Molecular cytogenetic map visualizes the heterozygotic genome and identifies translocation chromosomes in <i>Citrus sinensis</i>

Shipeng Song; Hui Liu; Luke Miao; Li He; Wenzhao Xie; Hong Lan; Changxiu Yu; Wenkai Yan; Yufeng Wu; Xiao-peng Wen; Qiang Xu; Xiuxin Deng; Chunli Chen

doi:10.1016/j.jgg.2022.12.003

Volume 50 Issue 6

Jun. 2023

Turn off MathJax

Article Contents

Article Navigation > Journal of Genetics and Genomics > 2023 > 50(6): 410-421

PDF( 2650 KB)

Molecular cytogenetic map visualizes the heterozygotic genome and identifies translocation chromosomes in Citrus sinensis

doi: 10.1016/j.jgg.2022.12.003

a. National Key Laboratory for Germplasm Innovation and Utilization for Fruit and Vegetable Horticultural Crops, Hubei Hongshan Laboratory, Wuhan, Hubei 430070, China;
b. College of Life Science and Technology, Huazhong Agricultural University, Wuhan, Hubei 430070, China;
c. National-local Joint Engineering Laboratory of Citrus Breeding and Cultivation/Horticulture Institute, Sichuan Academy of Agricultural Sciences, Chengdu, Sichuan 610066, China;
d. National Key Laboratory of Crop Genetics and Improvement, Huazhong Agricultural University, Wuhan, Hubei 430070, China;
e. Hubei Province Engineering Research Center of Legume Plants, College of Life Science, Jianghan University, Wuhan, Hubei 430056, China;
f. State Key Laboratory for Crop Genetics and Germplasm Enhancement, Bioinformatics Center, Academy for Advanced Interdisciplinary Studies, Nanjing Agricultural University, Nanjing, Jiangsu 210095, China;
g. Key Laboratory of Plant Resource Conservation and Germplasm Innovation in Mountainous Region (Ministry of Education), Institute of Agro-bioengineering, College of Life Science, Guizhou University, Guiyang, Guizhou 550025, China

Funds:

We thank Prof. Jianwei Zhang for helping DNA sequences editing and Dr. Simon moore, Dr. Sumeera Asghar, and Mr. Dengyue Zheng for language editing.

Received Date: 2022-10-24
Accepted Date: 2022-12-22
Rev Recd Date: 2022-12-19
Publish Date: 2023-01-04

Abstract

Abstract

Citrus sinensis is the most cultivated and economically valuable Citrus species in the world, whose genome has been assembled by three generation sequencings. However, chromosome recognition remains a problem due to the small size of chromosomes, and difficulty in differentiating between pseudo and real chromosomes because of a highly heterozygous genome. Here, we employ fluorescence in situ hybridization (FISH) with 9 chromosome painting probes, 30 oligo pools, and 8 repetitive sequences to visualize 18 chromosomes. Then, we develop an approach to identify each chromosome in one cell through single experiment of oligo-FISH and Chromoycin A3 (CMA) staining. By this approach, we construct a high-resolution molecular cytogenetic map containing the physical positions of CMA banding and 38 sequences of FISH including centromere regions, which enables us to visualize significant differences between homologous chromosomes. Based on the map, we locate several highly repetitive sequences on chromosomes and estimate sizes and copy numbers of each site. In particular, we discover the translocation regions of chromosomes 4 and 9 in C. sinensis “Valencia.” The high-resolution molecular cytogenetic map will help improve understanding of sweet orange genome assembly and also provide a fundamental reference for investigating chromosome evolution and chromosome engineering for genetic improvement in Citrus.
- Chromosomal marker,
- FISH,
- CMA banding,
- Genomic heterozygosity,
- Chromosomal translocation,
- Sweet orange

FullText(HTML)

References(62)

References

[1]	Albert, P.S., Zhang, T., Semrau, K., Rouillard, J.M., Kao, Y.H., Wang, C.J.R., Danilova, T.V., Jiang, J., Birchler, J.A., 2019. Whole-chromosome paints in maize reveal rearrangements, nuclear domains, and chromosomal relationships. Proc. Natl. Acad. Sci. U. S. A. 116, 1679-1685.
[2]	Belser, C., Baurens, F.C., Noel, B., Martin, G., Aury, J.M., 2021. Telomere-to-telomere gapless chromosomes of banana using nanopore sequencing. Commun. Biol. 4, 1047.
[3]	Beying, N., Schmidt, C., Pacher, M., Houben, A., Puchta, H., 2020. CRISPR-Cas9-mediated induction of heritable chromosomal translocations in arabidopsis. Nat. Plants 6, 638-645.
[4]	Bi, Y., Zhao, Q., Yan, W., Li, M., Liu, Y., Cheng, C., Zhang, L., Yu, X., Li, J., Qian, C., et al., 2020. Flexible chromosome painting based on multiplex PCR of oligonucleotides and its application for comparative chromosome analyses in cucumis. Plant J. 102, 178-186.
[5]	Braz, G.T., He, L., Zhao, H., Zhang, T., Semrau, K., Rouillard, J.M., Torres, G.A., Jiang, J., 2018. Comparative oligo-fish mapping: an efficient and powerful methodology to reveal karyotypic and chromosomal evolution. Genetics 208, 513-523.
[6]	Cao, H., Biswas, M.K., Yan, L., Amar, M.H., Tong, Z., Qiang, X., Xu, J., Guo, W., Deng, X., 2011. Doubled haploid callus lines of valencia sweet orange recovered from anther culture. Plant Cell Tissue Organ 104, 415-423.
[7]	Carvalho, R., Filho, W.S., Brasileiro-Vidal, A.C., Guerra, M., 2005. The relationships among lemons, limes and citron: a chromosomal comparison. Cytogenet. Genome Res. 109, 276-282.
[8]	Cheng, M., Li, X., Cui, H., Sun, H., Deng, T., Song, X., Song, R., Wang, T., Wang, Z., Wang, H., et al., 2022. FISH-based “pan” and “core” karyotypes reveal genetic diversification of roegneria ciliaris. J. Genet. Genom.
[9]	Chenqiao, Zhu, Xiongjie, Zheng, Yue, Huang, Junli, Peng, Chen, Chenglei, 2019. Genome sequencing and CRISPR/Cas9 gene editing of an early flowering mini citrus (Fortunella hindsii). Plant Biotechnol. J. 17, 2199-2210.
[10]	Deng, H., Cai, Z., Xiang, S., Guo, Q., Huang, W., Liang, G., 2019. Karyotype analysis of diploid and spontaneously occurring tetraploid blood orange [Citrus sinensis (L.) Osbeck] using multicolor fish with repetitive DNA sequences as probes. Front. Plant Sci. 10, 311.
[11]	Deng, H., Tang, G., Xu, N., Gao, Z., Lin, L., Liang, D., Xia, H., Deng, Q., Wang, J., Cai, Z., et al., 2020. Integrated karyotypes of diploid and tetraploid carrizo citrange (Citrus sinensis L. Osbeck x Poncirus trifoliata L. Raf.) as determined by sequential multicolor fluorescence in situ hybridization with tandemly repeated DNA sequences. Front. Plant Sci. 11, 569.
[12]	Dong, G., Shen, J., Zhang, Q., Wang, J., Yu, Q., Ming, R., Wang, K., Zhang, J., 2018. Development and applications of chromosome-specific cytogenetic bac-fish probes in S. Spontaneum. Front. Plant Sci. 9, 218.
[13]	Guerra, 1993. Cytogenetics of rutaceae. V. High chromosomal variability in Citrus species revealed by CMA/DAPI staining. Heredity 71, 234-241.
[14]	Guerra, M., Santos, K., Silva, A., Ehrendorfer, F., 2000. Heterochromatin banding patterns in rutaceae-aurantioideae - a case of parallel chromosomal evolution. Am. J. Bot. 87.
[15]	Han, Y., Zhang, T., Thammapichai, P., Weng, Y., Jiang, J., 2015. Chromosome-specific painting in cucumis species using bulked oligonucleotides. Genetics 200, 771.
[16]	Hao, Z., Lv, D., Ge, Y., Shi, J., Weijers, D., Yu, G., Chen, J., 2020. Rideogram: drawing SVG graphics to visualize and map genome-wide data on the idiograms. PeerJ Comput. Sci. 6, e251.
[17]	He, L., Zhao, H., He, J., Yang, Z., Guan, B., Chen, K., Hong, Q., Wang, J., Liu, J., Jiang, J., 2020. Extraordinarily conserved chromosomal synteny of citrus species revealed by chromosome-specific painting. Plant J. 103, 2225-2235.
[18]	Hofstatter, P.G., Thangavel, G., Lux, T., Neumann, P., Vondrak, T., Novak, P., Zhang, M., Costa, L., Castellani, M., Scott, A., et al., 2022. Repeat-based holocentromeres influence genome architecture and karyotype evolution. Cell 185, 3153-3168 e3118.
[19]	Huang, D., Wu, W., Zhou, Y., Hu, Z., Lu, L., 2004. Microdissection and molecular manipulation of single chromosomes in woody fruit trees with small chromosomes using pomelo (citrus grandis) as a model. I. Construction of single chromosomal DNA libraries. Theor. Appl. Genet. 108, 1366-1370.
[20]	Huang, X., Huang, S., Han, B., Li, J., 2022. The integrated genomics of crop domestication and breeding. Cell 185, 2828-2839.
[21]	Ibiapino, A., Baez, M., Garcia, M.A., Costea, M., Stefanovic, S., Pedrosa-Harand, A., 2022. Karyotype asymmetry in Cuscuta l. Subgenus Pachystigma reflects its repeat DNA composition. Chromosome Res. 30, 91-107.
[22]	Iovene, M., Wielgus, S.M., Simon, P.W., Buell, C.R., Jiang, J., 2008. Chromatin structure and physical mapping of chromosome 6 of potato and comparative analyses with tomato. Genetics 180, 1307.
[23]	Iwamasa, M. 1970. Chromosome Aberrations in Citrus in Relation to Sterility and Seedlessness.
[24]	Jiang, J., 2019. Fluorescence in situ hybridization in plants: recent developments and future applications. Chromosome Res. 27, 153-165.
[25]	Jiang, X., Song, Q., Ye, W., Chen, Z.J., 2021. Concerted genomic and epigenomic changes accompany stabilization of Arabidopsis allopolyploids. Nat. Ecol. Evol. 5, 1382-1393.
[26]	Lan, H., Chen, C.L., Miao, Y., Yu, C.X., Deng, X.X., 2016. Fragile sites of ‘valencia’ sweet orange (Citrus sinensis) chromosomes are related with active 45s rDNA. PLoS One 11, e0151512.
[27]	Leitch, A.R., Leitch, I.J., 2008. Genomic plasticity and the diversity of polyploid plants. Science 320, 481-483.
[28]	Liu, J., Seetharam, A.S., Chougule, K., Ou, S., Swentowsky, K.W., Gent, J.I., Llaca, V., Woodhouse, M.R., Manchanda, N., Presting, G.G., et al., 2020. Gapless assembly of maize chromosomes using long-read technologies. Genome Biol. 21, 121.
[29]	Liu, H., Wang, X., Liu, S., Huang, Y., Guo, Y.X., Xie, W.Z., Liu, H., Tahir Ul Qamar, M., Xu, Q., Chen, L.L., 2022. Citrus pan-genome to breeding database (CPBD): a comprehensive genome database for citrus breeding. Mol. Plant
[30]	Martins, L., Yu, F., Zhao, H., Dennison, T., Jiang, J., 2019. Meiotic crossovers characterized by haplotype-specific chromosome painting in maize. Nat. Commun. 10.
[31]	Melters, D.P., Bradnam, K.R., Young, H.A., Telis, N., May, M.R., Ruby, J.G., Sebra, R., Peluso, P., Eid, J., Rank, D., et al., 2013. Comparative analysis of tandem repeats from hundreds of species reveals unique insights into centromere evolution. Genome Biol. 14, R10.
[32]	Mendes, S., Moraes, A.P., Mirkov, T.E., Pedrosa-Harand, A., 2011. Chromosome homeologies and high variation in heterochromatin distribution between Citrus L. and Poncirus Raf. As evidenced by comparative cytogenetic mapping. Chromosome Res. 19, 521-530.
[33]	Meng, Z., Han, J., Lin, Y., Zhao, Y., Wang, K., 2020. Characterization of a saccharum spontaneum with a basic chromosome number of x = 10 provides new insights on genome evolution in genus saccharum. Theor. Appl. Genet. 133.
[34]	Meng, Z., Zhang, Z., Yan, T., Lin, Q., Wang, Y., Huang, W., Huang, Y., Li, Z., Yu, Q., Wang, J., 2018. Comprehensively characterizing the cytological features of saccharum spontaneum by the development of a complete set of chromosome-specific oligo probes. Front. Plant Sci. 9.
[35]	Mukai, Y., Nakahara, Y., Yamamoto, M., 1993. Simultaneous discrimination of the three genomes in hexaploid wheat by multicolor fluorescence in situ hybridization using total genomic and highly repeated DNA probes. Genome 36, 489-494.
[36]	Naish, M., Alonge, M., Wlodzimierz, P., Tock, A.J., Abramson, B.W., Schmucker, A., Mandakova, T., Jamge, B., Lambing, C., Kuo, P., et al., 2021. The genetic and epigenetic landscape of the Arabidopsis centromeres. Science 374, eabi7489.
[37]	Navratilova, P., Toegelova, H., Tulpova, Z., Kuo, Y.T., Stein, N., Dolezel, J., Houben, A., Simkova, H., Mascher, M., 2022. Prospects of telomere-to-telomere assembly in barley: analysis of sequence gaps in the morexv3 reference genome. Plant Biotechnol. J. 20, 1373-1386.
[38]	Nurk, S., Koren, S., Rhie, A., Rautiainen, M., Bzikadze, A.V., Mikheenko, A., Vollger, M.R., Altemose, N., Uralsky, L., Gershman, A., et al., 2022. The complete sequence of a human genome. Science 376, 44-53.
[39]	Oliveira, L.C., Torres, G.A., 2018. Plant centromeres: genetics, epigenetics and evolution. Mol. Biol. Rep. 45, 1491-1497.
[40]	Pedrosa, A., Schweizer, D., Guerra, M., 2000. Cytological heterozygosity and the hybrid origin of sweet orange [Citrus sinensis (l.) Osbeck]. Theor. Appl. Genet. 100, 361-367.
[41]	Prall, T.M., Neumann, E.K., Karl, J.A., Shortreed, C.G., Baker, D.A., Bussan, H.E., Wiseman, R.W., O'Connor, D.H., 2021. Consistent ultra-long DNA sequencing with automated slow pipetting. BMC Genom. 22, 182.
[42]	Ronspies, M., Schindele, P., Wetzel, R., Puchta, H., 2022a. CRISPR-Cas9-mediated chromosome engineering in Arabidopsis thaliana. Nat. Protoc. 17, 1332-1358.
[43]	Ronspies, M., Schmidt, C., Schindele, P., Lieberman-Lazarovich, M., Houben, A., Puchta, H., 2022b. Massive crossover suppression by CRISPR-Cas-mediated plant chromosome engineering. Nat. Plants.
[44]	Schmidt, C., Fransz, P., Ronspies, M., Dreissig, S., Fuchs, J., Heckmann, S., Houben, A., Puchta, H., 2020. Changing local recombination patterns in Arabidopsis by CRISPR/Cas mediated chromosome engineering. Nat. Commun. 11, 4418.
[45]	Schmidt, C., Pacher, M., Puchta, H., 2019. Efficient induction of heritable inversions in plant genomes using the CRISPR/Cas system. Plant J. 98, 577-589.
[46]	Silva, A., Marques, A., Santos, K., Guerra, M., 2010. The evolution of CMA bands in Citrus and related genera. Chromosome Res. 18, 503-514.
[47]	Soltis, D.E., Visger, C.J., Soltis, P.S., 2014. The polyploidy revolution then…and now: Stebbins revisited. Am. J. Bot. 101, 1057-1078.
[48]	Song, J.M., Xie, W.Z., Wang, S., Guo, Y.X., Chen, L.L., 2021. Two gap-free reference genomes and a global view of the centromere architecture in rice. Mol. Plant, S1674-2052(1621)00230-00236.
[49]	Talon, M.,Gmitter, F.G., Jr., 2008. Citrus genomics. Int. J. Plant Genom. 2008, 528361.
[50]	Wang, L., Huang, Y., Liu, Z., He, J., Jiang, X., He, F., Lu, Z., Yang, S., Chen, P., Yu, H., et al., 2021. Somatic variations led to the selection of acidic and acidless orange cultivars. Nat. Plants 7, 954-965.
[51]	Wendel, J.F., Jackson, S.A., Meyers, B.C., Wing, R.A., 2016. Evolution of plant genome architecture. Genome Biol. 17, 37.
[52]	Wu, G.A., Terol, J., Ibanez, V., Lopez-Garcia, A., Perez-Roman, E., Borreda, C., Domingo, C., Tadeo, F.R., Carbonell-Caballero, J., Alonso, R., 2018. Genomics of the origin and evolution of citrus. Nature 554, 311-316.
[53]	Xia, Wang, Yuantao, Xu, Siqi, Zhang, Li, Cao, Yue, Huang, 2017. Genomic analyses of primitive, wild and cultivated citrus provide insights into asexual reproduction. Nat. Genet. 49, 765-772.
[54]	Xia, Q.M., Miao, L.K., Xie, K.D., Yin, Z.P., Wu, X.M., Chen, C.L., Grosser, J.W., Guo, W.W., 2020. Localization and characterization of citrus centromeres by combining half-tetrad analysis and cenh3-associated sequence profiling. Plant Cell Rep. 39, 1609-1622.
[55]	Xin, H., Zhang, T., Wu, Y., Zhang, W., Zhang, P., Xi, M., Jiang, J., 2020. An extraordinarily stable karyotype of the woody populus species revealed by chromosome painting. Plant J. 101, 253-264.
[56]	Xu, Q., Chen, L.L., Ruan, X., Chen, D., Zhu, A., Chen, C., Bertrand, D., Jiao, W.B., Hao, B.H., Lyon, M.P., et al., 2013. The draft genome of sweet orange (Citrus sinensis). Nat. Genet. 45, 59-66.
[57]	Yu, C., Deng, X., Chen, C., 2019. Chromosomal characterization of a potential model mini-citrus (Fortunella hindsii). Tree Genet. Genomes 15.
[58]	Yu, F., Zhao, X., Chai, J., Ding, X., Li, X., Huang, Y., Wang, X., Wu, J., Zhang, M., Yang, Q., et al., 2021. Chromosome-specific painting unveils chromosomal fusions and distinct allopolyploid species in the Saccharum complex. New Phytol. 233, 1953-1965.
[59]	Yuan, Y., Scheben, A., Edwards, D., Chan, T.F., 2021. Toward haplotype studies in polyploid plants to assist breeding. Mol. Plant 14, 1969-1972.
[60]	Zhao, Q., Meng, Y., Wang, P., Qin, X., Cheng, C., Zhou, J., Yu, X., Li, J., Lou, Q., Jahn, M., et al., 2021. Reconstruction of ancestral karyotype illuminates chromosome evolution in the genus Cucumis. Plant J. 107, 1243-1259.
[61]	Zheng, J.S., Sun, C.Z., Zhang, S.N., Hou, X.L., Bonnema, G., 2016. Cytogenetic diversity of simple sequences repeats in morphotypes of Brassica rapa ssp. Chinensis. Front. Plant Sci. 7, 1049.
[62]	Zhou, C., Olukolu, B., Gemenet, D.C., Wu, S., Gruneberg, W., Cao, M.D., Fei, Z., Zeng, Z.B., George, A.W., Khan, A., et al., 2020. Assembly of whole-chromosome pseudomolecules for polyploid plant genomes using outbred mapping populations. Nat. Genet. 52, 1256-1264.