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Abstract: chinese bayberry (Myrica rubra Sieb. et Zucc.) is one of the important subtropical fruit crops native to the South of China and Asian countries. In this study, 107 novel simple sequence repeat (SSR) molecular markers, a powerful tool for genetic diversity studies, cultivar identification, and linkage map construction, were developed and characterized from whole genome shotgun sequences. M13 tailing for forward primers was applied as a simple method in different situations. In total, 828 alleles across 45 accessions were detected, with an average of 8 alleles per locus. The number of effective alleles ranged from 1.22 to 10.41 with an average of 4.08. The polymorphic information content (PIC) varied from 0.13 to 0.89, with an average of 0.63. Moreover, these markers could also be amplified in their related species Myrica cerifera (syn. Morella cerifera) and Myrica adenophora. Seventy-eight SSR markers can be used to produce a genetic map of a cross between ‘Biqi’ and ‘Dongkui’. A neighbor-joining (NJ) tree was constructed to assess the genetic relationships among accessions, and the elite accessions ‘Y2010-70’, ‘Y2012-140’, and ‘Y2012-145’, were characterized as potential new genotypes for cultivation.

基于杨梅基因组鸟枪法测序开发的107个SSR标记及其在品系鉴定中应用

开发多态性高的基因组简单重复序列（SSR）标记，为杨梅遗传多样性、遗传图谱构建及品种鉴定相关研究奠定基础。 从杨梅全基因组鸟枪法测序数据中新开发了107个SSR标记，并应用于品种间遗传多样性分析及品系鉴定。 应用生物信息学软件从基因组测序数据中搜索SSR位点并设计相应引物，添加通用的M13尾巴和荧光标识筛选多态性高的SSR标记。 开发的107个基因组SSR标记可以广泛应用于杨梅种质资源和遗传育种研究，鉴定出三个杨梅新品系。
杨梅；简单序列重复（SSR）标记；遗传多样性

1.  Introduction

 Chinese bayberry (Myrica rubra Sieb. et Zucc.), a member of the Myricaceae, is native to China and neighboring Asian countries and has been cultivated in southern China for more than two thousand years (Chen et al., 2004). Of the six Myrica species in China, only M. rubra is commercially cultivated for fresh fruit and processed products, especially in Zhejiang Province, which has the best known cultivars ‘Biqi’ and ‘Dongkui’. In 2013, the cultivated area was over 340 000 ha, and the annual production reached 1.2 million tons. Studies have shown that the fruit is rich in anthocyanins, which have a wide range of pharmacological properties (Zhang et al., 2011) and can also be used in the food industry to replace synthetic dyes (Bao et al., 2005). The bayberry fruit is usually consumed fresh, but can also be canned, and is used in products such as juice, jam, and wine. For the fresh market, Chinese bayberry is bred for good shipping qualities, color, appearance, size, and flavor, while the processing market always needs cultivars to meet product specifications such as low drip loss, exceptional flavor, and high soluble solid content. Chinese bayberry is regarded as a highly nutrient fruit, and the adverse reaction to this fruit is rare (Wang et al., 2012).

 There are abundant germplasm resources of Chinese bayberry in China, and 305 accessions have been recorded with 268 named as cultivars (Zhang et al., 2009b). However, less than 50 cultivars have been planted as a pure line on a large scale, and only two cultivars, ‘Biqi’ and ‘Dongkui’, have been widely exported from Zhejiang to other provinces. There are many lines within a cultivar group in some local regions. New cultivar selections of Chinese bayberry largely depend on seedling identification and sporadic sport mutations of current cultivars (Xie et al., 2011). Fruit color is a straightforward trait to select: bright red is usually more attractive than black, and nowadays, light yellow and pink fruit also attract consumers’ attention. Recent initiatives to select elite accessions from the natural individuals within a cultivar group, with variable fruit maturity date, high and stable yield, larger fruit size and different flavor, cater to the needs of fresh consumption or processing. Using a molecular marker to identify the new breeding line is needed before regional testing of cultivars.

 Through the long cultivation history of Chinese bayberry, there has been no report on cross-breeding for cultivar improvement. We have previously published a report on pollen from a mutated branch of cultivars (usually female plants), which allow crossing between cultivars (Jiao et al., 2013). As a result of crossing ‘Biqi’בDongkui’ in 2011, we obtained offspring plantlets in 2013. Because fruit trees are large and take many years to produce fruit, the identification of molecular markers linked to the gender would confer tremendous advantages in the breeding and female genotype selection. The construction of a genetic linkage map of Chinese bayberry is a first step towards this long-term goal.

 Simple sequence repeat (SSR) markers, which are co-dominant markers, are ubiquitous in genomes of plant species, highly polymorphic and reproducible, and follow the Mendelian inheritance. 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). M. rubra is dioecious, resulting in individual plants being highly heterozygous. Consequently, the SSR marker system would be useful in the construction of genetic maps. 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). However, the number of markers was insufficient to construct a high density genetic linkage map for breeding purposes. In this paper, we identify additional polymorphic SSR markers from whole genome shotgun sequences in M. rubra and use these markers to analyze the genetic diversity of Chinese bayberry and to identify new cultivars. Our objective is to develop more SSRs from the genomic sequences to construct an SSR-based linkage map.

2.  Materials and methods

2.1.  Plant materials and DNA extraction

 A set of 45 accessions was used to test polymorphism in newly developed SSR markers. This set comprised 43 accessions of the cultivated species (M. rubra) and two related species (M. adenophora and M. cerifera) collected from different provinces in China (Table 1).




 Young leaves were collected and frozen in liquid nitrogen and total genomic DNA was extracted from 1 g frozen leaf tissue with a modified version of the cetyltrimethylammonium bromide (CTAB) procedure described by Zhang et al. (2009b). The DNA concentration was diluted to 20 ng/μl based on the quality and quantity assessed on 0.01 g/ml agarose gels using standard DNA markers (TaKaRa, Dalian, China).

2.2.  SSR mining and primer design

 The MISA program (MIcroSAtellite identification tool; http://pgrc.ipk-gatersleben.de/misa/misa.html) was employed for microsatellite mining. 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. The expected polymerase chain reaction (PCR) product size of the markers was set from 120 to 300 bp and the primer size from 18 to 22 bp, with the annealing temperature set at 60 °C. The M13 tail (sequence: TGTAAAACGACGGCCAGT) was added to the 5' end of each forward primer pair, with an annealing temperature of 53 °C. The tail was alternatively labeled with the following four dyes: NED (yellow), PET (red), FAM (blue), and HEX (green). Primers were synthesized by Invitrogen Trading Co., Ltd., Shanghai, China. Two cultivars (‘Biqi’ and ‘Dongkui’) and M. cerifera were selected to check the polymorphism of the 400 SSR markers, and those heterozygous markers either in ‘Biqi’ and/or ‘Dongkui’ were analyzed for polymorphism, using the 45 accessions as described above.

2.3.  PCR reaction

 PCR was carried out as described previously by Jiao et al. (2012). The 20 μl reaction mixtures contained 10× PCR buffer (plus Mg²⁺), 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. DNA amplification was in an Eppendorf Mastercycler (Germany) programmed at 94 °C for 5 min for initial denaturation, 32 cycles at 94 °C (30 s)/58 °C (30 s)/72 °C (30 s), and then 8 cycles of 94 °C (30 s)/53 °C (30 s)/72 °C (30 s), followed by a final extension at 72 °C for 10 min. Each PCR product was run on 0.01 g/ml agarose gel to check the quality. PCR products with four different colors were diluted, mixed with an internal size standard LIZ500 (Applied Biosystems), loaded on an ABI 3130 genetic analyzer and analyzed using GeneMapper Version 4.0 software (Applied Biosystems, Foster City, CA, USA).

2.4.  Data analysis

 The number of alleles (N _a), number of effective alleles (N _e), observed heterozygosity (H _o), and expected heterozygosity (H _e) were calculated using GenAlEx 6.4 (Peakall and Smouse, 2006). The polymorphic information content (PIC) and Chi-square test for Hardy-Weinberg equilibrium were calculated using PowerMarker Version 3.25 (Liu and Muse, 2005). Cluster analysis was conducted using the neighbor-joining algorithm as implemented in TREECON ver.1.3 b (van de Peer and de Wachter, 1994). Bootstraps were performed for 1000 iterations to test the reliability of the branches (Felsenstein, 1985).

2.5.  Fruit characteristic analysis

 The width and length of each fruit were measured using a digital vernier caliper. Fruit and the fruit core were weighed and the edible ratio estimated. A texture analyzer (TA-XT2i Plus, Stable Micro System Ltd., Surrey, UK) fitted with a 5.0 mm-diameter head was used for fruit firmness analysis. Total soluble solids (TSSs) and titratable acids (TAs) were measured as described by Zhang et al. (2005). TSSs of thirty fruits (two measurements per fruit) were determined with a digital refractometer (PR-101, Atago, Japan). For TA analysis, 1 g of fruit mesocarp tissue derived from a segment of the flesh was ground with 5 ml of distilled water. After filtration and centrifugation for 5 min at 12 000×g, the supernatant was brought to 10 ml with distilled water. After heating for 5 min at 100 °C to eliminate CO₂, the water was titrated with fresh 10 mmol/L NaOH to pH 8.2. The results were expressed in mmol H⁺/g fresh weight (FW). The samples for TA analysis were in triplicate.

3.  Results

3.1.  Character of SSR markers

 The effectiveness of 400 primer pairs was analyzed in two cultivars (‘Biqi’ and ‘Dongkui’) and M. cerifera, and 354 (88.5%) SSRs were amplified in ‘Biqi’ and ‘Dongkui’ with expected sizes of PCR products, and 296 (74%) in M. cerifera. One hundred and seven markers identified as heterozygous either in ‘Biqi’ or ‘Dongkui’ were used for genotyping the set of 45 accessions. These sequence data have been deposited in the GenBank Data Library under the accession numbers from KF914760 to KF914866, which are accessible. The 107 SSR forward and reverse primers and characteristics in 45 bayberry accessions are listed in Table S1. The PCR product size ranged from 115 to 330 bp, and the detected N _a from 3 to 17, with an average of 8 alleles. N _e ranged from 1.22 to 10.41, with an average of 4.08. The PIC at each locus ranged from 0.13 to 0.89 with an average of 0.63. H _o was between 0.04 (ZJU190) and 0.96 (ZJU258), while H _e was between 0.18 (ZJU215) and 0.90 (ZJU181). There was a significant deviation from the Hardy-Weinberg equilibrium (P<0.05) of 92% of the SSR primers (98 primer pairs). Partial correlation analysis showed highly positive correlations between the repeat unit length and PIC (P<0.01, r=0.3301). This study showed that these 107 SSR markers had high rates of transferability (96.2% and 88.8%, respectively) for two related species, M. adenophora and M. cerifera, suggesting that these markers could be used for genetic diversity analyses in other Myrica species (Table S1). For constructing the bayberry molecular marker linkage maps, we selected a total of 78 primer pairs showing heterozygous loci either in ‘Biqi’ or ‘Dongkui’, two parents of the F₁ population.

3.2.  Genetic relationship analysis

 A total of 107 SSR markers were used to evaluate genetic diversity and relatedness among the 45 accessions. The SSR markers clearly discriminated between M. rubra, M. adenophora, and M. cerifera, separating them into distinct clusters (Fig. 1). According to neighbor-joining cluster analysis, the M. rubra was divided into five groups. These were labeled as ‘A-1’, ‘A-2’, ‘A-3’, ‘A-4’, and ‘A-5’.

Fig.1
Dendrogram for 45 Chinese bayberry accessions derived from neighbor-joining cluster analysis based on 107 highly polymorphic SSR markers
Accessions: underlined, male (androphyte) plant; in italic, C2010-4, monoecious plant in normal, common female plants. The numbers are bootstrap values based on 1000 iterations. Only bootstrap values greater than 50 are indicated

 The first cluster, ‘A-1’, consisted of 12 accessions from Zhejiang Province and one from Fujian Province. The two androphytes ‘Y2012-1’ and ‘Y2012-2’ clustered into this group were close to cultivars of the same geographic origin. As reported previously by Jiao et al. (2012), the rare monoecious individual ‘C2010-4’ was closely related to a main local cultivar ‘Biqi’. ‘Zaoshenganmei’ from Fujian Province was closely related to the Zhejiang accession ‘Zaoqimimei’. Sixteen accessions were in the subgroup ‘A-2’, ‘Daji’ and ‘Dafudayexidi’, two from Jiangsu Province, and 14 from Zhejiang Province. The pink fruit accession ‘Y2012-140’ was close to ‘Fenhong’, while ‘Shuijing’ and ‘Y2012-145’ (both light yellow or white fruit type) were clearly separated in the cluster, with a short genetic distance. The elite accession ‘Y2010-70’, with red fruit, was also clustered in this subgroup. The accessions ‘Songmaoli’ and ‘Xianghong’ (both from Hangzhou) were close together. The two accessions ‘Dafudayexidi’ and ‘Daji’, from Jiangsu Province, were clustered together, demonstrating a relationship between genetic background and geographic origin. Subgroup ‘A-3’ included nine accessions, from ‘W2011-1’ to ‘C2010-55’. Androphyte ‘W2011-1’ was close to a cultivar from the same geographic origin. It is worth noting that accessions from Taiwan of China (‘Heiruilin’) and Japan (‘Ruiguangmei’) were clustered with those cultivars from Zhejiang Province (China), as has been described previously (Zhang et al., 2009b; Xie et al., 2011). Two accessions originating from Guangzhou were included in subgroup ‘A-4’, implying these accessions may have independent genetic backgrounds. Subgroup ‘A-5’ included three accessions, two from Hunan Province and one from Jiangxi Province, indicating that these accessions may share a common ancestor.

3.3.  Cultivar identification

 SSR markers are a powerful tool for identification of Chinese bayberry cultivars. The black fruit cultivar ‘Biqi’, originating from Zhejiang Province, has been introduced to other provinces 20 years ago, although its yield and commercial quality are constantly declined. In addition, the light yellow (white) and pink fruit types, such as ‘Shuijing’ and ‘Fenhong’, have demanded the highest price of Chinese bayberry in recent years, due to their appealing color and delicious taste. The elite accessions ‘Y2010-70’, ‘Y2012-140’, and ‘Y2012-145’ with black-red, pink, and light yellow fruit, respectively, have been studied for more than five years, and introduced to other cities of Zhejiang Province due to their good fruit quality (Table 2, Fig. 2).

Table 2

Main fruit characters of the identified three new accessions and three local cultivars

Variety	Diameter (mm)	Weight (g)	Firmness (N)	TSS (°Brix)	TA (mmol H+/g FW)
Y2012-145	31.63±0.47A	16.97±0.82A	2.73±0.05AB	11.04±0.09AB	0.24±0.00C
Shuijing	26.33±0.70CD	9.33±0.50D	2.34±0.22B	11.39±0.01A	0.23±0.01CD
Y2012-140	29.48±0.23AB	13.52±0.14B	2.36±0.12B	10.12±0.04B	0.21±0.01CD
Fenhong	25.82±0.15D	8.95±0.09D	3.19±0.06A	8.63±0.05C	0.28±0.01B
Y2010-70	28.59±0.23BC	12.61±0.06BC	2.56±0.07AB	11.03±0.07AB	0.36±0.01A
Biqi	26.84±0.81BC	10.72±0.08CD	2.34±0.09B	9.16±0.13C	0.19±0.01D

TSS: total soluble solids; TA: titratable acid; FW: fresh weight. Data are expressed as mean±SD (n=3). Values followed by different letters (A–D) in the same column are significantly different at P<0.01

Fig.2
Fruit size and color of three selections and three reference cultivars
Each grid cell represents one square centimeter

 Based on our data, we can demonstrate that ‘Y2010-70’, ‘Y2012-140’, and ‘Y2012-145’ are genetically different lines of Chinese bayberry. The red fruit of line ‘Y2010-70’ has high acidity and the strong typical Chinese bayberry aroma, making it suitable for processing.

4.  Discussion

 In our study, we aimed to develop additional SSR markers for M. rubra and used 45 accessions to evaluate the character of the markers. These markers can be generally used in genetic diversity analysis of cultivars, androphyte and related species. These markers also can be used in construction of SSR-based linkage map in Myrica.

4.1.  SSR polymorphism

 SSRs from genome and transcriptome sequences have been widely used for genetic diversity analysis and map construction (Emanuelli et al., 2013; Frascaroli et al., 2013; Würschum et al., 2013). In our study, the mean value of PIC (0.63) was less than that (0.67) previously reported for genomic-SSRs (Jiao et al., 2012), but significantly higher than the figure (0.44) for EST-SSRs (Zhang et al., 2012). Our results are in agreement with that reported in lettuce (Rauscher and Simko, 2013). Compared with EST-SSRs, genomic-SSRs have relatively high polymorphism and good genome coverage, so they can be used in constructing a linkage map. Among 107 polymorphic SSRs, 98 were AG/CT repeat type, the same main dinucleotide repeat motifs of Chinese bayberry in the genome and transcriptome (Zhang et al., 2012). The SSRs developed from M. rubra could also be used in related species M. cerifera and M. adenophora, showing a high transferability. M. cerifera is from the USA, M. adenophora is grown as wild trees and the fruit is used for medical purposes. The bayberry genome has a high level of heterozygosity (Jiao et al., 2012), also reflected by over 70% of the SSR loci being heterozygous for most accessions. The most homozygous (58/107 SSRs) accession was found to be ‘Y2012-145’. This selection may be used as material for the whole genome sequencing project of Chinese bayberry.

4.2.  Genetic relationship

 The lowest similarity coefficient was observed between the species M. cerifera and M. rubra, as reported previously using amplified fragment length polymorphisms (AFLPs) (Zhang et al., 2009b) and SSRs (Xie et al., 2011; Jiao et al., 2012). Accessions from the same geographical region were clustered more closely than those from different regions. Moreover, some accessions, originating from different provinces in China, clustered within the same subgroups, suggesting the gene flow among those provinces (Zhang et al., 2009b; Xie et al., 2011; Jiao et al., 2012).

 The Taiwan cultivar ‘Heiruilin’ clustered into subgroup A-3 and was most closely related to ‘Wenlinbenmei’ from Zhejiang Province as reported previously (Zhang et al., 2009b; Xie et al., 2011). This indicates that these cultivars may share a common ancestor, although this remains to be further clarified. ‘Ruiguangmei’ from Japan has a close relationship with the Zhejiang Province cultivar ‘Dongkui’. Our data, along with previous analysis (Zhang et al., 2009b; Xie et al., 2011), support the hypothesis that the Japanese cultivar ‘Ruiguangmei’ (Zuiko) was introduced from China (Handa and Kajiura, 1991).

 The two famous cultivars ‘Biqi’ and ‘Dongkui’, widely cultivated in most regions, separated into different subgroups (‘A-1’ and ‘A-3’), consistent with the previous studies using SSR markers (Xie et al., 2011; Jiao et al., 2012), while they have been clustered together in previous studies using AFLP (Zhang et al., 2009b). This inconsistency might be due to the different molecular markers used.

 Androphyte accessions of M. rubra were distributed in two different subgroups (A-1 and A-3), which were closely related to the gynophyte accessions from the same geographic origin. This result suggests that the androphyte and gynophyte share a common ancestry.

4.3.  Cultivar identification

 Molecular technology is a reliable method to identify cultivars of Chinese bayberry. ‘Yongxuan56’, with bigger fruit and better fruit quality, has been shown to be a new line of ‘Biqi’ based on AFLP analysis (Zheng et al., 2006). In the new Chinese bayberry lines and cultivars, the fruit color is mainly black and red, such as ‘Zaoqimimei’ (Qi et al., 2003) and ‘Zijing’ (Huang et al., 2013). However, no new cultivar with light yellow (white) or pink fruit color has been reported. ‘Y2010-70’, with high quality and an early-mature date, and ‘Y2012-140’, with pink fruit and good flavor, are two elite selections within the red-colored bayberry group, while ‘Y2012-145’, with larger fruit than ‘Shuijing’, is derived from the light yellow (or white) color group. Based on our SSR data and the characteristics of the fruit, the elite accessions ‘Y2010-70’, ‘Y2012-140’, and ‘Y2012-145’ can be demonstrated as new varieties of Chinese bayberry.

4.4.  Cross-breeding perspective in Myrica

 Cross-breeding is a traditional breeding method, and use of a hybrid (F₁) to construct a genetic linkage map is essential for marker-assisted breeding and selection (MAS). Identification of viable pollens from authorized cultivars makes cross-breeding possible. Studies have shown that pollen characteristics of different bayberry plants grown in various regions are quite dissimilar. As the ‘Dongkui’ pollen has a high fruit setting rate, with an average of 21.47% (Chai et al., 2012; Jiao et al., 2013), we carried out a cross-breeding program between ‘Biqi’בDongkui’ cultivars using occasional pollens from the ‘Dongkui’ cultivar (usually only female followers). Polymorphic SSRs that are heterozygosis in either of the parents can be used in linkage mapping of F₁ populations. In previous studies, the numbers of SSRs from whole genome shotgun sequences and EST heterozygosis in ‘Biqi’ and ‘Dongkui’ were given as 135 and 80, respectively (Jiao et al., 2012; Zhang et al., 2012). In this study, we developed 78 SSR markers, which can be applied in linkage mapping of the F₁ populations resulting from the cross-breeding between ‘Biqi’ and ‘Dongkui’. Therefore, around 300 SSR markers are available for the linkage mapping work till now.

5.  Conclusions

 Chinese bayberry genome sequences are a good resource for developing SSR markers. In our study, 107 SSR markers were successfully used to evaluate the genetic diversity and identify cultivars, providing a valuable genetic and genomic tool for further genetic research and varietal development in the Chinese bayberry, such as diversity studies, construction of a genetic map, and molecular breeding.

Acknowledgements

We acknowledge Dr. Qi-kang GAO from the Bio-Macromolecules Analysis Lab, Analysis Center of Agrobiology and Environmental Sciences of Zhejiang University, China for his assistance in the lab work.

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* Project supported by the Ningbo Science and Technology Bureau (No. 2012C10012), the Science and Technology Project of Zhejiang Province (No. 2012C12904), and the Yuyao Financial Bureau for Chinese Bayberry Breeding Program, China# Electronic supplementary materials: The online version of this article (http://dx.doi.org/10.1631/jzus.B1400051) contains supplementary materials, which are available to authorized usersCompliance with ethics guidelines Guo-yun WANG and Zhong-shan GAO received research grants from the Ningbo Science and Technology Bureau (No. 2012C10012) and the Yuyao Financial Bureau for Chinese Bayberry Breeding Program; Sen-miao LIANG received research grant from the Science and Technology Project of Zhejiang Province (No. 2012C12904). Hui-min JIA, Yu-tong SHEN, Yun JIAO, Xiao DONG, Hui-juan JIA, Fang DU, Chao-chao ZHOU, and Wei-hua MAO declare that they have no conflict of interest.

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