UNREDUCED MEGAGAMETOPHYTE FORMATION VIA SECOND DIVISION RESTITUTION CONTRIBUTES TO TETRAPLOID PRODUCTION IN INTERPLOIDY CROSSES WITH ‘ORAH’ MANDARIN (CITRUS RETICULATA)

Highlights

• In addition to triploid progeny, tetraploid hybrids derived from the fertilization of 2n megagametophytes are frequently regenerated from 2x × 4x crosses that utilize ‘Orah’ mandarin as the female parent.

• Data here indicate that ‘Orah’ mandarin is a cultivar that readily produces 2n megagametophytes.

• Second division restitution is the mechanism underlying 2n megagametophyte formation in ‘Orah’ mandarin.

Abstract

Seedless fruits are desirable in the citrus fresh fruit market. Triploid production via diploid × tetraploid interploidy crosses is thought to be the most efficient and widely-used strategy for the breeding of seedless citrus. Although ‘Orah’ mandarin has desirable organoleptic qualities, seeds in the fruits weaken its market competitiveness. To produce new seedless cultivars that are similar to ‘Orah’ mandarin, we performed three 2x × 4x crosses using ‘Orah’ mandarin as the seed parent to regenerate triploid plantlets. A total of 182 triploid and 36 tetraploid plantlets were obtained. By analyzing their genetic origins using nine novel single nucleotide polymorphism (SNP) markers, all of the triploids and tetraploids derived from these three crosses were proven to be hybrids. Also, we demonstrated that 2n megagametophyte formation in ‘Orah’ mandarin result in tetraploid production in these three interploidy crosses. These tetraploid plantlets were genotyped using eight pericentromeric SNP markers and nine centromere distal SNP markers. Based on the genotypes of the 2n megagametophytes, the parental heterozygosity rates in 16 SNP loci and all 2n megagametophytes were less than 50%, indicating that second division restitution was the mechanism underlying 2n megagametophyte formation at both the population and individual levels. These triploid hybrids enrich the germplasm available for seedless breeding. Moreover, the tetraploid hybrids are valuable as parents for ploidy breeding for the production of seedless citrus fruits.

Graphical abstract

Keywords

Citrus / 2n gamete / interploidy hybridization / pericentromeric SNP marker / second division restitution

References

[1] Brownfield L, Köhler C. Unreduced gamete formation in plants: mechanisms and prospects. Journal of Experimental Botany, 2011, 62(5): 1659–1668

[2] Krug C A. Chromosome numbers in the subfamily Arantioideae, with special reference in the genus Citrus. Botanical Gazette, 1943, 104(4): 602–611

[3] Aleza P, Cuenca J, Juárez J, Navarro L, Ollitrault P. Inheritance in doubled-diploid clementine and comparative study with SDR unreduced gametes of diploid clementine. Plant Cell Reports, 2016, 35(8): 1573–1586

[4] Ollitrault P, Germana M A, Froelicher Y, Cuenca J, Aleza P, Morillon R, Grosser J W, Guo W W. Ploidy Manipulation for citrus breeding, genetics, and genomics. In: Gentile A, La Malfa S, Deng Z, eds. The Citrus Genome. Springer, 2020, 75–105

[5] Ollitrault P, Dambier D, Luro F, Froelicher Y. Ploidy manipulation for breeding seedless triploid citrus. In: Janick J, ed. Plant Breeding Reviews. Wiley, 2008, 30: 323–352

[6] Recupero G R, Russo G, Recupero S. New promising Citrus triploid hybrids selected from crosses between monoembryonic diploid female and tetraploid male parents. HortScience, 2005, 40(3): 516–520

[7] Aleza P, Juárez J, Cuenca J, Ollitrault P, Navarro L. Extensive citrus triploid hybrid production by 2x × 4x sexual hybridizations and parent-effect on the length of the juvenile phase. Plant Cell Reports, 2012, 31(9): 1723–1735

[8] Xie K D, Wang H Q, Wang X P, Liang W J, Xie Z Z, Yi H L, Deng X X, Grosser J W, Guo W W. Extensive citrus triploid breeding by crossing monoembryonic diploid females with allotetraploid male parents. Scientia Agricultura Sinica, 2013, 46(21): 4550–4557 (in Chinese)

[9] Xie K D, Wang X P, Wang H Q, Liang W J, Xie Z Z, Guo D Y, Yi H L, Deng X X, Grosser J W, Guo W W. High efficient and extensive production of triploid Citrus plants by crossing polyembryonic diploids with tetraploids. Acta Horticulturae Sinica, 2014, 41(4): 613–620 (in Chinese)

[10] Xie K D, Yuan D Y, Wang W, Xia Q M, Wu X M, Chen C W, Chen C L, Grosser J W, Guo W W. Citrus triploid recovery based on 2x × 4x crosses via an optimized embryo rescue approach. Scientia Horticulturae, 2019, 252: 104–109

[11] Cuenca J, Froelicher Y, Aleza P, Juárez J, Navarro L, Ollitrault P. Multilocus half-tetrad analysis and centromere mapping in citrus: evidence of SDR mechanism for 2n megagametophyte production and partial chiasma interference in mandarin cv ‘Fortune’. Heredity, 2011, 107(5): 462–470

[12] Kreiner J M, Kron P, Husband B C. Frequency and maintenance of unreduced gametes in natural plant populations: associations with reproductive mode, life history and genome size. New Phytologist, 2017, 214(2): 879–889

[13] Aleza P, Cuenca J, Hernández M, Juárez J, Navarro L, Ollitrault P. Genetic mapping of centromeres in the nine Citrus clementina chromosomes using half-tetrad analysis and recombination patterns in unreduced and haploid gametes. BMC Plant Biology, 2015, 15(1): 80–93

[14] Cuenca J, Aleza P, Juárez J, García-Lor A, Froelicher Y, Navarro L, Ollitrault P. Maximum-likelihood method identifies meiotic restitution mechanism from heterozygosity transmission of centromeric loci: application in citrus. Scientific Reports, 2015, 5(1): 9897–9908

[15] Xie K D, Wang X P, Biswas M K, Liang W J, Xu Q, Grosser J W, Guo W W. 2n megagametophyte formed via SDR contributes to tetraploidization in polyembryonic ‘Nadorcott’ tangor crossed by citrus allotetraploids. Plant Cell Reports, 2014, 33(10): 1641–1650

[16] Rouiss H, Cuenca J, Navarro L, Ollitrault P, Aleza P. Tetraploid citrus progenies arising from FDR and SDR unreduced pollen in 4x × 2x hybridizations. Tree Genetics & Genomes, 2017, 13(1): 10–24

[17] Rouiss H, Cuenca J, Navarro L, Ollitrault P, Aleza P. Unreduced megagametophyte production in lemon occurs via three meiotic mechanisms, predominantly second-division restitution. Frontiers in Plant Science, 2017, 8: 1211–1227

[18] Xie K D, Xia Q M, Peng J, Wu X M, Xie Z Z, Chen C L, Guo W W. Mechanism underlying 2n male and female gamete formation in lemon via cytological and molecular marker analysis. Plant Biotechnology Reports, 2019, 13(2): 141–149

[19] Barry G H, Gmitter F G Jr, Chen C X, Roose M L, Federici C T, McCollum G T. Investigating the parentage of ‘Orri’ and ‘Fortune’ mandarin hybrids. Acta Horticulturae, 2015, (1065): 449–456

[20] Guo W W, Prasad D, Serrano P, Gmitter F G Jr, Grosser J W. Citrus somatic hybridization with potential for direct tetraploid scion cultivar development. Journal of Horticultural Science & Biotechnology, 2004, 79(3): 400–405

[21] Grosser J W, Gmitter F G Jr. Protoplast fusion for production of tetraploids and triploids: applications for scion and rootstock breeding in citrus. Plant Cell, Tissue and Organ Culture, 2011, 104(3): 343–357

[22] Guo W W, Wu R C, Cheng Y J, Deng X X. Production and molecular characterization of Citrus intergeneric somatic hybrids between red tangerine and citrange. Plant Breeding, 2007, 126(1): 72–76

[23] Wang S M, Lan H, Jia H H, Xie K D, Wu X M, Chen C L, Guo W W. Induction of parthenogenetic haploid plants using gamma irradiated pollens in ‘Hirado Buntan’ pummelo (Citrus grandis [L.] Osbeck). Scientia Horticulturae, 2016, 207: 233–239

[24] Cheng Y J, Guo W W, Yi H L, Pang X M, Deng X X. An efficient protocol for genomic DNA extraction from Citrus species. Plant Molecular Biology Reporter, 2003, 21(2): 177–178

[25] Xia Q M, Miao L K, Xie K D, Yin Z P, Wu X M, Chen C L, Grosser J W, Guo W W. Localization and characterization of Citrus centromeres by combining half-tetrad analysis and CenH3-associated sequence profiling. Plant Cell Reports, 2020, 39(12): 1609–1622

[26] Xu Q, Chen L L, Ruan X, Chen D, Zhu A, Chen C, Bertrand D, Jiao W B, Hao B H, Lyon M P, Chen J, Gao S, Xing F, Lan H, Chang J W, Ge X, Lei Y, Hu Q, Miao Y, Wang L, Xiao S, Biswas M K, Zeng W, Guo F, Cao H, Yang X, Xu X W, Cheng Y J, Xu J, Liu J H, Luo O J, Tang Z, Guo W W, Kuang H, Zhang H Y, Roose M L, Nagarajan N, Deng X X, Ruan Y. The draft genome of sweet orange (Citrus sinensis). Nature Genetics, 2013, 45(1): 59–66

[27] Li H, Durbin R. Fast and accurate long-read alignment with Burrows-Wheeler transform. Bioinformatics, 2010, 26(5): 589–595

[28] Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, Marth G, Abecasis G, Durbin R. The Sequence Alignment/Map format and SAMtools. Bioinformatics, 2009, 25(16): 2078–2079

[29] Cingolani P, Platts A, Wang L, Coon M, Nguyen T, Wang L, Land S J, Lu X, Ruden D M. A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff: SNPs in the genome of Drosophila melanogaster strain w¹¹¹⁸; iso-2; iso-3. Fly, 2012, 6(2): 80–92

[30] Cuenca J, Aleza P, Navarro L, Ollitrault P. Assignment of SNP allelic configuration in polyploids using competitive allele-specific PCR: application to citrus triploid progeny. Annual of Botany, 2013, 111(4): 731–742

[31] Blary A, Jenczewski E. Manipulation of crossover frequency and distribution for plant breeding. Theoretical and Applied Genetics, 2019, 132(3): 575–592

[32] Fernandes J B, Wlodzimierz P, Henderson I R. Meiotic recombination within plant centromeres. Current Opinion in Plant Biology, 2019, 48: 26–35

[33] Dong C B, Suo Y J, Kang X Y. Assessment of the genetic composition of triploid hybrid Populus using SSR markers with low recombination frequencies. Canadian Journal of Forest Research, 2014, 44(7): 692–699

[34] Liesebach H, Ulrich K, Ewald D. FDR and SDR processes in meiosis and diploid gamete formation in poplars (Populus L.) detected by centromere-associated microsatellite markers. Tree Genetics & Genomes, 2015, 11(1): 801–811

[35] Cuenca J, Aleza P, Garcia-Lor A, Ollitrault P, Navarro L. Fine mapping for identification of Citrus alternaria brown spot candidate resistance genes and development of new SNP markers for marker-assisted selection. Frontiers in Plant Science, 2016, 7: 1948–1961

[36] Wang X, Xu Y, Zhang S, Cao L, Huang Y, Cheng J, Wu G, Tian S, Chen C, Liu Y, Yu H, Yang X, Lan H, Wang N, Wang L, Xu J, Jiang X, Xie Z, Tan M, Larkin R M, Chen L L, Ma B G, Ruan Y, Deng X, Xu Q. Genomic analyses of primitive, wild and cultivated citrus provide insights into asexual reproduction. Nature Genetics, 2017, 49(5): 765–772

Supplementary materials

The online version of this article at https://doi.org/10.15302/J-FASE-2021385 contains supplementary materials (Tables S1–S2; Fig. S1).

Acknowledgements

This research was funded by the National Key Research and Development Program of China (2018YFD1000200), the National Natural Science Foundation of China (31820103011), the Key Research and Development Program of Hubei Province (2020BBA036), and the Fundamental Research Funds for the Central Universities of China (2662019QD048). The authors thank their colleague Professor Robert M. Larkin for critical reading of the manuscript.

Compliance with ethics guidelines

Qiangming Xia, Wei Wang, Kaidong Xie, Xiaomeng Wu, Xiuxin Deng, Jude W. Grosser, and Wenwu Guo declare that they have no conflicts of interest or financial conflicts to disclose. This article does not contain any studies with human or animal subjects performed by any of the authors.

RIGHTS & PERMISSIONS

The Author(s) 2021. Published by Higher Education Press. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0)