Natural variations in the Cis-elements of GhRPRS1 contributing to petal colour diversity in cotton

Abstract

The cotton genus comprises both diploid and allotetraploid species, and the diversity in petal colour within this genus offers valuable targets for studying orthologous gene function differentiation and evolution. However, the genetic basis for this diversity in petal colour remains largely unknown. The red petal colour primarily comes from C, G, K, and D genome species, and it is likely that the common ancestor of cotton had red petals. Here, by employing a clone mapping strategy, we mapped the red petal trait to a specific region on chromosome A07 in upland cotton. Genomic comparisons and phylogenetic analyses revealed that the red petal phenotype introgressed from G. bickii. Transcriptome analysis indicated that GhRPRS1, which encodes a glutathione S-transferase, was the causative gene for the red petal colour. Knocking out GhRPRS1 resulted in white petals and the absence of red spots, while overexpression of both genotypes of GhRPRS1 led to red petals. Further analysis suggested that GhRPRS1 played a role in transporting pelargonidin-3-O-glucoside and cyanidin-3-O-glucoside. Promoter activity analysis indicated that variations in the promoter, but not in the gene body of GhRPRS1, have led to different petal colours within the genus. Our findings provide new insights into orthologous gene evolution as well as new strategies for modifying promoters in cotton breeding.

Keywords: Gossypium hirsutum; anthocyanin transport; fine mapping; petal colour; promoter variation.

Figure 1

Phenotyping and genetic analysis of petal colour in cotton genus. (a) Investigation of petal colour and geographic distribution in 29 diverse cotton species. The 29 species are categorized into 8 diploid chromosomal groups (A–G plus K) and the allotetraploid AD group. G. aridum (D₄), G. gossypioides (D₆), G. lobatum (D₇), G. laxum (D₉), G. schwendimanii (D₁₁), G. sturtianum (C₁), G. bickii (G₁), G. australe (G₂), G. nelsonii (G₃), and G. rotundifolium (K₂) exhibit a red petal colour. (b) Evolutionary relationships among cotton species with different petal colours. (c) Genetic analysis of the red petal by crossing G. hirsutum (ZYH) with G. hirsutum (ZM24), and G. barbadense (3–79), respectively.

Figure 2

Genetic mapping of red petal with red spot. (a) BSA analysis conducted on the red petal phenotype within an F₂ segregating population. The ΔSNP index (representing the SNP index difference between the red and white bulk populations) is illustrated by red lines, with the 99% confidence interval depicted in black. (b) Zoomed‐in the BSA signal on chromosome A07 from Figure 1a. (c) The polymorphism InDel markers locating on A07 signal interval. The digit within the bracket is the number of recombination individuals. (d) The signal interval was narrowed down between InDel9099 and InDel1535. (e) SNP density distribution on chromosome A07 for ZYH and ZM24. (f–h) The number of SNPs (f), Indels (g), and mapped reads (h) on chromosome A07. The TM‐1 genome was used as a reference genome for read mapping.

Figure 3

Validation of the RPRS1 locus deriving from introgression. (a) Comparison of phylogenetic trees analysis among RPRS1 locus, its upstream border and downstream border. SNPs from of 109 accessions were used for the tree construction (b) The read mapping depth of ZYH, ZM24, red petal pool, and white petal pool within 8–16 Mb interval on chromosome A07. All the data were mapping to the TM‐1 reference genome. (c) Mapped read number on chromosome A07 of G. hirsutum within 8–16 Mb. (d) The relative read mapping depth of ZYH, ZM24, red petal pool, and white petal pool within 8–16 Mb interval on chromosome A07. All the data were mapping to the integrated sequences of G. bickii Chr07 and TM‐1. (e) Mapped read number on chromosome Chr07 of G. bickii within 8–14 Mb.

Figure 4

Functional validation of two genotypes of GhRPRS1 in cotton. (a) The WMV067‐AADA vector was employed for overexpressing the GhRPRS1 in ZM24. (b) Two genotypes of GhRPRS1 cloned from white petal and red petal were overexpressed in ZM24. Lines RPRS1‐5 and RPRS1‐6 were generated by overexpression GhRPRS1 from white petal, while RPRS1‐9 and RPRS1‐10 were generated by the overexpression of GhRPRS1 from red petal. ZM24 served as controls for white petals. (c) The measurement of anthocyanin content in GhRPRS1 overexpression lines. (d–f) The expression levels of GhRPRS1 in overexpression lines were detected by qPCR and semi‐quantitative PCR. GhActin served as an internal control and western blotting of GhRPRS1 in transgene lines. (g) WMVC016 was utilized for gene editing of GhRPRS1 in ZYH. Three mutants with base deletion were observed at the target position. rprs1‐10, rprs1‐4, and rprs‐2 were the knockout lines in ZYH. (h) The phenotype of three GhRPRS1 knockout lines in ZYH, ZYH served as controls for red petals. (i) The measurement of anthocyanin content in GhRPRS1 knockout lines. (j) Comparison of petal colours between ZM24 and ZYH. A total of 13 stages were photographed from little bud to flowering. (k) Expression level of the GhRPRS1 in 13 stages as shown in panel j. (l) Comparison of petal colours before and after flowering between ZM24 and ZYH. (m, n) Comparison of expression level of GhRPRS1 between ZM24 and ZYH in four stages as showed in panel (k). Data were analysed using GraphPad Prism (v8.0.2, GraphPad Software, United States) software. Statistical testing was applied using a Student's t‐test, statistical significance is defined as P < 0.05.

Figure 5

Cis‐element variations in promoters of GhRPRS1 resulted in petal colour differentiation in cotton. (a) Six haplotypes were identified in 21 cotton species. (b) Transient expression of the two haplotypes of GhRPRS1 in the cotyledons of DC109. (c) Comparison promoter sequence similarities of GhRPRS1 from 26 cotton species. (d) A series of vectors carrying GUS were constructed for promoter activities analysis. F1, F2, F3, and F4 represent different length of truncated sequences. (e) Promoter activities of F1–F4 were tested in tobacco leaves by GUS staining. (f) Expression levels of GUS were detected 3 days after injection. Actin served as an internal control. (g) Dot plot analysis of promoter sequences between ZYH and ZM24. E1, E2, and E3 represent the Indels between the two promoters. (h) E1, E2, and E3 were identified −500 to −1500 regions. (i) E1, E2, and E3 were integrated into the pGreenII‐0800‐35smini‐LUC vector to test their regulated activity in tobacco leaves, respectively. (j–l) Transient expression E1 (j), E2 (k), E3 (l) using the pGreenII‐0800‐35smini‐LUC system in tobacco leaves. The luciferase (LUC) activity detected 3 days after injection. Fluorescence intensity is captured, and data represent means ± SE of three independent experiments. Data were analysed using GraphPad Prism (v8.0.2, GraphPad Software, United States) software. Statistical testing was applied using a Student's t‐test, statistical significance is defined as P < 0.05.

Figure 6

A work model for GhRPRS1 transport anthocyanin in both red and white petals. GhRPRS1 works as anthocyanin transporter in cotton. The presence of E3 element in promoter of GhRPRS1 from ZYH enhanced the expression of the GhRPRS1. As a result, the red petals have more anthocyanin transporter, which facilitates the transport of anthocyanins from the endoplasmic reticulum membrane to the tonoplast. While in ZM24, the absence of E3 resulted in weakly expressed of GhRPRS1, and only a small part of transporters is available for anthocyanin transporting. Since different amount of anthocyanin were deposited in vacuoles between ZHY and ZM24, they exhibited red and white colours in petals.