Gossypium arboreum L. is a diploid species introduced from India, which possesses many favorable traits that upland cotton cultivars lack, including drought tolerance as well as resistance to diseases and insect pests (Liu et al. 2006). In addition, some G. arboreum accessions produce strong fibers as well as seeds with high oil contents and seed indexes (Liu et al. 2006). Another major difference between the diploid and tetraploid cottons is that in most of G. arboreum lines, red spots develop at the proximal petals during flower development and persist lifelong (Zhou et al. 2011). In contrary, only sparse cases have been reported in G. hirsutum flowers (Jones 1996; Zhou et al. 2011; Abid et al. 2022). These spots are easily recognizable features, which could enhance the attractiveness for insect pollinators (Jones 1996; Abid et al. 2022). Despite the universally pronounced phenomenon observed, little researches regarding the molecular mechanisms or the genetic determinants have been conducted in G. arboreum.
Of the many internal and external factors influencing flower coloration, anthocyanin types and concentrations are considered to be major determinants in many species (Weiss 1995; Tanaka et al. 2008). Anthocyanins, which are water-soluble pigments, are a class of flavonoids responsible for the red, purple, and blue coloration of flowers, fruits, and leaves (Zhang et al. 2018). Although anthocyanins are structurally diverse chemicals, there are only six common chromophore forms (aglycones) of anthocyanins: cyanidin, delphinidin, pelargonidin, peonidin, petunidin, and malvidin (Jaakola, 2013). The anthocyanin biosynthesis pathway has been well characterized and the genes encoding proteins mediating anthocyanin biosynthesis have been identified in many plant species, including Arabidopsis thaliana (Li and Strid 2005), Petunia hybrida (Chen et al. 2021), spinach (Cai et al. 2018), alfalfa (Duan et al. 2020), and many fruits (Zhang et al. 2018). Meanwhile, the regulatory transcription factors of anthocyanin biosynthesis have also been identified (Saito et al. 2013). Specifically, the MYB transcription factors are crucial for the accumulation of anthocyanins in fruits, including apple, pear, peach, sweet cherry, and the fruits of Citrus species (Zhang et al. 2018). In Theobroma cacao L., the green/red pod coloration is likely regulated by the MYB transcription factor encoded by TcMYB113 (Motamayor et al. 2013). In G. barbadense, an R2R3 MYB113 transcription factor could control purple spot formation at the base of flower petals (Abid et al. 2022).
Aside from the biosynthetic genes and the regulatory factors, the anthocyanins are, after being synthesized in the cytosol, generally transported into vacuoles via specific transporters (Zhang et al. 2018), wherein the glutathione S-transferases (GSTs) are likely the most important. Earlier researches revealed visible changes to pigmentation in mutants with loss-of-function mutations of GST-encoding genes, including tt19 in A. thaliana, bz2 in maize, an9 in petunia, and fl3 in carnation (Marrs et al. 1995; Alfenito et al. 1998; Larsen et al. 2003; Kitamura et al. 2004; Sun et al. 2012). A recent study has demonstrated one GST that functions downstream of FvMYB10 is the main transporter of the anthocyanins responsible for strawberry foliage and fruit coloration; it can be modified to change strawberry fruit from white to red (Luo et al. 2018).
Although considerable researches on flower coloration have been performed in various species (Sasaki et al. 2015; Jiao et al. 2020; Morimoto et al. 2020; Pu et al. 2020), little is known regarding the formation of red spots on G. arboreum flowers. One way to narrow down the scope of candidate genes for downstream sifting and identification is the combination of genomic resequencing and bulked segregant analysis (BSA), which has been utilized to dissect color traits in numerous species. For instance, the BrTT1 responsible for the seed coat coloration in Brassica rapa was determined by combining genomic resequencing and a BSA (Wang et al. 2016). Similarly, the anthocyanin accumulation-related genes in pepper fruits (Wang et al. 2018) and the gene affecting the foliage and fruit coloration in strawberry (Luo et al. 2018) was also identified. Additionally, a next-generation sequencing technique for transcriptome analyses (RNA-seq) has facilitated the rapid and cost-effective identification of candidate genes for specific traits (Zhang et al. 2017; Hou et al. 2018; Meng et al. 2019; Jiang et al. 2020; Li et al. 2020). In this study, the red-spot-petal phenotype in G. arboreum was related to the content variation of anthocyanin chemicals by metabolic profiling, it was mapped onto a dominant locus on chromosome 7 through the combination of genomic resequencing and BSA. Next, a GST-coding gene was pinpointed as the likely candidate by further integrating the transcriptomic data that spans sequential developmental stages of flowers wherein contrast visual characters regarding colored spots are formed. At last, functional validation was fulfilled by the observation of scattered color spots on petals with inhibited expression via virus-induced gene silencing (VIGS) methodology. To this end, we have successfully identified a key gene involved in the red spot formation of G. arboreum flowers. The whole process represents a first, to the best of our knowledge, systematic evaluation of this trait in G. arboreum.