Chinese bayberry (Morella rubra, former name Myrica rubra), the only species in Morella commercially cultivated for fresh fruit, is a subtropical evergreen tree native to China and other Asian countries (Chen et al., 2004; Jia et al., 2014; Zhang et al., 2009a). Chinese bayberry is planted as an important fruit crop in China and commercial production of this fruit is mainly restricted to southern China, with a history of over 2000 years, but also in Japan and recently Australia. The fruit ripens in early summer, an off season for the fresh fruit market in the northern hemisphere, with a characteristic flavor welcomed by consumers. The fruit is also rich in bioactive compounds like anthocyanins, which have multiple benefits for human health, such as scavenging of reactive oxygen species and prevention of cancers (Liu et al., 2013; Sun et al., 2013; Zhang et al., 2005, 2015a).
There are as many as 305 cultivars or lines reported for Chinese bayberry (Zhang and Miao 1999), but the diversity and phylogenetic relationship are far from being well studied compared with many other fruit species (Zhang et al., 2009a). In industry, the availability of a molecular identity for each elite cultivar is frequently required. For example, some accessions are quite close to each other and cannot be distinguished morphologically. This is quite common especially for white-fruited cultivars, also named Bai or Shuijing in Chinese, with ripe fruit accumulating little or no anthocyanins.
The genetic diversity of Chinese bayberry germplasm has been analyzed with different molecular markers, initially using random amplified polymorphic DNA (Lin et al., 1999; Xie et al., 2008), inter simple sequence repeat (Pan et al., 2008; Qian et al., 2006; Qiu et al., 2002; Xie et al., 2008), and amplified fragment length polymorphism (AFLP) (Zheng et al., 2006; Zhang et al., 2009a) as well as simple sequence repeat (SSR) (Jia et al., 2015; Jiao et al., 2012; Terakawa et al., 2006; Xie et al., 2011a, 2011b; Zhang et al., 2015b; Zhang et al., 2012; Zhang et al., 2009b) recently. Occasionally, some other molecular markers such as sequence-related amplified polymorphism (Lin et al., 2013), inter primer binding site, and start codon targeted polymorphism (Chen and Liu, 2014) are used. Compared with other molecular markers, SSRs have certain advantages such as high abundance, high reproducibility, codominant inheritance, and their multiallelic nature and have been widely applied in parentage analysis, hybrid identification, cultivar discrimination, and genetic diversity studies in various fruits (Heidi and Andrew, 2007). However, despite the availability of SSR markers from previous studies, the number of high-quality and highly polymorphic markers, with only weak or no PCR stutter bands, is still limited. PCR stutter bands are common for some SSR markers, especially those with a high number of dinucleotide repeats, which results from enzyme slippage during SSR-PCR amplification, severely affecting the size discrimination of PCR products (Guichoux et al., 2011; Hite et al., 1996).
With advances in sequencing technology, data from RNA-Seq can offer a multitude of SSR loci. Previously, we developed 109 EST-SSR markers based on 41,239 unigene sequences generated from the transcriptome data of Chinese bayberry (Feng et al., 2012; Zhang et al., 2012). In this study, 21 high-quality and highly polymorphic EST-SSR markers were screened out from these previously obtained 109 markers and 17 new ones were developed. With these markers, 42 Chinese bayberry accessions were discriminated and their diversity and phylogenetic relationships were analyzed.
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