Citrus is a genus of flowering trees and shrubs nested in the Rutacea family, which includes our common oranges, lemons, and other citrus fruits. Although we frequently rely on citrus fruits for nutrition, very little is known about them. Even with our advancement in sequencing technology, Citrus phylogeny remain relatively unresolved and understudied.
Most citrus fruits are products of hybridization that are readily able to hybridize with one another (Ramadugu, 2013). Additionally, many ancient and modern civilizations have inconsistent propagation methods for citrus fruits, which makes studying lineage and taxonomy even more difficult. However, through comparisons of SNPs on chromosome 2, four clusters of ancestor were characterized (Curk, 2014). These ancestor species are Mandarin (C. reticulata), Pomelo (C. maxima), Citron (C. medica), and Papeda (C. micrantha). The hybrid cross between these four ancestors is what gave rise to most citrus fruits today. Many citrus hybrid crosses were documented by cultivators (Figure 1), but some were lost overtime and unknown to modern taxonomist.
The goal of this project is to gain a preliminary understanding of Citrus phylogeny and identify potential challenges in the field. In this project, eight samples of commonly available citrus fruits are characterized in the internal transcribed spacer 1 (ITS1) region.
Materials and Methods
Eight citrus samples were collected from the local grocery store.(Figure 2) DNA was extracted from the citrus rind using the FastDNA SPIN Kit for Soil. The ITS1 locus was amplified from sample DNA in 20 Ál PCRs with p5 forward and u2 reverse universal primers. PCR product were visualized on a 1% agarose gel. PCR products were purified then sequenced by Genewiz. Sequence alignment and phylogenetic tree were generated using Clustal Omega. In addition, to these samples, eight sequences from Citrus ancestors published on GenBank were added for analysis.
The gel image of PCR products shows that all samples have expected band size. Unexpectedly, the Persian Lime (sample C) and the Lemon/Citron (sample D) samples have double banding (Figure 3) and the trace file of their sequence also have double sequencing (not shown). This might due to the fact they are hybrids organisms.
The resulting phylogenetic tree shows that sample C and D are closely clustered with the ancestor sequences of citron, which is expected since they are known hybrids of citron and papeda. Interestingly, sample F (Mandarin) did not cluster with the known ancestor sequences of mandarin, which would suggest sample F may not indeed be mandarin. (Figure 4) Although not much is known about the hybrid crosses of blood oranges, the tree suggests it is closely related to sweet orange and grapefruit.
Phylogenetic tree and PCR product gel are reflective of the difficulties in Citrus phylogeny. The double banding of sample C and D suggests multiple alleles and their genetic characteristic as hybrid. However, the double bands may also reflect the ploidy of the fruit. Most citrus fruits are diploid, but limes and lemons are known to be tetra or polypoid due to propagation practices (Curk, 2016). It is impossible to know without further experimentation with digital PCR or molecular cloning.
Secondly, the Citrus Industry are also actively mislabeling and falsely distributing citrus fruits. The given name Lemon/Citron (sample D) is not consistent with the known fact that citrons are ancestor species while lemons are hybrids. Different varieties of same species can be mislabeled or distributed as another. Mandarin sample did not cluster with the known sequences. According to literature, mandarins are often sold as tangerines if the variety is slightly more red or smaller (Krezdorn, 2014).
Study of citrus phylogeny is complicated by false data and lack of documentation. For instance, hybrid sequences such as sweet orange (C. sinensis) and lemon (C. limon) are being published as unique species sequences even though they are not unique species. In addition, citrus fruits like blood oranges are not well documented. It is known that blood oranges are hybrids but the exact hybrid crosses were lost overtime. Although blood oranges and sweet oranges are genotypically and phenotypically unique from one another, they are both categorized as C. sinensis.
Curk et al., 2014 Next generation haplotyping to decipher nuclear genomic interspecific admixture in Citrus species: analysis of chromosome 2. BMC Genetic (2014) 15:152
Tao et al., 2016 Barcoding the kingdom of Plantae: new PCR primers for ITS regions of plants with improved universality and specificity. Molecular Ecology resources (2016) 16, 138-149
Ramadugu et al., 2013 A Six Nuclear Gene Phylogeny of Citrus (Rutacea) Taking into Account Hybridization and Lineage Sorting. PLOS 8:1
Xu et al., 2013 The draft genome of sweet orange (Citrus sinensis). Nature Genetics 45:59-66
Curk et al., 2016 Phylogenetic origin of limes and lemons revealed by cytoplasmic and nuclear markers. Annals of Botany 117(4):565-583
Krezdorn, 2014 Classification of Citrus 1-6