. BMC Plant Biol, 2018, 18(1): 110. doi: 10.1186/s12870-018-1325-2 pmid: 29879904
[30]	Lloyd Evans D, Joshi SV, Wang JP. Whole chloroplast genome and gene locus phylogenies reveal the taxonomic placement and relationship of Tripidium(Panicoideae: Andropogoneae)to sugarcane[J]. BMC Evol Biol, 2019, 19(1): 33. doi: 10.1186/s12862-019-1356-9