The genetic diversity analysis of citrus accessions based on molecular markers as revealed in this study could provide fundamental information in directing citrus breeding strategy and improvement. Present study compared the effectiveness of SCoT and SSR markers in identifying alleles and gene diversity in 53 citrus accessions. The use of SCoT markers could identify more alleles (137) than SSR markers (107). Additionally, SCoT markers could detect alleles with a higher maximum size of 2050 bp, while SSR markers could only detect alleles with a maximum size of 488 bp. Previous studies from Han et al. (2011) and Mahjbi et al. (2019), have also shown that SCoT markers can detect alleles with even larger sizes than those observed in this study. However, the SSR markers used in this study showed a greater average PIC value than SCoT markers. PIC value is a useful tool in determining the effectiveness of polymorphic loci in distinguishing genetic diversity among the genotypes (Ikten et al. 2023). Both SSR and SCoT markers used in this study showed an average PIC value of greater than 0.5, indicating their potential as highly informative markers in citrus breeding programs (Eltaher et al. 2018). Overall, the study suggests that genetic variation analysisi using a combination of SCoT and SSR markers can be useful in breeding and improving citrus fruit related traits.
SSR and SCoT markers target a different portions of the plant genome. SSR markers target a flanking region of repeat sequence that highly abundant in plant genome, while SCoT markers target a flanking region of START codon (ATG) that highly conserved in related plant genes (Zhang et al. 2015; Jian et al. 2021). SSR is a codominant marker and can be used to distinguished between heterozygotes and homozygotes genotypes (Jian et al. 2021). This makes SSR markers are useful for highly heterozygous plant such as citrus. On the other hand, SCoT markers are dominant markers, and they cannot distinguish heterozygote genotypes. However, converting SCoT markers to codominant SCAR markers can solve this problem (Rai 2023). Both SSR and SCoT markers are highly polymorphic and reproducible, making them suitable for genetic diversity analysis, fingerprinting construction, sex determination, and the construction of linkage or assosiation maps (Vierira et al. 2016; Rai 2023).
All of the SSR and SCoT markers utilized in this study demonstrated their robustness in identifying the genetic relationship from citrus accessions. The phylogenetic tree and PCoA plot clearly distinguished tangerine, mandarin, and outgroup citrus accessions. The SSR markers revealed a separation between tangerine and mandarin, while SCoT markers showed a mixture between mandarin and tangerine accessions. Based on SCoT markers, eleven mandarin accessions were grouped together with tangerine as presented in Figs. 7 and 8. In accordance to this study, Martasari et al. (2023) previously reported about the mixing among mandarin and tangerine accessions in the similar cluster based on ISSR and SSR markers. This study also found that both SSR and SCoT markers could distinguished the accessions with similar name, between Nipis (C. aurantifolia) accession that collected from local market in Bogor, West Java with Nipis Borneo (C. aurantifolia var. Borneo) accession that originated from Kalimantan island. The phylogenetic tree from SSR analysis also showed a separation from Kalamansi FR (C. microcarpa) with outgroup cluster, in line with previous study from Wu et al. (2018) that showed a separation of calamondin (C. microcarpa) from orange, lemon, pummelos, and lime group based on chloroplast genome. However, the study found that neither the SSR nor SCoT markers could separate orange jasmine (M. paniculata) accession from outgroup cluster. Orange jasmine is an ornamental plant commonly grown in home gardens. In contrast to our study, Penjor et al. (2013) and Nagano et al. (2018) reported separation of orange jasmine from true citrus cluster consisted of C. reticulata, C. maxima, and C. medica by using matK gene and RAD-seq respectively.
The SSR and SCoT markers used in this study could also classified two accessions, namely Sinta Ponsoe and Proksi-1 Agrihorti, derived from breeding activities using mandarin and tangerine as their parentals. Sinta Ponsoe was created by hybridizing Siam Pontianak (tangerine group) and Keprok SoE (mandarin group) and it was clustered with the tangerine group in the phylogenetic tree and PCoA plot. Proksi-1 Agrihorti, on the other hand, was formed from protoplast fusion between Siam Madu (tangerine group) and Satsuma mandarin (mandarin group) and was classified as an outgroup accession. In the phylogenetic tree and PCoA plot, this accession clustered in the mandarin group. In contrast to those results, SSR and SCoT markers used in this study showed a different robustness in ponkam accession clustering, of which SSR markers classified ponkam in a mandarin group along with Proksi-1 Agrihorti and Japansche citroen (C. limonia), while SCoT markers classified it in an outgroup with sunkist valencia Egypt (C. sinensis), lemon (C. limon), and nipis (C. aurantifolia). According to Velasco and Licciardello (2014), ponkam is a hybrid derived from hybridization between mandarin (C. reticulata) and pummelo (C. maxima). In this case, SSR markers showed a better robustness in distinguishing citrus accessions used in this study.
Furthermore, SSR and SCoT markers yielded different results in Nei’s genetic distance (1983). The genetic distance between tangerine and outgroup accessions was found to be close (0.570) using SSR markers, while SCoT markers revealed a closer genetic relationship between tangerine and mandarin accessions (0.206). According to Nicolosi et al. (2000), citrus taxonomy and phylogeny are still a very complex, disputable, and confusing problem, because of their cross compatibility between citrus species and related genera, high rate of bud mutations, the long history of cultivation, and wide dispersion. The taxonomy of citrus has gone through numerous system. The citrus taxonomy proposed by Scora (1975) and Barrett and Rhodes (1976) revealed that there were only three species constituted citrus ancestral, namely citron (C. medica L.), mandarin (C. reticulata Blanco), and pummelo (C. grandis (L.) Osb.). The hybridization among these three species inherited other genotypes including commercial citrus found today. Previous molecular analysis using RAPD and SCAR markers reported by Nicolosi et al. (2000) successfully clustered eight citrus group consisting of citron, mandarin, pummelo, ichang, fortunella, and micrantha clusters. The mandarin cluster consisted of all mandarin (C. reticulata) and mandarin-like accessions such as C. tachibana, C. paradisi, C. aurantium, C. sinensis, C. junos, including C. nobilis (tangerine citrus). The result obtained from this study and Martasari et al. (2023) support the previous study by Nicolosi et al. (2000) that tangerine citrus is the member of mandarin group.
The AMOVA from both SSR and SCoT markers showed that there was a higher variance within population than among population. This suggests that cross-pollination occured mostly in the citrus accessions used in this study. However, the results are contrary to those found by Barbhuiya et al. (2015) who discovered a higher variance among populations than within populations in C. medica populations from the Eastern Himalayan. This different of result could be due to the clonal propagation system by grafting, which may have increased the homogenity level at those populations.
Citrus plants have unique reproduction systems. Some genotypes, such as Valencia orange, are capable of self-pollination, while other like mandarin and mandarin-hybrid require cross-pollination (Halder et al. 2019). Since, citrus pollen is heavy and sticky, it cannot be carried by wind, making the presence of insect pollinators crucial for pollination (Chacoff and Aizen 2007). However, whether insect pollinators play a significant rolein citrus pollination still a matter of debate. Certain genotypes, such as tangelos and tangerines, are known to require pollinator for better fruit sets (Halder et al. 2019). Interestingly, citrus plants can also reproduce clonally through apomixis, a natural process that result in a polyembryony phenomenon (Zhang et al., 2018), or artificially by human through grafting (Warschefsky et al. 2016). This leads to high heterozigosity in citrus species, which makes their taxonomy and phylogeny challenging to determine.
The analysis of population structure from SSR markers demonstrated that the highest delta K value was obtained when K value was equal 2, indicating that all citrus accessions used in this study were best classified into two subpopulations. This result showed that tangerine and outgroup were classified in the same subpopulations, whereas the mandarin group was separated. Among these, 13 mandarin showed mixing color, which indicated gene flow between populations, leading to genetic recombination. On the other hand, SCoT markers showed that the highest delta K value was achieved when K was equal to3, indicating that three subpopulations were best classified for all of citrus accessions used in this study. The first subpopulation consisted of a mix of all Tangerine and ten Mandarin accessions. The second subpopulation consisted of 18 mandarin accessions including Proksi-1 Agrihorti and Japansche citroen from outgroup, while the third subpopulations consited of 15 outgroup accessions. According to this result, two tangerine accessions, namely Siam Pontianak and Siam Gunung Omeh, and also four Mandarin accessions namely Keprok Brasitepu, Keprok Rimau Gerga Lebong, Keprok Grabag, and Keprok Terigas showing mixing color representing their recombination with outgroup citrus accessions. The population structure obtained in this study corroborated with with AMOVA results, which showed that citrus accessions mostly underwent cross-polinaton between species and related genera, resulting in their heterogenity.
Mandarin citrus is believed to have originated from the east coast of China and then spread to Formosa Island (now Taiwan) and Japan (Krueger and Navarro 2007). Many mandarin landraces and wild mandarins have been found in China, supporting the hypothesis that China is the center of origin of this species (Li et al., 2007). However, based on genomic analysis, proposed by Wu et al. (2018) revealed that the Southeast Himalaya region, which includes the Eastern part of Assam, Northern Myanmar, and Western China, are the center of origin of citrus species. The process by which mandarin and tangerine citrus were disseminated to Southeast Asia, especially to Indonesia, still needs to be clarified. In contrast to mandarin, tangerine (C. nobilis) is thought to have originated from a hybrid between mandarin (C. reticulata) and sweet orange (C. sinensis) (Krueger and Navarro 2007). This finding is supported by the population structure from SCoT analysis, which showed an admixture between mandarin and tangerine accessions in one subpopulation. The mandarin and tangerine accessions used in this study are probably came from a similar ancestral or shared partial ancestral. This hypothesis is supported by the results of the phylogenetic tree, both from SSR and SCoT analysis, which showed that the clustering of tangerine and mandarin accessions did not depend on geographical origins, but rather on their genetic background. This suggests that similarities still exist between tangerine or mandarin accessions, even if they originated from different islands and have been separated geographically for an extended period.
Citrus genetic relationships are closely related to the dissemination pattern of citrus mother trees and seedlings, which has occurred for a long time, according to Agisimanto et al. (2007). Three locations in East Java Province, such as Tlekung-Batu, Purworejo, and Tulungagung have become the source of mother trees and scions for both distribution and production centers of citrus plants in Indonesia. Both tangerine and mandarin accessions used in this study might have undergone genetic improvement from their origin, either by hybridization, spontaneous mutations, adaptation to their geographical conditions, or human domestication, creating their new genetic diversity.
The information on genetic diversity obtained from this study could be highly useful in managing, utilizing, and breeding citrus germplasm. It could also help in resolving the complexity of their taxonomy and phylogeny. The SSR and SCoT markers applied in present study have shown a high level of polymorphism and reproducibility, thus they are recommended as promising tools to be used for future genetic diversity analyses of citrus species. Performing a combination analysis of data obtained from molecular markers with those from morphological and biochemical markers in future studies will lead to gain more comprehensive results.