Open Access

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

  • Yun Jiao1,
  • Hui-min Jia1,
  • Xiong-wei Li1,
  • Ming-liang Chai1,
  • Hui-juan Jia1,
  • Zhe Chen2,
  • Guo-yun Wang3,
  • Chun-yan Chai4,
  • Eric van de Weg5 and
  • Zhong-shan Gao1Email author
BMC Genomics201213:201

DOI: 10.1186/1471-2164-13-201

Received: 4 January 2012

Accepted: 3 April 2012

Published: 23 May 2012

Abstract

Background

Chinese 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.

Results

The 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.

Conclusion

Myrica 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.

Background

Chinese bayberry is an important commercial horticultural crop. It has been cultivated for more than 7,000 years in southern China, but is little known elsewhere. The production area is currently 340,000 ha, with an annual production of 1.1 million tons. The plant is diploid (2n = 16), generally dioecious, with the female plants cultivated for fruit[1], growing well on poor soils due to the association of nitrogen-fixing bacteria with the root system. It is rich in anthocyanins exhibiting a wide range of pharmacological properties, such as anti-inflammatory, antitumor and antioxidative effects[2].

There are four species within the genus Myrica in China, namely Myrica rubra Sieb. & Zucc., M. esculenta Buch.-Ham., M. nana Cheval., and M. adenophora Hance. M. rubra is widely distributed, with many local cultivars in the Zhejiang, Jiangsu, Fujian and Guangdong provinces and a few from Guizhou, Yunnan and Hunan provinces. The best known cultivars are Biqi and Dongkui, both from the Zhejiang province. Although there are abundant germplasm resources, studies on genetics and breeding of the species are still in their infancy. Molecular marker technology is a popular tool for breeding and genetic research, and with the construction of a genomic library, 13 polymorphic microsatellite loci have been developed in M. rubra[3] and 11 from an expressed sequence tag library[4]. Recently, 12 primer pairs have been temporarily developed by ISSR-suppression PCR[5] with GSG (GT)6 as the primer for enriching microsatellite sequences. Reports on the genetic diversity in Chinese bayberry using SSR markers have also recently been published[6, 7], but the number of markers for Chinese bayberry is limited.

The reproducibility, multiallelicism, co-dominance, relative abundance and good genome coverage of SSR markers have made them one of the most useful tools for genetic diversity and linkage mapping. Genomic SSRs and EST-SSRs, considered complementary to plant genome mapping, have been reported in many fruit crops, such as walnut[8], cherry[9], apricot[10] and coconut[11]. EST-SSRs are useful for genetic analysis, but their relatively low polymorphism and the high possibility of no gene-rich regions in the genome are limitations to their use. In contrast, genomic SSRs are highly polymorphic and tend to be widely distributed throughout the genome, resulting in better map coverage[12].

With genetic maps serving as the basis for future positional gene cloning, making map-based cloning and marker-assisted selection possible, the development of more SSRs is essential. As sequencing technologies advance, whole-genome shotgun (WGS) sequences are becoming increasingly available. These DNA sequences are valuable resources for SSR development in many plant species, such as rice[13] and papaya[14]. In addition, they can be used to evaluate the frequency and distribution of different types of SSRs in the genome, and even help to estimate genome size and characters such as heterozygosis and repeats.

As a way of reducing the cost of genotyping research, Schuelke[15] proposed a method for fluorescent dye labelling of PCR fragments with a sequence-specific forward primer: the universal fluorescent-labelled M13(-21) primer, at the 5 end, acts as the forward primer in a ‘one-tube’ reaction. As this method allows for high-throughput genetic analyses, with a high number of microsatellite markers widely used, we considered the possibility of using this approach for multiplex PCR, to improve the efficiency and save costs.

In this study, we mined and validated 158 SSR markers and describe their application for understanding the genetic relationship among 29 Chinese bayberry accessions and other Myrica species. These markers are useful for genotyping and genetic diversity analysis and linkage mapping of Myrica rubra and other Myrica species.

Results

Genome survey using whole genome shotgun data in Chinese bayberry

WGS generated 273,161 (>100 bp) high quality sequence reads from two DNA libraries (250 bp and 500 bp) of the androphyte individual ‘C2010-55’. We used 9.01 G raw data for K-mer analysis and heterozygous simulation. For the 17-mer frequency distribution (Figure1), the peak of the depth distribution was about 22. The estimated genome size was 323 Mb, using the formula: genome size = k-mer count/peak of the kmer distribution. The minor peak at 1/2 altitude of the main peak indicates the high level of heterozygosity in this genome (Figure1). A total of 739,969 contigs were assembled with a total sequence length of 255.7 Mb. The length of N50 was 295 bp in our assembly, and the longest contig and scaffold 7,593 and 127,008 bp, respectively.
https://static-content.springer.com/image/art%3A10.1186%2F1471-2164-13-201/MediaObjects/12864_2012_Article_4370_Fig1_HTML.jpg
Figure 1

The distribution of 17-mer depth analysis based on whole genome shotgun data in Chinese bayberry.

Frequency distribution of different types of SSR markers

A total of 17,172 out of 273,161 scaffolds (6%) retrieved from the genome survey sequence contained 28,602 SSRs (Table1), of which 5,401 contained more than one SSR, and 1,444 SSRs were present in compound format. Among the derived SSR repeats, the di-nucleotide was the most abundant repeat, accounting for 84.72% of total SSRs, followed by tri- (13.78%), tetra- (1.34%), penta- (0.12%), and hexa- (0.04%) nucleotides (Table1). There was a large proportion of both dinucleotides and trinucleotides while the rest amounted to less 2%. The average frequency of occurrence was about 10.47% (Table1).
Table 1

Occurrence of SSRs in the Genome Survey of Chinese bayberry

Type

Number

Proportion in all SSRs (%)

Frequency (%)

Dinucleotide

24,233

84.72%

8.87%

Trinucleotide

3,941

13.78%

1.44%

Tetranucleotide

383

1.34%

0.14%

Pentanucleotide

35

0.12%

0.013%

Hexanucleotide

10

0.04%

0.004%

Total

28,602

100%

10.47%

The SSR frequency of each motif is presented in Additional file1. The SSR motif consists of 69 types. Among the repeat motifs of the dinucleotide, the AG/CT repeat was the most common, representing 53.72%, followed by 39.20% AT repeats (Figure2), and the predominant motifs of trinucleotide (AAG/CTT and AAT/ATT) repeats accounted for 37.15% and 32.56%, respectively (Figure3).
https://static-content.springer.com/image/art%3A10.1186%2F1471-2164-13-201/MediaObjects/12864_2012_Article_4370_Fig2_HTML.jpg
Figure 2

Percentage of different motifs in dinucleotide repeats in Chinese bayberry genome.

https://static-content.springer.com/image/art%3A10.1186%2F1471-2164-13-201/MediaObjects/12864_2012_Article_4370_Fig3_HTML.jpg
Figure 3

Percentage of different motifs in trinucleotide repeats in Chinese bayberry genome.

Polymorphism of SSR markers

We first designed and synthesised 600 SSR primer pairs from those scaffolds more than 2Kb long. The majority of SSR loci were dinucleotide repeats (597, 99.5%), and the remainder trinucleotide. Initially, the effectiveness of these primer pairs was detected in two cultivars (Biqi and Dongkui) and M. cerifera through denaturing PAGE (Polyacrylamide gel electrophoresis), and 581 (96.8%) of these were amplified successfully in Biqi and Dongkui, and 400 (66.7%) in M. cerifera. The SSR loci (186, 31%) were identified as heterozygous loci either in Biqi or in Dongkui. Subsequently, they were used to screen 32 accessions, and detected an average of 8.25 alleles and from 3 to 15 alleles per locus (Table2).
Table 2

Characteristics of 158 SSR markers in this study

Locus

GenBank

Repeat motif

Primer sequence (5'-3')

Size range(bp)a

Na

Ho

He

PIC

P HW

 

Accession

        

ZJU001ab

JQ318696

(GA)10

F:<NED > <Tail-1 > CCTCTCCACCCATGAGAAAC

160-188

7

0.1667

0.4271

0.4002

0.0000

R:CAAATCATTCCCTGCTTTCC

ZJU002ac

JQ318697

(TC)13

F:<NED > <Tail-1 > TCAAAGAGACGTTGTGGCAG

219-229

4

0.2083

0.5257

0.4572

0.0005

R:TCCGCTCACAGACAGAGAGA

ZJU003ab

JQ318698

(AG)11

F:<NED > <Tail-1 > GTCACCTTGCTCTTCTTGGC

203-217

8

0.7407

0.8344

0.7949

0.0003

R:TCCTTGTACTTGTTCTGCTGGA

ZJU004ac

JQ318699

(GA)10

F:<NED > <Tail-1 > AACAGAACCATCGTCAAGGC

204-210

4

0.3571

0.7325

0.6704

0.0003

R:GGTACAGTCGCTCCGGTTTA

ZJU005ab

JQ318700

(AG)14

F:<NED > <Tail-1 > CTTTGGACATGGCAACACAC

200-228

11

0.3000

0.8679

0.8291

0.0000

R:TCCACTTTGACAGATTCCCA

ZJU006ab

JQ318701

(GA)10

F:<NED > <Tail-1 > CTCGCCCTCTCTCTCTACCA

193-205

5

0.2593

0.3305

0.3089

0.0000

R:AGTTTATCCACCCGTGTCGT

ZJU007ab

JQ318702

(AG)13

F:<NED > <Tail-1 > TGATCCATTGGAACCATGTG

193-209

8

0.5625

0.6617

0.6302

0.1868

R:TCAGTTGATGGTGCAGAAGC

ZJU008ab

JQ318703

(CT)10

F:<NED > <Tail-1 > GGAGAAATGAACGGTGGAGA

191-215

10

0.7931

0.7973

0.7563

0.0002

R:TCCAAAGCTAATACCCACGC

ZJU009ab

JQ318704

(CT)10

F:<NED > <Tail-1 > AATTGTCGCAAGTAGTCGCC

207-221

5

0.0741

0.3599

0.3371

0.0000

R:ATATCAACCCATGGGAGCAA

ZJU010ab

JQ318705

(CT)11

F:<NED > <Tail-1 > TGCAACATCGAAATTGGAAA

181-205

9

0.9032

0.8012

0.7614

0.0000

R:ATGCCGGCAAGTCTTAGTGT

ZJU011a

JQ318706

(GA)10

F:<NED > <Tail-1 > GGAGGCTCGTCAGTCATCTC

200-216

9

0.2692

0.7926

0.7554

0.0000

R:TTAGCGTCCCTTCTCTCTCG

ZJU012ab

JQ318707

(CT)12

F:<NED > <Tail-1 > CTTCACTCACCGCCTTTCTC

184-218

13

0.5000

0.8571

0.8251

0.0000

R:AATGGCCTCCACATCTCAAG

ZJU013ab

JQ318708

(CT)10

F:<NED > <Tail-1 > ACTTGTCATTCCCACGTTCC

211-221

6

0.4444

0.5199

0.4515

0.0094

R:CACTCCATCTCAACCACCCT

ZJU014ab

JQ318709

(AG)15

F:<NED > <Tail-1 > TGGAATGTCGATCTGAAACAA

186-212

13

0.6875

0.9033

0.8791

0.0251

R:ACCAGCTTATACGACGGTGG

ZJU015ab

JQ318710

(GA)11

F:<NED > <Tail-1 > TTGGTGTGGTGGTAATGGTG

199-221

6

0.6154

0.6614

0.5902

0.0585

R:AAATAATGCAAGCAGGTGGG

ZJU016ab

JQ318711

(TC)10

F:<NED > <Tail-1 > CCGTTGACTATTGCCCAGTT

196-216

11

0.6333

0.8469

0.8130

0.0179

R:GGCAATTTCCAAATCGCTAA

ZJU017ab

JQ318712

(CT)13

F:<NED > <Tail-1 > ACTGAAGAACCAAACGTGGG

180-200

6

0.6250

0.7093

0.6518

0.0003

R:GGTGTGTTTCTCTGTGTGCG

ZJU018ab

JQ318713

(CT)15

F:<NED > <Tail-1 > ACGAAATTTGACCAATCGCT

196-216

7

0.1429

0.7189

0.6667

0.0000

R:AGGGTTTCTTCTGGTTCGGT

ZJU019ab

JQ318714

(GA)12

F:<NED > <Tail-1 > TTTCATAACCCGTTGGCTTC

209-219

6

0.2800

0.6865

0.6317

0.0000

R:AAGGTGGAAACGTGTCAAGG

ZJU020b

JQ318715

(AG)10

F:<NED > <Tail-1 > CACAGGACATGTGATGGAGG

201-213

7

0.5172

0.7453

0.6983

0.0000

R:CCATCCTGAGCTTTGTCGAT

ZJU021a

JQ318716

(TG)10

F:<NED > <Tail-1 > TCGCCAGCTTCCTAATGTCT

190-212

8

0.7778

0.7428

0.7025

0.0663

R:GAGCGCATGTTGTTGCTAAA

ZJU022ab

JQ318717

(GA)10

F:<NED > <Tail-1 > AAGCTTAAGCAAGCGTCGAG

188-208

9

0.6923

0.8575

0.8227

0.0109

R:TGCGAAGGGAAATTTCAGAC

ZJU023ac

JQ318718

(AG)15

F:<NED > <Tail-1 > GTGTTTGGGCAGCACCTATT

200-226

14

0.6667

0.8840

0.8544

0.0251

R:AAAGAGTACAACAACGCGGG

ZJU024ab

JQ318719

(TC)10

F:<NED > <Tail-1 > CCGCATGTTTGATTGATGTC

180-196

6

0.6000

0.7345

0.6716

0.1624

R:GCGTTGAGCGGAGAGATTAC

ZJU025ab

JQ318720

(TC)10

F:<NED > <Tail-1 > TTTGAGCGATAGTACGGAGG

216-234

8

0.2667

0.7537

0.7044

0.0000

R:ATATGCTACGTTGGTTGCCC

ZJU026ab

JQ318721

(TC)10

F:<NED > <Tail-1 > CCAGACAGGTTAGCCACCAT

200-220

10

0.4545

0.8573

0.8199

0.0000

R:GCCTCTGGATCTCGATTACG

ZJU027

JQ318722

(TTC)8

F:<NED > <Tail-1 > GTTGCAATTTGCCTCCATTT

203-227

6

0.3125

0.6250

0.5321

0.0003

R:GGTGCCTATACTGCCAGCTC

ZJU028ab

JQ318723

(AG)10

F:<NED > <Tail-1 > CAACCATCCAAACCAAATCC

164-170

4

0.1724

0.2789

0.2566

0.0000

R:TCTACCAATCGTGGCTAGGG

ZJU029ab

JQ318724

(AG)10

F:<NED > <Tail-1 > TCTTCCGGGATGTCTACAGG

189-205

6

0.5312

0.6925

0.6296

0.0480

R:CAACAGCAATCGCAAAGAAA

ZJU030ab

JQ318725

(CA)13

F:<NED > <Tail-1 > AAGTGAGCTCTCCCTCCCTC

193-205

7

0.4286

0.7208

0.6676

0.0000

R:CACCGAAATACTTGCCGTTT

ZJU031ab

JQ318726

(GA)16

F:<NED > <Tail-1 > GCACAGGAACACCAGGATCT

179-195

8

0.8387

0.7948

0.7492

0.0000

R:CCAAGCCCTAATTCCCTTTC

ZJU032ab

JQ318727

(TC)11

F:<NED > <Tail-1 > ATTCCCACGTTCGTTCAGAC

204-226

8

0.6786

0.6442

0.5852

0.0220

R:GATGCCTAACTCCGAATCCA

ZJU033ab

JQ318728

(TC)10

F:<NED > <Tail-1 > GCACAAGTTGCTGACATGCT

195-207

6

0.0690

0.6655

0.5897

0.0000

R:AGTTGCATTCAACCCACACA

ZJU034ab

JQ318729

(CT)10

F:<NED > <Tail-1 > ATGGGAATGTGGAGAACGAG

191-209

8

0.4138

0.7762

0.7250

0.0000

R:GCTTTGCTTCTTTGCTTTGG

ZJU035ab

JQ318730

(GA)14

F:<NED > <Tail-1 > TTGGATCCTGGTTACCTTCG

201-217

8

0.1290

0.7425

0.6900

0.0000

R:AAACTGCATGCATGGTTCCT

ZJU036ab

JQ318731

(GA)10

F:<NED > <Tail-1 > CTGCCACTCTTACTGGCCTC

186-214

8

0.3333

0.5895

0.5516

0.0000

R:ATGTGCCCAATCTTGACTCC

ZJU037ab

JQ318732

(TC)10

F:<NED > <Tail-1 > GTGATTTCCCTCCCATTGAC

208-228

9

0.8125

0.7867

0.7429

0.0135

R:ACGAAGCGGGAAGTAGGATT

ZJU038b

JQ318733

(AG)10

F:<NED > <Tail-1 > CTTATGGCCCGTTTGTAACC

194-200

4

0.2273

0.5106

0.4646

0.0007

R:AACGATTGCTTTAAGCGGAA

ZJU039a

JQ318734

(CT)10

F:<NED > <Tail-1 > AAACGAAAGTGGGCGTATTG

219-229

6

0.3077

0.6161

0.5745

0.0004

R:CACCAGTGCGTCCTATGAGA

ZJU040

JQ318735

(TC)16

F:<NED > <Tail-1 > AAACTCCGTGCTGGAATCAA

198-220

10

0.3182

0.8192

0.7798

0.0000

R:GCAGACAAGCCTTCCTGTTC

ZJU041ab

JQ318736

(TC)11

F:<PET > <Tail-2 > TGATCACCTTTCAGTTGGCA

226-244

5

0.2258

0.3199

0.3031

0.0000

R:CACATTGGCAGAGTCCTGAA

ZJU042ab

JQ318737

(TC)10

F:<PET > <Tail-2 > AGGATTTCTCCAGAGGGACG

220-242

5

0.3571

0.5331

0.4880

0.0000

R:GGTTCCGCATCAAACTACAAA

ZJU043b

JQ318738

(CT)10

F:<PET > <Tail-2 > AAACCGAGCTCTCCTAAGCC

225-245

4

0.5714

0.6383

0.5667

0.2655

R:CTCGCAATTTCTCGGGATAC

ZJU044ab

JQ318739

(GA)12

F:<PET > <Tail-2 > GATGGTGGCTTGTCTTGGTT

235-255

8

0.2500

0.5091

0.4853

0.0000

R:AAGTGGGACGTCAATTCCTG

ZJU045ab

JQ318740

(CT)10

F:<PET > <Tail-2 > GAGAGAGGGAGAGAGGCCAT

228-258

13

0.6129

0.8821

0.8544

0.0007

R:GGAAGATTCATGGGAGAGGG

ZJU046ab

JQ318741

(AG)10

F:<PET > <Tail-2 > TTGCTGTAAGCATCGCAATC

226-242

7

0.3871

0.6256

0.5824

0.0000

R:AAGCTCCGGTAACACACACC

ZJU047ab

JQ318742

(GA)13

F:<PET > <Tail-2 > TTCGATCATTCATGAGGCTG

247-259

7

0.7097

0.7615

0.7074

0.0019

R:TTAATTGCATCCCGGATTTC

ZJU048ab

JQ318743

(CT)14

F:<PET > <Tail-2 > AGCGGACCGAGTTGTAGAGA

230-254

12

0.2903

0.8493

0.8166

0.0000

R:CCAACCCTACAAAGCGAGAG

ZJU049ab

JQ318744

(GAA)8

F:<PET > <Tail-2 > GTGTCTGCAGCAACTTCCAC

234-267

10

0.8125

0.7262

0.6797

0.0000

R:GTCGGAACCGAAGATGGTTA

ZJU050ab

JQ318745

(AG)11

F:<PET > <Tail-2 > GTCACAGCCTGGATAGCTCC

233-245

7

0.3000

0.7288

0.6916

0.0000

R:GTCTCTCCTGGATGAGCTGC

ZJU051ab

JQ318746

(AG)12

F:<PET > <Tail-2 > AGAGAAAGACCGGGACCAAT

229-233

3

0.4333

0.4198

0.3594

0.0012

R:GAGAAATAAAGCCGAGCGTG

ZJU052ab

JQ318747

(AG)16

F:<PET > <Tail-2 > CCCGAGCTGAACGAAATAGA

230-248

9

0.4348

0.8628

0.8261

0.0000

R:GGATCAAAGCGTTGTCGTTT

ZJU053ab

JQ318748

(AG)10

F:<PET > <Tail-2 > AAATCCGAAACACCTCTCCC

222-240

8

0.5000

0.5655

0.5211

0.0001

R:ATGTGGAGACTTCCACTGGG

ZJU054ab

JQ318749

(CT)13

F:<PET > <Tail-2 > TTGATTTGCTTTGTGCATTTG

232-250

9

0.3000

0.8667

0.8268

0.0003

R:CAAACTACCGTGCCCAACAT

ZJU055ab

JQ318750

(CT)10

F:<PET > <Tail-2 > TTATGGGTTTCATTGGGCAG

238-254

6

0.2500

0.7006

0.6357

0.0000

R:TCACCAGGCTACTGCATGTC

ZJU056ab

JQ318751

(GA)13

F:<PET > <Tail-2 > GACAAAGTGGGTGCCATTCT

230-246

7

0.5714

0.7643

0.7122

0.0068

R:TGCATGCTTCCTTTCTTCCT

ZJU057ab

JQ318752

(CT)10

F:<PET > <Tail-2 > TCATGTGGAGATTGAAGCCA

230-244

6

0.1579

0.6814

0.6283

0.0000

R:CGTCCCGAATGAAGATTTGT

ZJU058ab

JQ318753

(GT)10

F:<PET > <Tail-2 > TCCGGAGCTTTCAATCTCAT

252-274

11

0.7500

0.8274

0.7900

0.8036

R:GCCTACGAACTCAGGTTCCA

ZJU059b

JQ318754

(TC)14

F:<PET > <Tail-2 > TGTTTGTTTCTTGCTATTTCCATC

217-235

7

0.5200

0.7935

0.7505

0.0016

R:GACAGTTCCCACCAGCATTT

ZJU060ab

JQ318755

(GT)8(GA)9

F:<PET > <Tail-2 > TGGCCAGGAACTTTGTATCC

223-243

7

0.6562

0.8110

0.7691

0.0000

R:GAAAGATTGGGAATGCTGGA

ZJU061ab

JQ318756

(TC)11

F:<PET > <Tail-2 > TTTGGAGGAAGCAAACAAGC

204-232

11

0.2812

0.7922

0.7506

0.0000

R:TCCTGCGCCAACAATCTAAT

ZJU062

JQ318757

(TC)10

F:<PET > <Tail-2 > GTCGAGAGAACAAAGCGACC

240-252

7

0.2400

0.3282

0.3135

0.0004

R:GTCCAATGCCGCACTAACTT

ZJU063ab

JQ318758

(TC)12

F:<PET > <Tail-2 > ACTCAGCAGGACCACCAACT

232-250

10

0.7000

0.8593

0.8270

0.1320

R:TTAGACGGAAATTGGGCTTG

ZJU064b

JQ318759

(GA)10

F:<PET > <Tail-2 > ACCATGAAGCTGACCTGGAG

226-244

6

0.4348

0.7256

0.6666

0.0001

R:TTTCGTGGTCCCACCTACTC

ZJU065ac

JQ318760

(CA)13

F:<PET > <Tail-2 > TCCAGAATATCATCTCTTGCCA

214-236

9

0.6333

0.7706

0.7219

0.0001

R: ATATTCCTAACGTGTGCGGG

ZJU066ab

JQ318761

(GA)10

F:<PET > <Tail-2 > CTTTCCCTTGTCGCTTTCAG

221-235

8

0.2593

0.6450

0.6075

0.0000

R:GGTCGCAGATCAGGTCAAGT

ZJU067ab

JQ318762

(CT)10

F:<PET > <Tail-2 > CAGACAGCGAGGAGACAACA

217-263

11

0.6923

0.8273

0.7861

0.0070

R:GGTCTTTCGAACTTTGCTCG

ZJU068ab

JQ318763

(CT)10

F:<PET > <Tail-2 > GAAGCTAAACGCCAGAAACG

227-239

6

0.2917

0.7535

0.6913

0.0000

R:ACTCCTCACACGAATGGGTC

ZJU069bc

JQ318764

(GA)10

F:<PET > <Tail-2 > TGCCATAATCCTGAGAGCCT

224-258

8

0.2609

0.5594

0.5235

0.0004

R:TGTTCTGTAATGGCGTGGAA

ZJU070ab

JQ318765

(CT)11

F:<PET > <Tail-2 > GTGCTCGAGATGTCCTCCAT

221-247

7

0.5200

0.7861

0.7364

0.0000

R:ACAATCCCATCGCATACAGG

ZJU071ab

JQ318766

(GA)10

F:<PET > <Tail-2 > CTAAGGTTGGTCCCTGTCCA

228-234

3

0.3704

0.6157

0.5305

0.0110

R:CTTGTGTGGTGATGGTTTGG

ZJU072ab

JQ318767

(AG)10

F:<PET > <Tail-2 > AGTCAGCGTGGGAATGTACC

223-237

7

0.5625

0.7604

0.7117

0.0000

R:TTTCAGAACAAGTTCGTCGC

ZJU073a

JQ318768

(AG)12

F:<PET > <Tail-2 > TACGCCAAGATCCAAAGACC

222-242

7

0.2105

0.7568

0.7087

0.0000

R:TCTCGAGTTGAGTTTGGGCT

ZJU074ab

JQ318769

(CT)15

F:<PET > <Tail-2 > TGCAGAGGAACTGGTGACTG

215-239

10

0.5517

0.8234

0.7831

0.0007

R:GAGAAGGCTCAGTGGGTGAG

ZJU075b

JQ318770

(CT)17

F:<PET > <Tail-2 > AATAAACACACAGGGCGAGG

239-255

9

0.0769

0.8650

0.8307

0.0000

R:ATCGGGCAGACCAGAATATG

ZJU076ab

JQ318771

(AG)9

F:<FAM > <Tail-3 > ATGGTTACCGACGTCCTCTG

131-169

11

0.8438

0.8353

0.8034

0.0000

R:AGTGCAGAGTGCGAGATCAA

ZJU077ab

JQ318772

(AC)9

F:<FAM > <Tail-3 > TTTGGAATTCAACAACATTTAGAC

137-153

8

0.2000

0.6590

0.6079

0.0000

R:TGCAGCCTTGCTCCTCTTAT

ZJU078ab

JQ318773

(TC)10

F:<FAM > <Tail-3 > ACACCACGGTTCTTCGATTC

130-146

6

0.5500

0.7513

0.6881

0.1339

R:GTAACGAGGCTCTTGCTTGC

ZJU079ab

JQ318774

(TC)13

F:<FAM > <Tail-3 > AAGGCTAGACCGCAATCTGA

116-134

9

0.8438

0.8596

0.8291

0.0008

R:GGGCAACAGTTTACTTCCCA

ZJU080ab

JQ318775

(CT)9

F:<FAM > <Tail-3 > CTTGACGAAATGCAGACGAA

124-134

5

0.2903

0.3411

0.3172

0.0103

R:TCCGGATCAGGGTCAAATAG

ZJU081ab

JQ318776

(GA)8

F:<FAM > <Tail-3 > TGCTCTTGCAGAGAGTCGAG

137-157

6

0.5517

0.5820

0.5379

0.0003

R:TCATAATACCCTTGGCAAACA

ZJU082ab

JQ318777

(CT)10

F:<FAM > <Tail-3 > TATATCGAATCCCAAAGGCG

129-141

5

0.3438

0.4043

0.3792

0.0169

R:AAGATATTGGTCCGGCTCCT

ZJU083ab

JQ318778

(AG)10

F:<FAM > <Tail-3 > TAGCCTTGGAGATTTAGGGC

133-157

11

0.8667

0.8960

0.8692

0.0000

R:TTGAAATTTCGCAGCCTCTT

ZJU084ab

JQ318779

(AG)9

F:<FAM > <Tail-3 > TTTCGATTGGTGGTCTGTGA

124-138

6

0.1379

0.5197

0.4766

0.0000

R:TTATTAACTTCACTTTGTTTATTCGG

ZJU085ab

JQ318780

(AG)9

F:<FAM > <Tail-3 > GCTTTAACCGAGTGATGGGA

150-184

8

0.6875

0.5992

0.5383

0.6352

R:TAAAGGAGCGCTGGAAAGAA

ZJU086ab

JQ318781

(TC)10

F:<FAM > <Tail-3 > TCCTCTCTTTCACACTTCCGA

118-152

13

0.9062

0.8720

0.8445

0.0005

R:GGTCGATCATTTCTCTCCCA

ZJU087ab

JQ318782

(GA)9

F:<FAM > <Tail-3 > CGAGTGTAGCTAGGAACGGC

135-149

8

0.4688

0.7748

0.7273

0.0204

R:AATTGGACCTGCAAATCTCG

ZJU088ab

JQ318783

(CT)9

F:<FAM > <Tail-3 > GAGCTCCGAACTTCTTCCCT

126-150

13

0.9677

0.8773

0.8490

0.0053

R:CTTCTCCACAGGACTCTGCC

ZJU089ab

JQ318784

(GA)8

F:<FAM > <Tail-3 > CGTTAGGATTCGGGAACAGA

138-152

7

0.8065

0.7382

0.6778

0.0000

R:CAGGGCTAATGTGGACCAGT

ZJU090ab

JQ318785

(AG)9

F:<FAM > <Tail-3 > GGAAATCTCCGAATGTGATCC

118-134

8

0.2903

0.6642

0.6089

0.0000

R:TGGTGGATGAACCACTCAAA

ZJU091bc

JQ318786

(TC)15

F:<FAM > <Tail-3 > AAAGAGCACACAGCCCTAGC

124-146

10

0.4615

0.8695

0.8358

0.0012

R:GGCAGTGTCGCAGTGATAGA

ZJU092ab

JQ318787

(TG)10

F:<FAM > <Tail-3 > CTCTTGCCGACCTCATTGTT

127-151

11

0.6875

0.8264

0.7916

0.0041

R:CGGGACTCGCATAAATCACT

ZJU093ab

JQ318788

(GA)10

F:<FAM > <Tail-3 > ATGCCATGTTGCATGAGTGT

130-156

12

0.9355

0.8662

0.8367

0.3689

R:TATCCCGTAAGCAATCAGGG

ZJU094ab

JQ318789

(CT)10

F:<FAM > <Tail-3 > ATCACGGGTTCTGCTGTTCT

124-150

10

0.9062

0.8646

0.8332

0.0000

R:CAGAAGAAGCCATTTCTGCC

ZJU095ab

JQ318790

(AG)9

F:<FAM > <Tail-3 > TACCCACCGTACCAAAGGTC

114-130

7

0.4839

0.7070

0.6420

0.0004

R:GAATGAACCCAGGCGATAGA

ZJU096ab

JQ318791

(CT)10

F:<FAM > <Tail-3 > CATACTGCAATGCATCTCCC

126-154

13

0.8000

0.8757

0.8479

0.0310

R:TCAATTTGTGTGCCCTTACG

ZJU097ab

JQ318792

(AG)10

F:<FAM > <Tail-3 > AATTGTTAGGGAGGGCTCGT

118-134

8

0.8438

0.7778

0.7297

0.0009

R:TGCGTTGTGGAGACCATTTA

ZJU098ab

JQ318793

(CT)9

F:<FAM > <Tail-3 > GACGCTCCATCTCTGGTCTC

145-167

10

0.9355

0.8831

0.8549

0.0483

R:CCCAAACCGCACTAGAGAAA

ZJU099ab

JQ318794

(GA)10

F:<FAM > <Tail-3 > TTGTTGCACTTGTGGGTGAT

130-150

9

0.7742

0.7763

0.7299

0.0000

R:AACTACAAACAGCCCAACCG

ZJU100ab

JQ318795

(TC)9

F:<FAM > <Tail-3 > ACTTGTCCGGATTCCACAAC

128-154

5

0.8333

0.6316

0.5629

0.2930

R:TCAAGGCACACAATAATGCAA

ZJU101ab

JQ318796

(AG)9

F:<FAM > <Tail-3 > TGATTGAGCTGCCAACAAAG

134-154

7

0.6667

0.7062

0.6527

0.8110

R:TTTAACATTTGGCACCGACA

ZJU102ab

JQ318797

(GA)10

F:<FAM > <Tail-3 > GAACCACGAACTTCAACCGT

118-132

8

0.4231

0.5890

0.5441

0.0111

R:AACCACCAAACTTAGCTTCCA

ZJU103ab

JQ318798

(AG)9

F:<FAM > <Tail-3 > TGAGGAGGGAGTTGAGTTGG

121-139

10

0.7097

0.7731

0.7359

0.0003

R:GCGTCTTCCTCCTCCTTCTT

ZJU104ab

JQ318799

(TA)9

F:<FAM > <Tail-3 > ACGTGGCAGTTGAGTTGTTG

114-128

6

0.3704

0.6296

0.5702

0.1383

R:TCAGATCTCCGTTGGAGCTT

ZJU105ab

JQ318800

(GA)11

F:<FAM > <Tail-3 > TGAGAAACGCAGCAAGAGAA

135-157

11

0.5806

0.8165

0.7801

0.0000

R:CATCTCTCCCAAGCATCCTC

ZJU106ab

JQ318801

(GA)8

F:<FAM > <Tail-3 > GCAGTCGGATAGAGAGACGG

134-146

7

0.3636

0.7717

0.7203

0.0000

R:TGTTGATCAAACACACCGAGA

ZJU107ab

JQ318802

(TC)10

F:<FAM > <Tail-3 > TGGTGTCACGATTCACTGGT

114-130

8

0.4375

0.5322

0.5012

0.3632

R:CTGCATGTAATGGCAGTTCAA

ZJU108b

JQ318803

(CT)9

F:<FAM > <Tail-3 > TTGGTAGTGCACTGCAGGAG

132-160

13

0.3929

0.8253

0.7909

0.0000

R:CGAGGGTCGAGTTCAGAGAG

ZJU109ab

JQ318804

(TC)10

F:<FAM > <Tail-3 > TCCGCTCTCCTCTCTGTCTC

138-164

11

0.8000

0.8441

0.8082

0.0003

R:GTGAGTTGTGCTGCTGCAAT

ZJU110ab

JQ318805

(AG)9

F:<FAM > <Tail-3 > TTGCACGGTGGTAGCTGTAG

143-159

5

0.7667

0.6486

0.5844

0.0000

R:ACTGTGGTCCGTCGAACTCT

ZJU111ab

JQ318806

(TC)8

F:<FAM > <Tail-3 > TTTCTAATGTTGTTCGCCCA

122-136

5

0.9000

0.5701

0.4652

0.0000

R:TCATTCTCCTTGCAGATCCC

ZJU112ac

JQ318807

(GA)8

F:<FAM > <Tail-3 > GGAGAGTGAGAGATCGCAGC

133-147

8

0.4839

0.6557

0.6212

0.0009

R:GGCAACACCCTCAGTATCGT

ZJU113ab

JQ318808

(AG)9

F:<FAM > <Tail-3 > AAACGCACCAGAGAAAGACG

138-154

6

0.6667

0.6588

0.5987

0.0130

R:TCCATCTCTGGTCTCCATCC

ZJU114a

JQ318809

(GA)10

F:<FAM > <Tail-3 > CTAGAGCGCTCCACGATACC

132-160

12

0.8214

0.8740

0.8448

0.0388

R:AGAACGCTTGGAGAATCGAA

ZJU115ab

JQ318810

(AG)14

F:<FAM > <Tail-3 > GGTCTGAGGCCTTCACTCTG

126-156

14

0.9677

0.9022

0.8775

0.0068

R:GAGACCCAATAACCCATCCA

ZJU116ab

JQ318811

(AG)15

F:<FAM > <Tail-3 > CTTTCTCCGTCTGCTCCATC

110-136

13

0.6875

0.8199

0.7846

0.0001

R:GTCCAAACTTGGAGCCCATA

ZJU117ab

JQ318812

(AAG)9

F:<FAM > <Tail-3 > TCTCAGATCCCTCCACGTTC

118-133

6

0.4688

0.6944

0.6426

0.0000

R:CCACTGGATCAGGACAACCT

ZJU118ab

JQ318813

(CT)9

F:<FAM > <Tail-3 > CAAGCCACGTGCATACCTATT

120-144

11

0.8750

0.8502

0.8171

0.0001

R:CAGCTGGCTTCTAACTGCAA

ZJU119a

JQ318814

(AG)11

F:<FAM > <Tail-3 > CTTTCGACTTCAGAGGCAGC

136-152

9

0.4828

0.8348

0.7975

0.0000

R:TCCCTCTCAAACTTTGCCAC

ZJU120ab

JQ318815

(GA)8

F:<HEX > <Tail-4 > TTGGTTTCGTTTGCAAGTCA

164-180

6

0.9355

0.7012

0.6354

0.0073

R:GTCATCCATCCAATCCATCC

ZJU121a

JQ318816

(CT)11

F:<HEX > <Tail-4 > AATCACCGAAGAAATCCACG

164-186

11

0.8621

0.8705

0.8426

0.0000

R:ATTGCCCTCCCTTCTGTTCT

ZJU122ab

JQ318817

(TC)8

F:<HEX > <Tail-4 > TGACGGAAGGATACTGGCTC

164-180

7

0.7742

0.7509

0.7012

0.0000

R:CCATCAGACATGGCTTTCCT

ZJU123ab

JQ318818

(CT)8

F:<HEX > <Tail-4 > TGAATTATTCGGTTCCCTGG

172-176

3

0.4667

0.6367

0.5499

0.2152

R:TGCTTCAGTTCCAAACGAAA

ZJU124ab

JQ318819

(CT)10

F:<HEX > <Tail-4 > GTGGCAGCCTCTCTATCGTC

161-187

12

0.9355

0.8778

0.8498

0.0001

R:ATGACGTACTGCCCTTGCTT

ZJU125ab

JQ318820

(TC)8

F:<HEX > <Tail-4 > TAAGGGCAGTCAGACCAACC

164-186

4

0.2188

0.3884

0.3453

0.0000

R:CTGCAGCCTACAATGATCCA

ZJU126ab

JQ318821

(GA)10

F:<HEX > <Tail-4 > CCAATGTGGACAGGTGTGAG

173-193

11

0.9677

0.8535

0.8228

0.0000

R:GGCAGTAGTCGCTTCCCATA

ZJU127

JQ318822

(GC)10

F:<HEX > <Tail-4 > AGGATCCTTGTCACCACCAG

165-189

11

0.9259

0.8288

0.7900

0.0079

R:AATTCTTCCTTCCCAGCCTC

ZJU128ab

JQ318823

(AG)14

F:<HEX > <Tail-4 > CCCAATTGACACAAATTCCC

145-161

5

0.4194

0.5019

0.4496

0.1981

R:TTGGCATAGCATTGTTCGTC

ZJU129ab

JQ318824

(CT)10

F:<HEX > <Tail-4 > GAGGTGCAATTACGTGGCTT

161-189

10

0.7500

0.8031

0.7611

0.0234

R:TCAAGCATCAGCTGCTCAGT

ZJU130ab

JQ318825

(GA)8

F:<HEX > <Tail-4 > GATTGCATGCACCAAATCAC

160-176

5

0.3478

0.4599

0.4131

0.2852

R:GAATGTCCACGACGTGAATG

ZJU131ab

JQ318826

(CT)14

F:<HEX > <Tail-4 > TTGAGAATCACAAACGCCTG

153-187

13

0.8710

0.8990

0.8735

0.0009

R:GGTGGGTGAAATGCCTAGAA

ZJU132ab

JQ318827

(CT)11

F:<HEX > <Tail-4 > AGGCACCTTTCTTTCCTCTC

164-178

5

0.6452

0.6568

0.5834

0.6586

R:CAAGGAAGGAGGTGACGAAG

ZJU133ab

JQ318828

(TC)11

F:<HEX > <Tail-4 > GCCCTGCAGTCTTTGTCAAT

171-195

8

0.8710

0.7731

0.7267

0.0000

R:CAGCTTGCAGTGTTCATTCA

ZJU134ab

JQ318829

(GA)11

F:<HEX > <Tail-4 > AGTGCCCAAGCATGACTTCT

172-190

8

0.9688

0.7907

0.7507

0.0004

R:AATCAGTTGTCCGAGGATGG

ZJU135ab

JQ318830

(AG)10

F:<HEX > <Tail-4 > AATTTACGGCTGTCCGTGAG

173-191

10

0.9688

0.7966

0.7557

0.0000

R:CCTTGGGCTTCATGAACATT

ZJU136ab

JQ318831

(GA)10

F:<HEX > <Tail-4 > TCCCACAGATCTCTAGCCGT

173-201

13

0.7742

0.8953

0.8692

0.0004

R:CGCTCAGTTCTTAATTTCTTACTGTC

ZJU137ab

JQ318832

(TC)8

F:<HEX > <Tail-4 > TGGATCTTGCTGCAGTTGTC

140-168

12

0.1875

0.6930

0.6612

0.0000

R:AGCTAGCACTGGCCTAACCA

ZJU138ab

JQ318833

(CT)10

F:<HEX > <Tail-4 > GCACAGTTGAGTTATGGGCA

152-170

8

0.3333

0.7746

0.7261

0.0001

R:CTCTTTCAAATCCACGCACA

ZJU139ab

JQ318834

(GA)12

F:<HEX > <Tail-4 > CCGAGCTTCGTTAGGACTTG

138-164

6

0.3667

0.4418

0.4043

0.0000

R:CCAACAATACCCGAACCATC

ZJU140b

JQ318835

(CT)14

F:<HEX > <Tail-4 > TGTGCTCATCTTGGATCCTG

172-198

9

0.6538

0.6139

0.5474

0.0000

R:ACATCAGCTTGCATCCCTCT

ZJU141ab

JQ318836

(CT)13

F:<HEX > <Tail-4 > CACAATCAGCTGCAGAATCAA

175-201

11

0.6774

0.7996

0.7600

0.0002

R:AATGGCCGCTTGCAATATAA

ZJU142ab

JQ318837

(TC)13

F:<HEX > <Tail-4 > CATTCACCTCCTTTCGCAAT

166-184

9

0.6774

0.6912

0.6498

0.0231

R:ATCCAACGGCTCAAAGAATG

ZJU143ab

JQ318838

(CT)12

F:<HEX > <Tail-4 > GTAGAGTAGATGCGCCTCGG

181-197

7

0.6923

0.7044

0.6397

0.0000

R:ACGTACGAGCCATACACACG

ZJU144ab

JQ318839

(AG)12

F:<HEX > <Tail-4 > GCCACTCTTCCCTCAACGTA

148-164

7

0.5161

0.6864

0.6252

0.0430

R:CAGGTCAGTCCTGATGGGAT

ZJU145ab

JQ318840

(CT)10

F:<HEX > <Tail-4 > TGTGGCTGTGTTCCTCCATA

155-175

7

0.6875

0.7351

0.6912

0.0000

R:CAATGTTGGGTGCTTTGTTG

ZJU146ab

JQ318841

(AG)10

F:<HEX > <Tail-4 > TGGAAACTTTGTCGTGTGGA

154-168

6

0.2258

0.6663

0.6187

0.0000

R:TTATATCGGGCAGCCAGAAC

ZJU147ab

JQ318842

(AG)10

F:<HEX > <Tail-4 > TTAGGAACCAAACTGGACGG

173-195

10

0.8333

0.7169

0.6811

0.0005

R:TCAAATGCCGTGCTATTGAG

ZJU148ab

JQ318843

(AG)18

F:<HEX > <Tail-4 > AAGAGCAGGAACCGAACCTT

160-190

15

0.9375

0.9067

0.8829

0.4973

R:ACCGAAAGACGAAGAAACGA

ZJU149ab

JQ318844

(TC)8

F:<HEX > <Tail-4 > AGCCCTCCATGTGTGCTTAT

139-163

11

0.8333

0.8718

0.8417

0.0022

R:AGGGAGAGAGTGGTTCTGCC

ZJU150ab

JQ318845

(AG)10

F:<HEX > <Tail-4 > ACTTAACTGAGAGGCTGCGG

163-201

10

0.9000

0.8469

0.8123

0.0053

R:GTGGAAACCGAACGTCCTAA

ZJU151ab

JQ318846

(CA)9

F:<HEX > <Tail-4 > GAATTGGAAATCCCTAGCCC

156-170

6

0.3750

0.5511

0.5188

0.0001

R:CATTTGCGCATGTCTCCTTA

ZJU152ab

JQ318847

(AG)11

F:<HEX > <Tail-4 > AAACGAAGTCGTTCAATGCC

163-181

7

0.9355

0.7578

0.7040

0.0161

R:CTTGATTTGGGCCTTCGATA

ZJU153ab

JQ318848

(AG)10

F:<HEX > <Tail-4 > CCAGCTCCGAATTAGCAAAC

173-191

6

1.0000

0.6667

0.5927

0.0000

R:GTGGCGGTTTATCTCATCGT

ZJU154ab

JQ318849

(AG)11

F:<HEX > <Tail-4 > TTGTCAATTGCCCTTCCTTC

156-184

10

0.9333

0.6847

0.6184

0.0000

R:TTCCTCCCTTTCCCACTTCT

ZJU155ab

JQ318850

(TC)9

F:<HEX > <Tail-4 > GAGAGCAATCAGTGAAGCCC

160-188

8

0.8438

0.6731

0.6037

0.0000

R:GGGAGACGGATGTCGATTTA

ZJU156ab

JQ318851

(TA)8

F:<HEX > <Tail-4 > ATACGTCGAAAGATCCACCG

166-184

7

0.5484

0.6626

0.6063

0.0000

R:TTCTGGAATCCTTCCCATTG

ZJU157ab

JQ318852

(AG)9

F:<HEX > <Tail-4 > CACTCACAACCAAAGCCAGA

154-186

13

0.9677

0.9064

0.8823

0.0171

R:GTGCATAATCACAGGCATGA

ZJU158ab

JQ318853

(AT)10

F:<HEX > <Tail-4 > CCAGATGATCACGCAGCTTA

156-174

9

0.6452

0.8292

0.7917

0.0000

R:CGTCCTCCAATACGTTCCTC

Mean

8.25

0.5636

0.7178

0.6730

 

Note: a b c These SSRs are transferable for M. adenophora, M. nana and M. cerifera, respectively. SSR markers are listed according to ascending order in different fluorescent dyes. Shown for each primer pair are the repeat motif, primer sequences, size range (bp), number of alleles detected (Na), observed heterozygosity (Ho), expected heterozygosity (He), polymorphism information content (PIC) and Chi-square test for Hardy-Weinberg equilibrium (PHW). The annealing temperature was 60 ° C; a, including length of tail sequences (18 bp total). PHW over 0.05 are underlined.

The PIC at each locus ranged from 0.256 to 0.883 with an average of 0.67 loci. The PCR product size ranged from 110 to 274 bp. All the primers produced amplicons in agreement with the expected sizes. Most of the SSR primers (139 primer pairs) showed significant deviation from HW equilibrium (P < 0.05). Partial correlation analysis showed that significant positive correlations existed between the repeat unit length and PIC (P < 0.01, r = 0.2747). This showed that these SSRs had high rates of transferability for M. adenophora (91.14%) and M. nana (89.87%) and low rates for M. cerifera (46.84%), indicating that these markers are suitable for genetic diversity analyses in other Myrica species.

One of the objectives of this study was to develop potential suitable SSR markers for genetic mapping using Biqi and Dongkui as crossing parents. We selected 99 heterozygous loci in Biqi and 105 in Dongkui (Table3): 135 primer pairs can be used together in linkage mapping of the planned F1 populations between Biqi and Dongkui.
Table 3

Distribution of the segregation types expected for the mapping population (Biqi × Dongkui)

Segregation type

Alleles

Number

Mapping in F1

aa × aa

1

12

No

aa × bb

2

11

No

aa × ab

2

24

Yes

ab × aa

2

18

Yes

ab × ab

2

8

Yes

aa × bc

3

12

Yes

ab × cc

3

12

Yes

ab × ac

3

41

Yes

ab × cd

4

20

Yes

Total

135

 

Genetic relationship analysis

The 32 accessions were divided into three groups (A, B and C, Figure4), based on Nei’s genetic distance coefficient[16] using UPGMA cluster analysis. The similarity among all the accessions varied from 0.54 to 0.84. At the species level, the UPGMA dendrogram produced clusters separating M. nana and M. cerifera into two distinct groups. The genetic similarity between M. cerifera and M. rubra was 0.54, lower than the 0.74 previously reported by Xie[6].
https://static-content.springer.com/image/art%3A10.1186%2F1471-2164-13-201/MediaObjects/12864_2012_Article_4370_Fig4_HTML.jpg
Figure 4

Dendrogram for 32 Chinese bayberry accessions derived from UPGMA cluster analysis based on 158 SSR markers. The symbols before the accession codes indicate the sex: , androphyte plant, , common cultivars, and , monoecious plant. The numbers are bootstrap values based on 1000 iterations. Only bootstrap values larger than 50 are indicated.

The main cluster ‘A’ included the subgroups A-1 and A-2. Subgroup A-1 includes 16 accessions, mainly from the cities of Ningbo (12) and Hangzhou (3), where the popular and dominant cultivar is Biqi. This demonstrates that these natural elite seedling selections are truly distinct from the local cultivars. For their genetic relationships (Figure4), the rare monoecious individual (C2010-4) is closely related to Biqi, while the accessions ‘Shuijing’ and ‘Y2010-72’ (both white fruit type) are clearly separated in the cluster, with low genetic distance.

Subgroup A-2 includes 14 accessions, with four from Wenzhou, two from Taizhou, and one each from the cities of Hangzhou and Ningbo, and the Hunan, Guangxi, Guizhou and Jiangsu provinces. This group includes the popular cultivar Dongkui. The four accessions from Wenzhou distributed in this cluster have genetic similarity. The accession ‘Tongzimei’ from the Hunan province is on an independent branch, showing that it is genetically distinct. ‘Xiaolejiangchonghei’ and ‘M. adenophora’ grouped together, and are possibly in the same population. Six androphyte accessions, distributed in group A, are close to cultivars of the same geographic origin.

The accessions ‘Myrica nana’ from Yunnan and ‘Myrica cerifera’ from the USA were independently classified as the ‘B’ and ‘C’ group, indicating a distant relationship with cultivated Myrica rubra.

Discussion

Our major aims were to find a large set of SSR markers for Myrica rubra and understand the genetic diversity and relationship among representative cultivars, androphyate and related species. This research paves the way for constructing an SSR-based linkage map in Myrica.

The genome characteristics of genus Myrica

Shotgun sequencing is suitable for simple genomes, with no or few repeat sequences, such as Chinese bayberry. For such genomes, the genome can largely be assembled simply by merging together reads containing overlapping sequence[17]. We report the genome survey of Chinese bayberry using whole genome shotgun sequencing. The 17-nucleotide depth distribution suggests a genome size of 323 Mb, larger than peach (220 Mb,http://www.rosaceae.org/peach/genome), but close to our estimate of 250 Mb from flow cytometry using rice as the reference (date not shown). Although the highly homozygous material was selected on a limited number of SSR loci assays, the actual heterozygous rate, as revealed by 185 new SSR markers, was very high (63%). To overcome the key issue of heterozygosity and allow us to generate a high-quality genome sequence, we can use a unique homozygous form such as monoploid, derived using tissue culture or from nature and worth further study.

Marker development for under-utilised fruit crops

SSRs have been widely used for high-throughput genotyping and map construction as they have the advantage of high abundance, random distribution within the genome, high polymorphism information content and stable co-dominance[1820]. They can be developed from either genomic or expressed sequence tag (EST) libraries. Although EST-SSRs are useful for genetic analysis, their disadvantages of relatively low polymorphism and high concentration in gene-rich regions of the genome may limit their usage, especially for the construction of linkage maps[21]. In this study, a total of 600 SSR primer pairs were designed from 28,602 SSR sites, and 581 (96.8%) primer pairs were effective. Due to the self-complementary nature to form dimers, AT/TA is not usually used to develop markers[12]. Our findings are in agreement with that published for peach, where the dinucleotide repeat motifs were also found to be the most common, and (CT)n as the most common repeat unit[22].

In the present study, the mean value of PIC was higher than the previously reported 0.62[7], but the consistent relationship was observed between SSR polymorphism and repeat unit length. There are some reports of a positive relationship between degree of polymorphism and repeat unit length[23, 24]. However, those polymorphic SSRs that are homozygous in both parents cannot be mapped in F1 populations, although they are useful for mapping in F2 or backcross populations[25], while heterozygous SSRs can be used for mapping in F1 populations (Table2). The estimated number of SSRs that can be mapped in the F1 populations between Biqi and Dongkui was about 85%.

Recently, based on mass sequence data of Chinese bayberry obtained by RNA-Seq, 41,239 UniGenes were identified and approximately 80% of the UniGenes (32,805) were annotated, which provides an excellent platform for future EST-SSR development and functional genomic research[26].

High efficient test methods

Normally, a universal M13 primer is labelled with one of a number of fluorescent dyes. The tailed primer provides a complementary sequence to the fluorescent labelled M13 primer, leading to the amplification of fluorescent PCR products, and then the PCR products of different sizes and/or labelled with different fluorescent dyes are mixed and tested[27]. In this research, a multiplex PCR strategy was designed using the universal M13-tailed primer and three additional tail primers, designed arbitrarily, in presumed four-plex amplifications in single PCR, for a major reduction in cost and time. However, as only a few primer combinations were successful, most resulting in weak bands, we did the PCR individually and mixed the PCR products. Further optimisation of multiplex PCR is needed to evaluate its general applicability.

Evolution of Myrica species

In this study, both cultivated species and wild species were analysed and their genetic diversity could easily be differentiated. M. nana and M. cerifera were clearly genetically distant to M. rubra. M. nana, also known as the dwarf or Yunnan arbutus, is indigenous to the Yunnan and Guizhou provinces, and has a plant height of < 2 m. The juvenile period of fruit tree can be shortened for breeding purposes. Studies on embryo culture in vitro of the F1 seeds of crosses between M. rubra and M. nana,[28], has shown good cross compatibility between M. rubra and M. nana, resulting in 70.5% normal seeds with intact embryo. M. adenophora and M. nana grow as wild trees, with the fruit of M. adenophora only suitable for medical purposes and not edible.

Our findings on the genetic similarity between M. adenophora and M. rubra, which are considered a progenitor-derivative species pair, are consistent with a previously published figure of 0.897[29]. An earlier study observed little change in allelic diversity along the chronosequence and no evidence for heterosis, although there was a moderate change in genotypic diversity[30]. The markers developed in this study can be very useful in future population structure analysis.

Conclusions

In summary, the genome size of Myrica genus is small, about 320 Mb. A large set of SSRs were developed from a genome survey of Myrica rubra. The results suggest that they have high rates of transferability, making them suitable for use in other Myrica species.

Materials and methods

Plant materials and genome survey

We selected an androphyte ‘C2010-55’ for the genome survey because it was the most homozygous (10 out of 14 SSRs) individual among 230 accessions. Two DNA libraries of 250 and 500 bp insert size were constructed and sequenced by Illumina Hi-Seq 2000.

Twenty-nine accessions of the cultivated species (M. rubra) and 3 related species (M. adenophora, M. nana, M. cerifera), collected from different provinces in China (Table4), were used to evaluate the suitability of the SSRs for genetic distance analysis. Young leaves were collected and frozen in liquid nitrogen prior to genomic DNA extraction using CTAB methods[4]. DNA concentrations were measured spectrophotometrically at 260 nm, and the extracts electrophoresed on 1% agarose to confirm the quality. The purified DNAs were standardised at 40 ng/μl and stored at -40°C.
Table 4

The 32 bayberry accessions included in this study

No.

Accession

Fruit/Flower coloura

Fruit maturity date

Region

1

Biqi

black

Late June

Cixi, Ningbo, Zhejiang

2

Dongkui

red

Early July

Taizhou, Zhejiang

3

Dayehuang

red

Mid-June

Hangzhou, Zhejiang

4

Dingaomei

black

Mid to late June

Wenzhou, Zhejiang

5

Huangshanbai

white

Early July

Hangzhou, Zhejiang

6

Jiazhaizao

black

Mid-June

Wenzhou, Zhejiang

7

Jianmei

red

Late June

Cixi, Ningbo, Zhejiang

8

Muyemei

black

Late June

Jinhua, Zhejiang

9

Putaoli

black

Mid June

Hangzhou, Zhejiang

10

Shuijing

white

Late June/Early July

Yuyao, Ningbo, Zhejiang

11

Tongzimei

black

Mid-June

Hunan

12

Wandao

black

Early July

Zhoushan, Zhejiang

13

Xiaolejiangchonghei

black

May

Guizhou

14

Biqi12

black

Late June

Yuyao, Ningbo, Zhejiang

15

Y2010-70

red

Late June/Early July

Yuyao, Ningbo, Zhejiang

16

Y2010-71

black

Mid to late June

Yuyao, Ningbo, Zhejiang

17

Y2010-72

white

Late June/Early July

Yuyao, Ningbo, Zhejiang

18

Y2010-73

red

Late June

Yuyao, Ningbo, Zhejiang

19

Y2010-74

red

Late June/Early July

Yuyao, Ningbo, Zhejiang

20

Y2010-75

black

Late June

Yuyao, Ningbo, Zhejiang

21

Y2010-76

white

Late June/Early July

Yuyao, Ningbo, Zhejiang

22

Y2010-77

red

Late June/Early July

Yuyao, Ningbo, Zhejiang

23

C2010-4

red

Late June

Cixi, Ningbo, Zhejiang

24

*C2010-55

red

-

Cixi, Ningbo, Zhejiang

25

*W2011-1

yellow- red

-

Wenzhou, Zhejiang

26

*W2011-5

red

-

Wenzhou, Zhejiang

27

*H2011-12

yellow-green

-

Hangzhou, Zhejiang

28

*JS2011-16

red

-

Suzhou, Jiangsu

29

*T2011-30

red

-

Taizhou, Zhejiang

30

Myrica adenophora

red

February to May

Guilin, Guangxi

31

Myrica nana

red

June to July

Yunnan

32

*Myrica cerifera

yellow-green

-

Cixi, Ningbo, Zhejiang

Note: fruit colour for cultivar and flower colour for androphyte. * selected androphytes.

SSR identification and primer design

We used MISA scripting language (http://pgrc.ipk-gatersleben.de/misa/misa.html) to identify microsatellite repeats in our sequence database. The SSR loci containing perfect repeat units of 2-6 nucleotides only were considered. The minimum SSR length criteria were defined as six reiterations for dinucleotide, and five reiterations for other repeat units. Mononucleotide repeats and complex SSR types were excluded from the study.

The SSR primers were designed using BatchPrimer3 interface modules (http://pgrc.ipk-gatersleben.de/misa/primer3.html). We selected 600 primers that met the following parameters: 110–230 final product length, primer size from 18 to 22 bp with an optimum size of 20 bp, and the annealing temperature was set at 60°C. The repeat units over eight were used.

Tail-1(M13 universal sequence-TGTAAAACGACGGCCAGT), Tail-2(CGAGTCAGTATAGGGCAC), Tail-3(ATCACGAAGCAGATGCAA) and Tail-4(GAGCATCTCGTACCAGTC) were added to the 5’ end of each 150 forward primers of pairs respectively. Tail-2, Tail-3 and Tail-4 were designed so that the primer size was 18 bp and the annealing temperature was 53°C. Primers were synthesised by Invitrogen Trading (Shanghai) Co., Ltd. We primarily tested two cultivars (Biqi and Dongkui) and M. cerifera for 600 SSR loci by PAGE (polyacrylamide denaturing gel) to confirm their suitability. Tail-1, Tail-2, Tail-3 and Tail-4 labelled with one of the following dyes: NED, PET, FAM, and HEX, respectively.

Polymerase chain reaction and gel electrophoresis

Each 20 μl reaction mixture contained 10 × PCR buffer (plus Mg2+), 0.2 mM of each dNTP, 5 pmol of each reverse, 4 pmol of the tail primer, 1 pmol of the forward primer, 0.5 units of rTaq polymerase (TaKaRa, China) and 40 ng genomic DNA template. Each primer pair had an interval of 20 bp according to the expected size of amplicons.

DNA amplification was in an Eppendorf Mastercycler (Germany) programmed at 94°C for 5 min for initial denaturation, then 32 cycles at 94°C (30 s)/58°C (30 s)/72°C (30 s), followed by 8 cycles of 94°C (30 s)/53°C (30 s)/72°C (30 s). The final extension step was 10 min at 72°C. Each PCR product was run on 1% agarose gel at 110 V for a quality check.

Subsequently, PCR products were electrophoresed on 8% denaturing PAGE, according to Myers et al.[31], at 60 W in a Sequi-Gen GT Nucleic Acid electrophoresis cell (BioRad) for 4 h, depending on the fragment sizes to be separated, and visualised by silver staining[32]. Genotypes showing one and two bands were scored as homozygous and heterozygous, respectively, and the results recorded and photographed.

Multiplex PCR was designed and tested with products of different sizes and labelled with different fluorescent dyes. Each 20 μl reaction mixture contained 10 × PCR buffer (plus Mg2+), 0.8 mM of each dNTP, 1 unit of rTaq polymerase, 40 ng genomic DNA template and a total of four primer pairs with 5 pmol of each reverse primer, 4 pmol of each tail primer, and 1 pmol of each forward primer. The PCR products were diluted, mixed with the internal size standard LIZ500 (Applied Biosystems) and loaded on an ABI 3130 Genetic Analyzer. Alleles were scored using GeneMapper version 4.0 software (Applied Biosystems, Foster City, CA, USA).

Data analysis

The raw genome sequence data was first filtered to obtain high quality reads, then assembled using SOAP (http://soap.genomics.org.cn/soapdenovo.html) denovo software to contig, scaffold and fill in gaps. In addition, we used SSPACE software to build the scaffold. K-mer analysis was to help estimate the genome size and characters, such as heterozygosis and repeats.

The number of alleles (A), observed heterozygosity (Ho) and expected heterozygosity (He) were calculated using POPGENE version 1.32 (http://www.ualberta.ca/~fyeh/popgene_download.html). Chi-square test for Hardy-Weinberg equilibrium allele frequencies and polymorphism information content (PIC) was calculated using PowerMarker version 3.25[33] (http://statgen.ncsu.edu/powermarker/downloads.htm). Microsoft office Excel 2007 was used to analyse the correlation. Genetic similarity among all the accessions was calculated according to Dice’s coefficients using Nei's coefficient index[16] with the Free Tree 0.9.1.50[34] (http://www.natur.cuni.cz/~flegr/programs/freetree.htm) software, and the dendrogram constructed using the unweighted pair-group method with arithmetic averaging (UPGMA) option. The confidence of branch support was then evaluated by bootstrap analysis with 1,000 replicates. The dendrogram was printed using MEGA version 5.05 software[35] (http://www.megasoftware.net/mega.php).

Declarations

Acknowledgements

This work was supported by grants from the Zhejiang Province (2006 C14016) and Special Research Fund for International Cooperation with European Union (1114) and Public Welfare in Chinese Agriculture (contract no. 200903044) We thank Dr Rangjin Xie for technical assistance in the PAGE experiment, and Dr. Shirley Burgess for correcting the English.

Author details

1Department of Horticulture, The State Agriculture Ministry Laboratory of Horticultural Plant Growth, Development and Quality Improvement, Zhejiang University, Hangzhou 310058, China. 2BGI-Shenzhen, Beishan Industrial Zone, Yantian District, Shenzhen 518083, China. 3Fruit Research Institute, Yuyao, Ningbo 315400, China. 4Forestry Technology Extension Center, Cixi Ningbo 315300, China. 5Plant Breeding-Wageningen University and Research Centre, P.O. Box 166700 AA, Wageningen, The Netherlands.

Authors’ Affiliations

(1)
Department of Horticulture, The State Agriculture Ministry Laboratory of Horticultural Plant Growth, Development and Quality Improvement, Zhejiang University
(2)
BGI-Shenzhen
(3)
Fruit Research Institute
(4)
Forestry Technology Extension Center
(5)
Plant Breeding-Wageningen University and Research Centre

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