Development of simple sequence repeat (SSR) markers from a genome survey of Chinese bayberry (Myrica rubra)

Abstract

BackgroundChinese bayberry (Myrica rubra Sieb. and Zucc.) is a subtropical evergreen tree originating in China. It has been cultivated in southern China for several thousand years, and annual production has reached 1.1 million tons. The taste and high level of health promoting characters identified in the fruit in recent years has stimulated its extension in China and introduction to Australia. A limited number of co-dominant markers have been developed and applied in genetic diversity and identity studies. Here we report, for the first time, a survey of whole genome shotgun data to develop a large number of simple sequence repeat (SSR) markers to analyse the genetic diversity of the common cultivated Chinese bayberry and the relationship with three other Myrica species.ResultsThe whole genome shotgun survey of Chinese bayberry produced 9.01Gb of sequence data, about 26x coverage of the estimated genome size of 323 Mb. The genome sequences were highly heterozygous, but with little duplication. From the initial assembled scaffold covering 255 Mb sequence data, 28,602 SSRs (≥5 repeats) were identified. Dinucleotide was the most common repeat motif with a frequency of 84.73%, followed by 13.78% trinucleotide, 1.34% tetranucleotide, 0.12% pentanucleotide and 0.04% hexanucleotide. From 600 primer pairs, 186 polymorphic SSRs were developed. Of these, 158 were used to screen 29 Chinese bayberry accessions and three other Myrica species: 91.14%, 89.87% and 46.84% SSRs could be used in Myrica adenophora, Myrica nana and Myrica cerifera, respectively. The UPGMA dendrogram tree showed that cultivated Myrica rubra is closely related to Myrica adenophora and Myrica nana, originating in southwest China, and very distantly related to Myrica cerifera, originating in America. These markers can be used in the construction of a linkage map and for genetic diversity studies in Myrica species.ConclusionMyrica rubra has a small genome of about 323 Mb with a high level of heterozygosity. A large number of SSRs were identified, and 158 polymorphic SSR markers developed, 91% of which can be transferred to other Myrica species.

Development of 107 SSR markers from whole genome shotgun sequences of Chinese bayberry (Myrica rubra) and their application in seedling identification

“…In this study, the SSRs were considered to contain motifs with two nucleotides and a minimum of eight contiguous repeat units. Based on MISA results, sequences already present in the public database were removed (Jiao et al, 2012). Primer3 v2.23 (http:// primer3.sourceforge.net) was used to design the primer pairs.…”

“…PCR was carried out as described previously by Jiao et al (2012). The 20 μl reaction mixtures contained 10× PCR buffer (plus Mg 2+ ), 0.2 mmol/L of each deoxyribonucleoside triphosphate (dNTP), 5 pmol of each reverse, 4 pmol of tail primer, 1 pmol of the forward primer, 0.5 U of recombinant Taq (rTaq) polymerase (TaKaRa, Dalian, China), and a 20-ng genomic DNA template.…”

“…Nowadays, it is much easier to develop SSR markers from genome or transcriptome databases (Jiao et al, 2012;Yue et al, 2014). They have been routinely used in the construction of molecular linkage maps, and in genetic diversity analysis, marker-assisted selection, fingerprinting and cultivar identification in fruit trees such as apple (Celton et al, 2009) and peach (Testolin et al, 2000).…”

“…Thirteen polymorphic microsatellite loci have been developed from a genomic library in M. rubra (Terakawa et al, 2006), and eleven from a library of expressed sequence tags (ESTs) (Zhang et al, 2009a). With the development of next-generation sequencing technology, a fast and cost-effective approach to develop SSR markers from a whole genome survey has been reported (Jiao et al, 2012). One hundred and nine EST-SSRs developed from the transcriptome have been published (Zhang et al, 2012).…”

Application of Molecular Tools in Animal Breeding, Crop Science, Food Control and Agrobiodiversity in the Republic of Macedonia

“…The main types of "linked" molecular markers are DNA microsatellites or simple sequence repeats (SSR), and single nucleotide polymorphisms (SNP) [3]. The reproducibility, co-dominance, relative abundance and complete genome coverage of SSR markers have made them one of the most useful tools for detecting genetic diversity, genetic linkage mapping, association mapping and evolution analysis [7].…”