Chia (Salvia hispanica L.), which belongs to the Lamiaceae family, is an emerging industrial crop (Ayerza and Coates, 2011). The seeds of S. hispanica have been used as food since 3500 BC in Mexico (Sosa et al., 2016). This species was a cash crop in the centre of Mexico from 1500 to 900 BC. Chia was cultivated in the Central Valley of Mexico between 2000 and 2600 and was an important component of the Aztec diet (de Falco et al., 2017; Dincoglu and Yesildemir, 2019). Chia is a hardy annual plant and grows up to two meters high. This plant adapts to a wide range of soils, climates and minimal rainfall (Dincoglu and Yesildemir, 2019). It starts flowering at the age of 3 months. Flowers are blue in colour. The spikes grow to 10 cm long, set on terminal stems, and fill out to a seed head (Caruso et al., 2018). The seeds are pin-head sized, and shiny. Two months after flowering, the seeds can be harvested. Chia seeds contain soluble and insoluble fibre, high omega-3 content, proteins, minerals, vitamins, and phytochemicals, that have great potential as nutraceutical compounds, which benefit human health (Dincoglu and Yesildemir, 2019; Grancieri et al., 2019; Reyes-Caudillo et al., 2008). Chia seeds supply excellent ingredients to consider in the formulation of functional foods, such as bakery products, cereal bars (Munoz et al., 2013; Sargi et al., 2013). The United States Department of Agriculture (USDA) has promoted the plantation of Chia as an industrial crop (Valdivia-López and Tecante, 2015). In USA, Mexico, Argentina, Australia, Bolivia, India and other countries, Chia is grown as a commercial crop and its seeds are available in supermarkets and health food shops (Dincoglu and Yesildemir, 2019). The annual yield of Chia seeds is around 100–1700 kg/ha/round (Ayerza, 2016; Grimes et al., 2018; Mack et al., 2018). Chia seeds are increasingly popular and are an important ingredient among consumers and manufacturers (Iglesias-Puig and Haros, 2013; Kuznetcova et al., 2020; Pizarro et al., 2013; Ribes et al., 2021; Zettel and Hitzmann, 2018). The production of Chia seeds has increased in recent years (Grancieri et al., 2019; Jamshidi et al., 2019). Through the centuries, humans have modified Chia, like all crops, by selective breeding. Early Mesoamerican breeders produced lines with well-developed agronomic characteristics including good, uniform seed yield and retention (Jamboonsri et al., 2012; Miranda-Ramos et al., 2020; Ullah et al., 2016). While most commercially available Chia is a mixture of different seeds, some companies offer seeds derived from a single cultivar, which they claim boosts the nutritional value and ensures consistency. However, the yield of Chia is low although conventional breeding has been conducted (Grimes et al., 2018). Further genetic improvement through molecular breeding is essential to make the production of Chia profitable and sustainable.
Polymorphic DNA markers are an essential tool in molecular breeding programs and genetic diversity studies (Nadeem et al., 2018; Tanksley, 1983). RAPD (Random Amplified Polymorphic DNA) (Welsh and McClelland, 1990) has been used in analysing genetic diversity in Chia populations. The study using RAPD found a near lack of diversity in modern commercial Chia varieties (Cahill, 2004). However, RAPD is laboratory dependent and is not highly reproducible (Wilkie et al., 1993). Thus, the RAPD results are difficult to interpret. Among all available molecular markers, microsatellites or simple sequence repeats (SSRs) have played an important role in plant breeding (Nadeem et al., 2018). Microsatellite sequences consist of tandemly repeated short motif with a length of two to six base pairs (bp) (Litt and Luty, 1989). Microsatellites are highly polymorphic, co-dominant and easy to genotype. They are randomly distributed across a genome of interest. They can be easily amplified with PCR using primers flanking the repeat DNA sequences (Guichoux et al., 2011; Yue and Xia, 2014). Genotyping of microsatellites can be conducted with automated DNA sequencers, which enables high-throughput analysis (Guichoux et al., 2011; Yue and Xia, 2014). Therefore, microsatellites are one of the most powerful DNA marker systems in analysing genetic diversity, population relationships in plant germplasm, and in genetic traceability of plant products (Kalia et al., 2011). However, there is currently no polymorphic and codominant DNA marker in Chia.
The purposes of this study were (1) to identify some microsatellites from publicly available DNA sequences in the Chia crop (Salvia hispanica), (2) to optimize PCR conditions of selected microsatellites, (3) to characterize 14 microsatellites using automated DNA sequencers and (4) to apply the developed microsatellites to analyse genetic diversity and population relationships in six Chia cultivars. The 14 microsatellites characterized are polymorphic and easily genotyped with PCR and an automated DNA sequencer. The novel information on genetic diversity and population relationships would facilitate the initiation of a molecular breeding program to increase the production of Salvia hispanica.