Genetic diversity and differentiation of South African cactus pear cultivars (Opuntia spp.) based on simple sequence repeat (SSR) markers

Tsholofelo Jastina Modise University of the Free State Bloemfontein Campus: University of the Free State Mathabatha Frank Maleka (  malekamf@ufs.ac.za ) University of the Free State Bloemfontein Campus: University of the Free State https://orcid.org/0000-0001-6748-5362 Herman Fouché University of the Free State Bloemfontein Campus: University of the Free State Gesine M. Coetzer University of the Free State Bloemfontein Campus: University of the Free State


Introduction
Cactus pear is a widely used and most economically valuable member of Cactaceae -a plant family that contains approximately 1,600 species of cacti (Wallace and Gibson 2002). The family is currently classed into four subfamilies, and thus, Maihuenioideae, Pereskioideae, Cactoideae and Opuntioideae (Simpson 2010). The genus Opuntia (subfamily Opuntioideae) comprises taxa that are predominantly used in the agriculture industry as livestock feed (Dubeux et al., 2021;Pastorelli et al., 2022;Sipango et al., 2022). However, humans also consume cactus pear fruits (also called 'prickly pear') and young cladodes as vegetables (Barba et al. 2022). Fruit is highly nutritious as it contains numerous bioactive compounds (including betalains, carotenoids, avonoids and phenols) along with proteins, minerals, vitamins, fatty acids, sterols, carbohydrates and bres (Daniloski et al. 2022). Apart from nutritional bene ts, cactus pear may further serve an important socio-economic role in the livelihood of human societies (Moshobane et al. 2022) and, as such, make it a crop of agricultural signi cance.
The genus Opuntia is highly diverse and comprises up to 250 different species (da Silva et al. 2022a). Members of Opuntia show different levels of ploidy, ranging from 2n = 2x to 2n = 8x (Segura et al. 2007). Polyploidy in plants has been construed as the main driver of diversity in addition to supporting geographic expansion, climatic niche exchange and lineage longevity ( (Snyman et al. 2007). The capacity to withstand severe environments has made Opuntia species some of the important production crops in the South African agriculture industry (de Wit and Fouché 2021).
Opuntia breeding over the years has produced many cultivars and South Africa hosts one of the largest germplasm collections in the world (Chapman et al. 2002). However, cultivar identi cation tends to be challenging when plants are in a vegetative state due to inadequate distinctive features (Felker et al. 2006). Therefore, novel methods are required for differentiating Opuntia cultivars and species too. Modern high-throughput sequencing techniques have facilitated the discovery of genome-wide DNA markers that can be used to characterise germplasm pools and assess genetic diversity therein. To this end, simple sequence repeats (SSRs) have been established as molecular markers of choice as a result of their abundance in plant genomes (Feng et  As molecular markers, SSRs are more informative than the biallelic single nucleotide polymorphism (SNP) markers as the former tend to have many alleles per locus (Hamblin et al. 2007). Further, being co-dominant means that SSRs are valuable over dominant molecular markers as they will be relatively variable and may easily differentiate between homozygous and heterozygous individuals (Csencsics et al. 2010;Dutta et al. 2011). In the past, SSRs have been used to assess the genetic diversity in many diploid (Wen et (Caruso et al. 2010) and even determine gene ow incidents among individuals of different populations or closely related species (Fava et al. 2020). Then, SSRs are ideal markers for discerning and assessing the diversity in species and cultivars that form the South African Opuntia germplasm.
In this study, we aimed to distinguish and evaluate the genetic diversity in cultivars that make up the South African Opuntia germplasm. This was done using SSR markers that were previously identi ed in the Opuntia cus-indica genome (Maleka et al., unpublished data). Similar to other genotypes all over the world (Caruso et al. 2010), some cultivars in the South African germplasm are phenotypically indistinguishable. Therefore, the current study may assist in genetically resolving the local germplasm in addition to enabling assessments of genetic diversity within breeding pools across South Africa.

Plant material and DNA extraction
In 2015, an orchard including 44 Opuntia cultivars was established at the University of the Free State in Bloemfontein, Free State Province, Republic of South Africa (29°6'27.08"S; 26° 11'32.90"E). The plants were propagated clonally from a 10-year-old evaluation trial that was planted at the Waterkloof farm, which is located roughly 30 km outside Bloemfontein. The Waterkloof orchard was established in 2005 using cladodes collected from other conservation sites located all over South Africa.
For the current study, a single ower was harvested from each of the 44 Opuntia cultivars (Table 1) and used for extracting genomic DNA. Speci cally, genomic DNA was extracted from 50-100 mg of liquid nitrogen-powdered samples following the NucleoSpin Plant II Kit (Macherey-Nagel, Düren, Germany). However, cell lysis was completed at 65°C for 30 minutes. DNA samples were quanti ed with a spectrophotometer (NanoDrop ND-1000, Thermo Fisher Scienti c, Waltham, USA), whereas DNA quality was analysed by electrophoresis via 1% (w/v) TAE agarose gels. Gels were stained with the GelRed Nucleic Acid Gel Stain (Biotium, Hayward, USA) and visualised under UV light using a G:Box Gel Documentation System (Syngene, Cambridge, UK). The nal extension was done at 72°C for 1 minute. All reactions were effected on an Applied Biosystems 2720 Thermal Cycler (Applied Biosystems, Foster City, CA, USA). Post-PCR, selected products were stained with GelRed (Biotium) and electrophoresed on 3% (w/v) TBE agarose gels that contained a 100-3,000 bp PCR DNA ladder (Ampliqon, Odense, Denmark). Once veri ed by gel visualisation (G:Box Gel Doc. System), amplicons were analysed by capillary electrophoresis on an ABI3500xl Genetic Analyzer (Applied Biosystems). The polymer POP-7 (Thermo Fisher Sci.) was applied as a separation matrix, while fragment sizes were determined with a GeneScan 500 LIZ Size Standard (Applied Biosystems) during these analyses.  Genotypic diversity was computed using various indices, and these included the Simpson's index (λ; Simpson, 1949), the Evenness index (E.5; Grünwald et al., 2003) and Nei's unbiased gene diversity (Hexp; Nei, 1978). The software poppr was also used to create a genotype accumulation curve, which shows the power of random loci to discern unique individuals. Linkage disequilibrium (LD) between pairs of loci was determined via the standardized index of association test (rbarD; Agapow and Burt, 2001 According to the poppr package, a total of 40 MLGs were expected (SE = 0.0) and realised based on the current data. The genotype accumulation curve, however, showed that ve loci were su cient to distinguish 90% of the 40 MLGs (Fig. 1). Each of the four remaining MLGs (10%) occurred twice in the samples (data not shown). The Simpson's index was high, and this indicated that most genotypes are unique (λ = 0.973), while the high Evenness score (E.5 = 0.959) suggested that the population has equally abundant genotypes. Genetic variation in Opuntia cultivars was assessed with Nei's unbiased genetic diversity index. To this end, locus L38909 emerged as the most diverse (Hexp = 0.66) in the study, followed by both L23031 (0.62) and L161092 (0.62). The least diverse polymorphic locus was L37320, with Hexp = 0.46 (Table 2). Across all loci, the Hexp index = 0.422 and this shows that the South African Opuntia germplasm harbours modest levels of genetic variation. Indices of the two LD tests deviated signi cantly from the null hypothesis of individuals in the population mating randomly (I A = 0.763, p-value = 0.02; rbarD = 0.173, p-value = 0.02). Hence, the null hypothesis is rejected and the alternative hypothesis (that the germplasm is asexual or clonal) is deemed to be valid. Four of the eight loci diverged from the HWE signi cantly (Table 2). Signi cant deviation from the HWE may be ascribed to population genetic effects including arti cial selection, non-panmixia or even random genetic drift.

Genetic relationships among Opuntia cultivars
Relationships among the 44 Opuntia cultivars were analysed via clustering (UPGMA) and ordination (PCoA) methods. Clustering analysis produced a dendrogram with three main clades (Fig. 2). Overall, cultivars in clade I tended to be genetically farther distant from each other than those within clades II and III. However, the two cultivars of O. robusta (Monterey and Robusta) in clade I were genetically closer to each other as expected. Cultivars that historically come from the same country, surprisingly, did not cluster together. Also, the three cultivars from Botswana (R1251, R1259 and R1260) occurred in each of the three clades, while those from Israel (Ofer, Messina and Sharsheret) grouped into clades II and III. Clades II and III each comprised two branched tips relating to the four shared MLGs de ned earlier. One of the divided tips showed Roedtan to be genetically related to Rossa, a cultivar of Italian origin (Fig. 2). As with the dendrogram, PCoA (Fig. 3) did not show any distinctive clustering, even when considering the commercial usage of cultivars as depicted in Table 1. Overall, the SSR loci in this study contain su cient polymorphisms that effectively segregated cultivars in the South African Opuntia germplasm.

Discussion
Members of the Cactaceae attract research interest globally because of their specialised physiological traits that allow survival in severe habitats (Nobel 2002 fragment length polymorphisms (AFLPs) as molecular markers to discern cultivars in the germplasm. Similarly, the current study sought to differentiate and measure the genetic diversity within the South African Opuntia germplasm using eight genomic SSR markers. Information on the relationships and genetic diversity in cultivars will assist in the selection of superior genotypes toward the development of new cultivars and preservation of cultivars as distinct genotypes. Overall, the studied SSR markers were able to distinguish 90% of the Opuntia cultivars, and evidently, the genotypes harbour moderate levels of genetic variability.
DNA extraction from cacti is infamously challenging due to the large quantity of mucilage, secondary metabolites and polysaccharides (Nobel et al. 1992). As such, several protocols have been developed and improved over the years to try overcoming this challenge ( Our study also sought to simplify DNA isolation from cactus pear by using small amounts of ower tissues because they are soft and amenable to crushing with minimal effort. While the amount of DNA extracted from ower tissues was below that obtained from cladodes using current protocols, the quality was, however, similar (Martínez-González et al. 2017). In retrospect, we suppose that the ower tissues were not crushed su ciently, and this impacted DNA recovery. Even so, our approach establishes owers as an alternative source of quality DNA for molecular studies in Opuntia. Tissue sampling, however, should be planned around the limited annual owering season.
To our knowledge, this is the rst study to describe estimates of genetic diversity in the South African Opuntia germplasm using SSRs. The The studied plants may, therefore, be adapting to the local environment -a notion that can only be con rmed by analysing the genetic diversity in Opuntia cultivars grown at other sites in South Africa. Overall, our ndings a rm the South African Opuntia germplasm as one of the diverse and key breeding pools of cactus pear in the world. kiwifruit (Liao et al. 2019). The South African Opuntia germplasm, in essence, appears to not only support the ex-situ conservation of cactus pear, but also holds the genetic capacity for breeding novel cultivars that will bene t the agriculture industry.

Conclusions
South Africa hosts one of the major germplasms of Opuntia cus-indica in the world. The germplasm understandably derives from the Burbank collection that was established using material sourced from all over the world. Consequently, the diverse history and possibly adaptation to the local environment over many years may have in uenced the moderate genetic variation found in South African cultivars. Genetic variability in the South African Opuntia cultivars means that the germplasm may be used for breeding new cultivars for the agriculture industry. Alternatively, the cultivars may serve as a source population for deriving new alleles that can rescue less diverse Opuntia breeding programmes around the world. Last, the SSR markers used in this study will be useful toward distinguishing and verifying unknown samples used by farmers and researchers all over South Africa. Figure 2 A UPGMA tree demonstrating the relationships among the 44 cultivars of Opuntia cus-indica and O. robusta(Robusta and Monterey