Recovery and characterization of a Citrus clementina Hort. ex Tan. 'Clemenules' haploid plant selected to establish the reference whole Citrus genome sequence

Abstract

Background: In recent years, the development of structural genomics has generated a growing interest in obtaining haploid plants. The use of homozygous lines presents a significant advantage for the accomplishment of sequencing projects. Commercial citrus species are characterized by high heterozygosity, making it difficult to assemble large genome sequences. Thus, the International Citrus Genomic Consortium (ICGC) decided to establish a reference whole citrus genome sequence from a homozygous plant. Due to the existence of important molecular resources and previous success in obtaining haploid clementine plants, haploid clementine was selected as the target for the implementation of the reference whole genome citrus sequence.

Results: To obtain haploid clementine lines we used the technique of in situ gynogenesis induced by irradiated pollen. Flow cytometry, chromosome counts and SSR marker (Simple Sequence Repeats) analysis facilitated the identification of six different haploid lines (2n = x = 9), one aneuploid line (2n = 2x+4 = 22) and one doubled haploid plant (2n = 2x = 18) of 'Clemenules' clementine. One of the haploids, obtained directly from an original haploid embryo, grew vigorously and produced flowers after four years. This is the first haploid plant of clementine that has bloomed and we have, for the first time, characterized the histology of haploid and diploid flowers of clementine. Additionally a double haploid plant was obtained spontaneously from this haploid line.

Conclusion: The first haploid plant of 'Clemenules' clementine produced directly by germination of a haploid embryo, which grew vigorously and produced flowers, has been obtained in this work. This haploid line has been selected and it is being used by the ICGC to establish the reference sequence of the nuclear genome of citrus.

Figure 1

a. Small seeds of 'Clemenules' obtained from pollination with irradiated pollen. b. Embryo present in seeds. c. Embryogenic calli originating from embryo culture. d. Cluster of embryos obtained from embryogenic calli. e. Shoots produced by embryos regenerated from embryogenic calli. f. Regenerated plant from direct germination of embryo without a callus phase. g, h. In vitro micrograft of haploid shoot.

Figure 2

Flow cytometry analysis. a. Histogram of the G haploid plant (peak 1) and control triploid plant (peak 2). b. Histogram displaying a control diploid plant (peak 1), B.1 aneuploid plant (peak 2) and control triploid plant (peak 3).

Figure 3

DAPI stained chromosomes at the metaphase stage. a. G haploid plant (2n = x = 9). b. C.1 double haploid plant (2n = 2x = 18). c. B.1 aneuploid plant (2n = 2x+4 = 22).

Figure 4

a. Ci03C08 SSR marker genetic analysis. b. TAA 15 SSR marker genetic analysis. 1. 'Clemenules', 2. 'Fortune', 3. Haploid H, 4 - 11 Haploids obtained from embryogenic callus A, 12. Haploid G, 13. Haploid D.1, 14. Haploid E, 15. Haploid F, 16. Double haploid C.1, 17 - 19. Aneuploid plants obtained from embryogenic callus B.

Figure 5

a. C.1 double haploid plant of 'Clemenules'. b. G haploid plant of 'Clemenules'. c. Detail of blossom of the G haploid plant. d. Haploid and diploid flower of 'Clemenules'. e. B.1 aneuploid plant of 'Clemenules'.

Figure 6

Histological sections of haploid and diploid anthers, ovaries, styles and stigmas of 'Clemenules'. a. Transversal section of haploid anther. b. Transversal section of diploid anther. c. Transversal section of haploid ovary. d. Transversal section of diploid ovary. e. Transversal section of haploid style. f. Transversal section of diploid style. g. Transversal section of haploid stigma. h. Transversal section of diploid stigma. C cortex, CA central axis, CT connective tissue, DB dorsal bundle, E epidermis, EN endothecium, II inner integument, JSP juice sac primordia, L locules, MB marginal bundle, N nucellus, O ovule, OI outer integument, P papilla, PG pollen grains, S septa, SB septal bundle, SC stylar canal, SSC stigmatic secretion canal, SZ stigmatic zone, T tapetum and VB vascular bundle.