In angiosperms or flowering plants, “Florogenesis” is the transitioning process in a plant’s apical meristem from vegetative tissue to reproductive organs or flowers . This transition is governed by flowering genes in which expression is influenced by factors such as vernalization, photoperiod, gibberellins, an autonomous pathway and ambient temperature . In Arabidopsis, several flowering genes have been discovered which are involved in flowering and act as floral integrators. These flowering genes include FT, SOC1, CO, VRN1, PPD, FCA, FLD, and FLK [3–4]. These floral integrator genes specifically upregulate flowering by promoting transition from vegetative to flowering or repressing floral repressor genes. Repressor genes act as repressors of floral integrator genes and upregulate the expression of repressors, including genes such as FLC, FRI, FLX, VRN2, and SVP [3, 5–6].
FLOWERING LOCUS C (FLC) and FLC-like are floral repressors found in many dicotyledon plants, such as Malus , Rosa , Coffea  and Brassica . The FLC gene is regulated by temperature changes throughout the year, both in annuals and perennials. In summer, FLC expression is upregulated through FRIGIDA (FRI) by binding the FLC promoter through the DNA-binding protein SUPPRESSOR OF FRIGIDA4 SUF4 . In addition, FRI expression is upregulated by the FLOWERING LOCUS C EXPRESSOR (FLX); both SUF4 and FLX are in the FRI-specific pathway . In winter, FLC is down-regulated through a process of vernalization as prolonged exposure of low temperature in winter in the meristem gradually reduces the expression of FLC . In addition to the vernalization pathway, the autonomous pathway reduces the expression of FLC both in the meristem and leaves . Gradual reduction of FLC allows FLOWERING LOCUS T (FT) to be expressed in the leaves and transported through phloem to reach to the meristematic tissue to stimulate the MADS box genes, thereby inducing flowering in Arabidopsis .
In wheat and barley, the flowering pathway is regulated by photoperiod, vernalization and the circadian clock . Vernalization gene-2 (VRN2) is a dominant repressor or inhibitor of flowering in winter wheat (Triticum aestivum; a monocot grass) that is down-regulated by vernalization (a cold period; winter) . Subsequently, the floral integrator leads to flowering in winter wheat while spring wheat doesn’t require vernalization due to a non-functional VRN2 gene. However, vernalization is a facultative stimulus for earlier flowering in spring wheat . In contract, maize (Zea mays) and rice (Oryza sativa) rely on plant age to build up sufficient energy requirements in order to transition to flowering through epigenetic action of miR172 . In monocot geophytes (defined as herbaceous perennial plants with underground storage organs, e.g. bulbs, corms, tubers, etc., that promote winter survival), such as Gladiolus, Lilium, Tulipa, Narcissus and Crocus, the flowering pathway is poorly understood. Factors of plant growth influencing flowering in commercial geophytes for commercial production are well known  and include photoperiod, light intensity, the autonomous pathway, gibberellins, ambient and cool temperatures (vernalization) . A clearly delineated genetic pathway for monocots (ornamental and otherwise) is still in the early stages of the discovery and characterization for many floricultural crops, such as gladiolus. In contrast, the Arabidopsis model is readily applicable to temperate dicotyledon plants  and may be only partially useful for monocots. Only a few flowering genes have been discovered in monocot ornamental geophytes , such as FT-like in Allium cepa [20–21], FT in Narcissus , NLF in Narcissus , LFY in Allium sativum [24–25] and in Lilium . Recently, many flowering genes have been discovered in Lilium ×formolongi (FT, CO-like, AP2, GA1, SOC1) and/or proposed for L. formosanum (VER1, VER2) [27–28]. The discovery of flowering genes in geophytes serve as valuable resources to model flowering pathway(s) therein.
Geophytes such as Gladiolus, Lilium, Tulipa, Narcissus and Crocus are floricultural crops with ornamental value wherein flowering is essential to maintain the marketing value for these crops. Gladiolus ×hybridus Rodigas, commonly known as gladiolus(-i), is commercially cultivated as a cut flower and as garden or landscape plants. Gladioli are geophytic plants with underground modified stem structures known as corms, producing cormels as a means of vegetative propagation . Flower initiation and development are crucial steps for its success as a cut flower. Therefore, understanding the flowering pathway is vital for genetics and breeding to improve the floral market value.
Gladiolus has a genome size of 1100 Mbp, although it is unclear whether this is for haploid or diploid and the species is unknown . The genome weight for gladiolus was recently measured in G. communis as 0.67-0.68 pg for monoploid G.s. (1Cx, pg) and in G. italicus at 0.61 pg for monoploid G.s. (1Cx, pg) . The limited knowledge of the gladiolus genome is also reflected in lack of knowing gladiolus flowering genes, there are no flowering genes discovered in gladiolus except for the gibberellin receptor gene GID1a in gladiolus . However, the relationship of GID1a with flowering has not been established. In Arabidopsis, gibberellin binds to the gibberellin receptor forming GID1 complex that binds to DELLA, causing its degradation, thereby enabling SOC1 and LFY to be upregulated, leading to flowering [32–33].
Understanding the flowering pathway and gene expression is important for efficient selective breeding of gladiolus for rapid generation cycling (RGC) or early flowering types that flower in ≤1 year from seed . An important flowering gene is FLC, a major flowering repressor found in Arabidopsis and many dicot species; FLC plays vital role in the control of flower initiation . Gladiolus is a monocot genus (Iridaceae) with both summer and winter flowering species; FLC has not been identified herein. It had been hypothesized that there is no FLC gene in monocot species until the FLC homologue was discovered in some cereal crops, such as Triticum aestivum , Hordeum vulgare  and Brachypodium distachyon . These FLC homologue studies did not discover FRI genes, which upregulate FLC expression in Arabidopsis thaliana . A hypothesis to test would be that some monocots do not possess the flowering repressor FLC gene and rely on alternative gene(s) to acts as a repressor(s) miR172 through epigenetic in plant age-dependent of Zea and Oryza . Additionally it remains unknown whether there is FLC-dependent pathway in all monocots.
In Arabidopsis, FLC is located between two flanking genes, UPSTREAM OF FLOWERING LOCUS C (UFC; located 4.7 Kb upstream of FLC) and DOWNSTREAM OF FLOWERING LOCUS C (DFC; found 6.9 Kb downstream of FLC) . The UFC gene expression is repressed by vernalization, independent of FLC repression due to vernalization . Thus, both FLC and UFC are repressed by vernalization, yet are not dependent on each other for expression; the suppression is through chromatin modification in an epigenetic manner . The VRN1 gene is expressed with vernalization and acts as a floral integrator whereas UFC is repressed and required by VRN1 expression dependently . The potential role for UFC in flowering has yet to be discovered and it may not involve flowering at all since vernalization only represses UFC in seeds while DFC is repressed by vernalization of the plant . Insertion of the NPTII gene between the UFC and FLC region confirmed NPTII response to cold as the whole cluster region of FLC responded to cold . In the UFC protein of A. thaliana, a conserved domain, DUF966, has 92 amino acids, although its function is still unknown . This lack of knowledge in DUF966 function creates a challenge to identify the function of UFC protein. However, a recent study shows the role of UFC in A. thaliana, with the gene SOK2, which appears to have a role in embryogenesis, root initiation, growth and branching of the primary and lateral roots . The conserved domain DUF966 is reported to be present in the OsDSR gene family of Oryza sativa . The promoter for genes that contain DUF966 have a defense-stress response to pathogens, salicylic acid, jasmonate, drought or salinity . The ZmAuxRP1 gene which promotes the biosynthesis of indole-3-acetic acid (IAA) to increase resistance against pathogens in Zea mays . Thus, all the genes that contain DUF966 vary in function but all of them are triggered by environmental or stress stimuli to overcome an undesirable change influencing plant growth and development.
The FLX gene encodes a putative leucine zipper domain which is required for FRI-mediated activation of FLC in Arabidopsis  (Fig. 1). Up-regulating FLC occurs in winter annual Arabidopsis , while late flowering phenotypes exhibit strong expression of FLX which indicates a role of FLX in the suppression of flowering . Several genes have been discovered in the FLX gene family, e.g. FLX-LIKE1 (FLL1), FLX-LIKE2 (FLL2), FLX-LIKE3 (FLL3), FLX-LIKE4 (FLL4) [11, 33]. FLX and FLL4 are the most crucial genes in flowering time control in Arabidopsis .
In order to test whether FLC is present in gladiolus, the adjacent gene (UFC) will also be probed, along with FLX which is part of the FLC-dependent mechanism. Therefore, the objective of this study is to identify whether UFC and FLX genes occur in the genetically diverse gladiolus germplasm of the University of Minnesota Gladiolus Breeding Program. The null hypotheses tested are: Ho1 = There is no difference among gladiolus genotypes in the existence of the UFC gene; Ho2 = There is no difference among gladiolus genotypes in the presence of FLX.