Natural variation in a CENTRORADIALIS homolog contributed to cluster fruiting and early maturity in cotton

Abstract

Background: Plant architecture and the vegetative-reproductive transition have major impacts on the agronomic success of crop plants, but genetic mechanisms underlying these traits in cotton (Gossypium spp.) have not been identified.

Results: We identify four natural mutations in GoCEN-D_t associated with cluster fruiting (cl) and early maturity. The situ hybridization shows that GhCEN is preferentially expressed in cotton shoot apical meristems (SAM) of the main stem and axillary buds. Constitutive GhCEN-Dt overexpression suppresses the transition of the cotton vegetative apex to a reproductive shoot. Silencing GoCEN leads to early flowering and determinate growth, and in tetraploids causes the main stem to terminate in a floral bud, a novel phenotype that exemplifies co-adaptation of polyploid subgenomes and suggests new research and/or crop improvement approaches. Natural cl variations are enriched in cottons adapted to high latitudes with short frost-free periods, indicating that mutants of GoCEN have been strongly selected for early maturity.

Conclusion: We show that the cotton gene GoCEN-Dt, a homolog of Antirrhinum CENTRORADIALIS, is responsible for determinate growth habit and cluster fruiting. Insight into the genetic control of branch and flower differentiation offers new approaches to develop early maturing cultivars of cotton and other crops with plant architecture appropriate for mechanical harvesting.

Keywords: CEN/TFL1; Cluster fruiting; Cotton; Determinate growth habit; Early maturity; Map-based cloning; Plant architecture.

Fig. 1

Phenotypic analyses of the first fruiting branch in wild-type and cl-type G. hirsutum and G. barbadense. a, e Phenotypes of the first fruit branch between reference and cl plants in G. hirsutum and G. barbadense. Scale bar, 10 cm. b, f Length of the first fruiting branch from stem to SAM. (c, g) Number of leaves on the first fruiting branch, including fully expanded young leaves. d, h Number of bolls and flower buds on the first fruiting branch. All data are means ± SD from 2012 to 2016 (n = 10 plants); * and ** represent significant differences determined by the Student’s t-test at P < 0.05 and P < 0.01

Fig. 2

Map-based cloning of the Gb-cl gene. a, b Coarse linkage map. Scale bar, 5 cM and 1 cM in A and B, respectively. c High-resolution linkage map. Scale bar, 0.1 cM. d, e Delimitation to 139.4 kb and 69 kb genomic regions in G. hirsutum and G. raimondii reference genomes, each containing two or one predicted gene. Scale bar, 30 Kb. f Sequence diversity of GoCEN revealed four mutations associated with cluster fruiting in tetraploid cotton. Broken lines indicates sites of mutations. Red letters indicate non-synonymous mutations. Scale bar, 100 bp

Fig. 3

Spatiotemporal expression pattern and subcellular location of GoCEN. a Expression levels of GhCEN in 11 G. hirsutum tissues using real-time PCR. b Expression levels of GoCEN in the shoot apical meristem (SAM) of G. barbadense genotypes Pima S-6, 3–79, Hai 170, Xinhai18* and Junhai 1*; and G. hirsutum genotypes CCRI35, Yumian1, Chaozao3*, Duan063* and Xiaoxian2*; * indicates cl mutants. The error bars are SD of three biological replicates. c In situ hybridization of GoCEN in shoot apical meristems (SAM). Hybridization was detected with antisense probes (c1, c2) but not sense probes (c3, c4) in the SAM of Pima S-6. c2 and c4 are the enlargement of the boxes in c1 and c3, respectively. (am) apical meristem of the main stem; (lp) leaf primordium which formed leaf on the main stem; (xm) axillary meristem which formed the fruiting branch. Scale bars, 200 μm. d Subcellular localization of GhCEN-GFP in tobacco plants. Scale bars, 50 μm

Fig. 4

Morphologies of GhCEN-Dt over-expression plants. a Jimian 14 (wild-type) with an indeterminate phenotype. Scale bars, 15 cm. b Jimian 14 overexpressing GbCEN-Dt from Hai 170 with normal flowering time. Scale bars, 15 cm. c Jimian 14 overexpressing GhCEN-Dt from CCRI 35 with delayed flowering time. Scale bars, 15 cm. d Relative GhCEN transcript levels in transgenic lines and WT. **P < 0.01, error bars are SD of three biological replicates. e Position (node) of the first fruiting branch formed in WT, 35S::cen and 35S::CEN cottons. **P < 0.01, error bars are SD of three biological replicates; n.s., not significant. f and g Branching phenotypes of the 10th node in 35S::cen (f) and 35S::CEN (g) over-expressed cotton. Scale bars, 60 cm

Fig. 5

Functional characterization of GoCEN by virus induced gene silencing (VIGS). a-c Phenotypes of WT and GoCEN-silenced by VIGS in G. hirsutum (a), G. barbadense (b) and G. arboretum c; WT and GoCEN-silenced are on left and right, respectively. Insets are close-up views of the top of the main stem. Black arrows show the earliest floral buds in GoCEN-silenced cottons. d Relative transcript levels of GoCEN in WT and VIGS silenced cottons. **P < 0.01. Error bars are SD of three biological repeats

Fig. 6

Transcriptomic comparison of SAM between WT and VIGS-silenced GoCEN plants in G. hirsutum and G. barbadense. a The SAM from wild type and VIGS-silenced GoCEN Pima S-6 at the three-leaf stage. Red arrows show growing points differentiated into fruit or fruiting branches. Scale bars, 100 μm. b Venn diagram showing the number of differentially expressed genes in WT and VIGS-silenced GoCEN G. hirsutum and G. barbadense. c Heatmap visualization of 37 MADS box transcription factors that are significantly differentially expressed in WT and VIGS-silenced GoCEN plants in G. hirsutum and G. barbadense. Red indicates up-regulated and blue indicates down-regulated expression values

Fig. 7

Varieties of G. barbadense in relation to geographic distribution and maturity. a Geographic distribution of the main growing areas of G. barbadense in the world. Colors represent the lengths of frost-free periods. Triangles and circles represent normal and cl fruiting branches. The world map was drawn by the authors based on the data from Resource and Environment Data Cloud Platform, DOI: 10.12078/2018110201. The data from Resource and Environment Data Cloud Platform are open and free. b-e Different types of fruiting branch of G. barbadense have a definite relationship with maturity. Dotted lines with corresponding colors show the average from two replicates in each year and ten mutant lines planted in a randomized complete block design. CEN and cen show the indeterminate and determinate growth habit, respectively. Error bars are SD of two biological replicates