Molecular Breeding

, Volume 23, Issue 1, pp 85–97

Development, characterization and cross-species/genera transferability of EST-SSR markers for rubber tree (Hevea brasiliensis)

Authors

  • S. P. Feng
    • Institute of Tropical Bioscience and BiotechnologyChinese Academy of Tropical Agricultural Science
    • Key Laboratory of Tropical Horticulture Plant Resources & Genetic Improvement, Ministry of Education, Agricultural CollegeHainan University
  • W. G. Li
    • Rubber Cultivation Research InstituteChinese Academy of Tropic Agricultural Science
  • H. S. Huang
    • Rubber Cultivation Research InstituteChinese Academy of Tropic Agricultural Science
  • J. Y. Wang
    • Institute of Tropical Bioscience and BiotechnologyChinese Academy of Tropical Agricultural Science
    • Key Laboratory of Tropical Horticulture Plant Resources & Genetic Improvement, Ministry of Education, Agricultural CollegeHainan University
    • Key Laboratory of Tropical Horticulture Plant Resources & Genetic Improvement, Ministry of Education, Agricultural CollegeHainan University
Article

DOI: 10.1007/s11032-008-9216-0

Cite this article as:
Feng, S.P., Li, W.G., Huang, H.S. et al. Mol Breeding (2009) 23: 85. doi:10.1007/s11032-008-9216-0

Abstract

Expressed sequence tags derived simple sequence repeats (EST-SSRs), are different from traditional genomic SSR (gSSR) markers. They are more likely to be embedded in the functional gene sequences, less costly and time effective, and may provide abundant information. By analysis of 10,018 expressed sequence tags (ESTs) out of 10,829 for rubber tree (Hevea brasiliensis) available in public domain DNA databases, 799 SSR loci were found in the 643 non-redundant SSR-ESTs (SSR-containing ESTs), corresponding to one SSR in every 2.25 kb of the ESTs in rubber tree transcriptome. Of the total 799 SSRs in these ESTs, 673 (84.2%) contained simple repeat motifs while 126 (15.8%) represented compound motif types. Of the total EST-SSRs, 45.3% (362/799) were mononucleotide repeats (MNRs), 42.2% (337/799) were dinucleotide repeats (DNRs), 11.9% (95/799) were trinucleotide repeats (TNRs) and 0.6% (5/799) were tetranucleotide repeats (TTNRs) and hexanucleotide repeats (HNRs). The repeat motifs AAG and AG were the most abundant without regard to single nucleotide repeat. A total of 184 primer pairs were successful designed based on the non-redundant SSR-ESTs. Using 55°C as annealing temperature, 110 primer pairs successfully amplified 12 H. brasiliensis cultivated varieties and four related species. Analysis on 74 alleles amplified by 30 randomly selected primer pairs indicated the medium polymorphism of the EST-SSRs developed. Based on 272 alleles detected by 87 EST-SSR markers, an assessment of genetic diversity was carried out on 12 H. brasiliensis cultivated varieties and four related species. In addition, investigation based on five selected EST-SSRs by cloning and sequencing cross some cultivated species and related species provided evidence for cross-species/genera transferability of the EST-SSR markers developed in this study.

Keywords

Expressed sequence tag derived simple sequence repeats (EST-SSRs)Rubber tree (Hevea brasiliensis)PolymorphismGenetic diversityCross-species/genera transferability

Introduction

In modern studies of plant genetics and breeding, molecular markers have become more and more important and efficient tools for genetic diversity assessment, QTL and/or gene mapping, variety protection, and marker assisted selection. The common molecular markers are RAPD (random amplified polymorphic DNA), SSR (simple sequence repeat or microsatellite), AFLP (amplified fragment length polymorphism), RFLP (restriction fragment length polymorphism) and SNP (single nucleotide polymorphism). Among these, SSRs have become the most favorable molecular markers because of their multi-allelic nature, reproducibility, codominant inheritance, high abundance and extensive genome coverage (Gupta and Varshney 2000).

EST-SSRs (expressed sequence tag-simple sequence repeats) may be mined from EST sequence collections. As markers, they differ from traditional genomic SSR (gSSR) markers in that they are more likely embedded in functional gene sequences, less costly to identify and may provide more abundant information. Linkage of EST-SSR markers with desired characters may lead to the identification of genes controlling these traits (Bozhko et al. 2003). In addition, EST-SSRs are universal and can be applied in comparative mapping and linkage map construction (Schubert et al. 2001; Cordeiro et al. 2001; Varshney et al. 2005). Therefore, in recent years, EST-SSRs have already been developed for various crops such as wheat (Stack et al. 2000), barley (Kota et al. 2001; Thiel et al. 2003; Holton et al. 2002), grape (Scott et al. 2000), tomato (Areshchenkova and Ganal 2002), sugar cane (Cordeiro et al. 2001) and coffee (Combes et al. 2000; Rovelli et al. 2000; Baruah et al. 2003; Moncada and McCouch 2004; Bhat et al. 2005; Aggarwal et al. 2007).

Hevea brasiliensis is the most widely cultivated tree species for producing natural rubber latex. The tree is a perennial cross-pollinating and monoecious plant of the family Euphorbiaceae. Because the rubber tree’s breeding period is very long, high costs are usually incurred in the development of new tree varieties. Marker assistant selection (MAS) provides an indirect but efficient measure to choose the target genotype, thereby enhances the breeding efficiency, shortens the breeding period, and thus reduces the field workload. However, there are few reports of application of molecular markers in genetics and breeding of the rubber tree (Besse et al. 1994; Luo and Boutry 1995; Low and Gale 1996; Lespinasse et al. 2000; Guen et al. 2003; Venkatachalam et al. 2006).

Prior to the present study, there had been not many reports on EST-SSR marker development and application in the rubber tree. Therefore, we report our work on EST-SSRs derived from 10,829 entries of rubber tree ESTs in the National Centre of Bioinformatics Information, USA database, based on (1) the frequency and distribution of SSRs in rubber tree ESTs, (2) the establishment and validation of EST-SSR markers for detection of polymorphisms in cultivated and related rubber tree species, and (3) the assessment of intraspecies genetic diversity by EST-SSR markers and their cross-species/genera transferability.

Materials and methods

Plant material and EST retrieval

Fresh leaf samples were collected from 12 varieties of the cultivated rubber tree species (Hevea brasiliensis), four related species (H. spruceana, H. nitida, H. benthamiana and H. pauciflora), and three species from other Euphorbiaceae genera (Ricinus communis L., Manihot utilissima and Phyllanthus emblica) growing in the Rubber Cultivation Research Institute, Chinese Academy of Tropic Agricultural Science (Danzhou) (Table 1). Leaf genomic DNA was extracted following the CTAB protocol (Venkatachalam et al. 2002).
Table 1

Plant materials used for marker validation and cross-species transferability

Materials

Scientific name

Pedigree/source

Castor-oil plant

Ricinus communis L.

CATASa

Cassava

Manihot utilissima

CATASa

Myrobalan

Phyllanthus emblica

CATASa

Sebao rubber

Hevea spruceana

Brazil

Guangye rubber

Hevea nitida

Brazil

Bianqin rubber

Hevea benthamiana

Brazil

Shaohua rubber

Hevea pauciflora

Brazil

PR107

Hevea brasiliensis

Original clone

RRIM600

Hevea brasiliensis

Tjir1 × PB86

IAN873

Hevea brasiliensis

PB86 × FA1717

Reyan88-13

Hevea brasiliensis

RRIM600 × Pilb84

Reyan8-79

Hevea brasiliensis

RY88-13 × ReYan217

Reken525

Hevea brasiliensis

Unknown

Reken523

Hevea brasiliensis

Unknown

Reyan7-33-97

Hevea brasiliensis

RRIM600 × PR107

GT1

Hevea brasiliensis

Original clone

Yunyan77-2

Hevea brasiliensis

GT1 × PR107

Wenchang217

Hevea brasiliensis

HaiKen1 × PR107

Haiken2

Hevea brasiliensis

PB86 × PR107

aRubber Cultivation Research Institute, Chinese Academy of Tropic Agricultural Science, Danzhou

EST sequences were obtained via the ENTREZ search tool of the EST database at the NCBI (http://www.ncbi.nlm.nih.gov/dbest/). A total of 10,829 ESTs availability on May 1, 2007 were obtained for this study.

The EST-trimmer software (http://www.pgrc.ipk-gatersleben.de/misa/download/est_trimmer.pl) was used to remove the 5′ or 3′ end of poly A or poly T stretches until there were no (T)5 or (A)5 within the range of 50 bp, EST sequences less than 100 bp in length were discarded while ESTs greater than 700 bp in length were retained their 5′ end 700 bp.

Data mining

After pre-treatment, the MISA software (http://www.pgrc.ipk-gatersleben.de/misa) was used to search for SSRs from the rubber ESTs. The search criteria were: mononucleotide repeats ≥10, dinucleotide to hexanucleotide repeats ≥6, meanwhile, those interrupted composite SSR had also been selected (interval bases ≤100). Dinucleotide repeats such as AT/TA, CT/GA were treated as the same type of repeat motif.

The SSR-containing ESTs (SSR-ESTs) were clustered with procedures stackPACK v 2.2 program in order to get the non-redundant sequences (Miller et al. 1999).

Non-redundant SSR-ESTs were used to design primers with PRIMER5.0 (Wetmur 1991) where the main parameters were: GC content of 40–60%, annealing temperature (Tm) of 53–57°C and expected amplified products size of 100–300 bp. All primers were synthesized by the Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. EST-SSRs were amplified using the Whatman Biometra T1 Thermocycler. Each PCR reaction consisted of: 2 μl of 10× buffer, 0.25 μl of 10 M dNTP, 1 μl each of forward and reverse primer (20 μmol), 2 μl of template leaf genomic DNA (20 ng/μl), 0.15 μl of Taq polymerase (5 U/μl) (Shanghai Sangon Biological Engineering Technology & Services Co., Ltd.), in a total reaction volume of 20 μl. The PCR reaction profile was: pre-denaturation at 94°C for 2 min followed by 30 cycles of 94°C for 30 s, 55°C for 45 s and 72°C for 1 min and finally, 72°C for an extension of 5 min.

SSR-ESTs were assigned putative function by comparison with the non-redundant sequence database at NCBI using the BLASTX2.2.17 software (Altschul et al. 1997) at http://www.ncbi.nlm.nih.gov/BLAST/Blast.cgi.

Primers designed based on non-redundant SSR-ESTs were used to amplify materials from 16 Hevea species before-mentioned and the NTSYSpc ver 2.1 software (http://www.exetersoftware.com/) was used to calculate genetic distances and UPGMAM (Unweighted Pair Group Method with Arithmetic Mean) values in order to construct interspecific dendrograms. Allelic polymorphic information content (PIC) was calculated in each case using the formula, PIC = 1 − ∑(pi)2, where pi is the frequency of the ith allele in the set of analyzed genotypes calculated for each SSR locus (Botstein et al. 1980).

Thirty primer pairs were selected to detect cross-species/genera transferability. The recovered expected PCR products from 10 materials that represented the intraspecies, interspecies and intergenera respectively of five randomly selected markers (HBE014, HBE117, HBE140, HBE156, HBE206) were cloned to PMD-18T vector and transformed into E. coli DH5 α. The cloned inserts were sequenced with the M13 universal primer using the ABI PRISM 3730 sequencing equipment in Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. The ClustalX program (http://www.ftp-igbmc.u-strasbg.fr/pub/ClustalX/) was used to compare the amplified SSR alleles with the SSR-containing ESTs investigate the cross-species/genera transferability and the repeat motif conservation of the SSRs.

Results and analysis

Distribution and frequencies of SSRs in rubber tree transcriptome

Of all the ESTs, 9,681 were RRIM600 latex ESTs that originated from 15-year-old H. brasiliensis trees (Chow et al. 2007) while 858 were submitted by Han (Ko et al. 2003) using the SMART (Switching Mechanism At 5′ end of the RNA Transcript) strategy from mature rubber plants latex of the clone RRIM 600. Of the remainder 110 ESTs, 18 were from leaf, 33 from bark and 59 from latex.

Subsequently, 10,018 rubber tree ESTs were obtained after pre-treatment analysis of 10,829 sequences downloaded from NCBI. This represented approximately 5,173.73 kb of putative functional rubber tree transcriptome. SSR screening with the MISA program resulted in the isolation of 1233 SSR-ESTs which contained 1524 SSRs (Fig. 1). Therefore, the frequency of SSR-ESTs in the rubber EST collection analysed (the ratio of the number of SSR-ESTs to the number of searched ESTs) was 12.31%, this representing an average frequency of one SSR every 3.39 kb or one SSR every 8.12 ESTs in the analysed rubber tree transcriptome.
https://static-content.springer.com/image/art%3A10.1007%2Fs11032-008-9216-0/MediaObjects/11032_2008_9216_Fig1_HTML.gif
Fig. 1

Scheme used for data exploring and development of EST-SSRs markers from rubber tree ESTs

The SSRs may be overestimated because of the possibility of the redundancy of ESTs from NCBI database. In order to get non-redundant data, stackPACK v 2.2 was used to cluster the 1233 SSR-ESTs. As shown in Fig. 1, 643 non-redundant ESTs were obtained, this consisting of 461 singletons and 182 clusters. The MISA software identified 799 SSR loci from the 643 non-redundant SSR-ESTs (Fig. 1) among which 112 (17.4%) contained more than one SSR. Of the 799 SSRs loci, 673 (84.2%) were simple repeat motifs while 126 (15.8%) represented the compound type.

The distribution and frequencies of non-redundant EST-SSRs in rubber tree transcriptome were ~1SSR/2.25 kb and most of them were smaller repeat-unit size (Table 2). 362 (45.3%) of the 799 SSR loci were mononucleotide repeats (MNRs) which are proved to be uninformative. 337 (42.2%) of SSR loci were dinucleotide repeats (DNRs). AG/TC motif was the most common among DNRs, accounted for 83.7%, followed by AT/TA (16.0%) and AC/TG (0.3%), and CG/GC was not seen. 95 (11.9%) of SSR loci were trinucleotide repeats, the motif of which were mainly AAG/TTC (38.9%) and AAT/TTA (14.7%). The motif of the two tetranucleotide repeats (TTNRs) was AAAG/TTTC and AAAT/TTTA, respectively. And the three hexanucleotide repeats (HNRs) contained AACCAC/TTGGTG, ACCAGC/TGGTCG and ACCTCC/TGGAGG motifs.
Table 2

Frequency and distribution of SSRs in the analysed 10018 rubber tree ESTs

Repeats motif

Number of repeat units

Total repeats

6

7

8

9

10

11

12

13

14

15

>15

A/T

83

39

36

13

17

19

138

345

C/G

5

3

1

2

2

1

3

17

AC/TG

1

1

AG/TC

30

9

17

13

15

19

8

9

11

7

144

282

AT/TA

19

5

1

2

2

2

1

1

4

17

54

AAC/TTG

1

1

1

3

AAG/TTC

10

7

7

6

3

2

1

1

37

AAT/TTA

2

5

1

1

1

1

2

1

14

ACC/TGG

5

2

2

9

ACG/TGC

3

3

6

ACT/TGA

4

2

1

7

AGC/TCG

7

1

8

AGG/TCC

3

1

3

7

AGT/TCA

2

1

3

CCG/GGC

1

1

AAAG/TTTC

1

1

AAAT/TTTA

1

1

AACCAC/TTGGTG

1

1

ACCAGC/TGGTCG

1

1

ACCTCC/TGGAGG

1

1

N (MNR)

88

42

37

15

19

20

141

362

NN (DNR)

50

14

18

15

17

21

9

10

15

7

161

337

NNN (TNR)

37

21

13

10

4

3

1

2

2

2

95

NNNN (TTNR)

1

1

2

NNNNNN (HNR)

2

3

Development of EST-SSR markers

362 MNRs were excluded from primer design and only 184 (accounted for 42.1%) among the remaining 437 EST-SSRs could be used for designing primers (Fig. 1). The remaining 253 EST-SSRs could not be used for designing primers due to their short flanking regions or unsuitable criteria of the primer design. The 184 SSR loci were embedded within 177 ESTs (85 singletons and 92 clusters) (see Fig. 1), indicating that only 1.8% of the ESTs in this study could be used to develop SSR markers.

Based on BLASTX analysis of 184 SSR loci against the NCBI non-redundant sequence database, putative functions could be assigned to 123 (66.8%) potential markers at a threshold of <1.00E-08, 81 (44%) markers at a threshold of <1.00E-30 and 47 (25.5%) markers at a more stringent threshold of <1.00E-50 (ESM Table 1). Of the 123 annotated SSR-ESTs, 17 (13.8%) were shown to have putative functions in rubber tree, 39 (31.7%) were matched to the protein of Arabidopsis thaliana, 30 (24.4%) were matched to some genes in grape, 15 (12.2%) were matched to genes in alfalfa, and 18 (14.6%) were matched to genes of other dicotyledonous plants such as poplar and only 5 (4.3%) were match to genes in the monocotyledons such as rice, bananas etc.

The polymorphism of developed EST-SSR loci

As mentioned, 184 EST-SSRs were used for primer design. Genomic leaf DNA amplification with these primers showed that 110 primer pairs (59.8%) produced clear bands from 12 cultivated rubber-tree species and four related species. The PCR results showed that 295 alleles were detected by 110 primer pairs among which 23 primer pairs were monomorphic and 87 primer pairs (47.3%) were polymorphic.

Statistical analysis on the products with expected size amplified by the 30 primer pairs on the above-mentioned DNA samples (Table 3) were carried out. 74 alleles were detected in cultivated species and 72 alleles were detected in related species, with an average of 2.47 and 2.4, respectively. The PIC values of cultivated species were 0–0.684 (average 0.383). HBE077, HBE190 and HBE192 detected four polymorphic alleles in cultivated species, respectively, while HBE063 and HBE126 detected four polymorphic alleles in related species. In addition, HBE001 and HBE103 amplified monomorphic bands in the cultivated species while HBE022, HBE056 and HBE090 amplified monomorphic bands in both cultivated species and related species. In summary, the EST-SSR alleles amplified by the 30 primer pairs had moderate level of polymorphism.
Table 3

Marker validation and inter-specific/generic transferability of the 30 working SSR markers

Sequence ID

Marker examined

Tm (°C)

Expected product size (bp)

Marker validation/cross species transferability

Cultivated species (n = 12)

Related species (n = 4)

Intergeneric species (n = 3)

Allele no.

Size range (bp)

PIC

Allele no.

Size range (bp)

Amplification

Allele size range (bp)

EC609907.1

HBE001

55

263

1

262–293

0

2

262–283

1 (1.3)a

265

EC609720.1

HBE004

55

105

3

105–140

0.5938

3

105–140

2 (3)

110–200

EC609118.1

HBE014

55

183

3

161–175

0.559

2

161–171

2 (1)

145–148

EC608908.1

HBE017

55

199

3

170–200

0.5417

3

170–230

3

169–192

EC608805.1

HBE021

55

162

3

149–170

0.5

3

149–175

3

144–267

EC608800.1

HBE022

55

284

1

284

0

1

284

3

284–313

EC608405.1

HBE034

55

222

2

210–250

0.5

2

210–250

3

208–215

EC608110.1

HBE043

55

273

3

243–280

0.4062

2

245–288

3

243–289

EC607870.1

HBE051

55

207

3

190–220

0.6111

2

190–215

3

187–204

EC607524.1

HBE056

55

153

1

153

0

1

153

0

 

EC607362.1

HBE063

55

210

3

192–217

0.6424

4

194–227

2 (3)

194–227

EC607289.1

HBE067

55

239

2

246–252

0.1528

2

252–272

3

252–292

EC606911.1

HBE077

55

255

4

232–262

0.5104

3

230–260

3

240–260

EC606350.1

HBE090

55

196

1

196

0

1

196

3

196–310

EC606060.1

HBE103

55

270

1

270

0

2

250–265

2 (1.3)

219.00

EC605512.1

HBE117

55

238

3

200–233

0.3924

3

200–247

3

148–207

EC605312.1

HBE122

55

212

3

205–228

0.4861

3

210–259

3

150–234

EC605124.1

HBE126

55

242

2

220–245

0.2188

4

225–242

3

223–313

EC604443.1

HBE140

55

192

2

181–191

0.375

2

180–187

3

149–226

EC603602.1

HBE156

55

153

2

148–157

0.4444

2

153–159

1 (2.3)

150.00

EC603146.1

HBE164

55

150

2

110–150

0.0799

2

128–165

3

135–217

EC603048.1

HBE167

55

128

2

110–128

0.0799

2

110–131

3

108–145

EC601817.1

HBE187

55

155

3

154–175

0.5382

2

159–170

3

118–185

EC601635.1

HBE189

55

113

3

100–125

0.5174

3

100–110

3

105–131

EC601511.1

HBE190

55

278

4

277–300

0.684

2

290–308

3

295–320

EC601354.1

HBE192

55

216

4

180–225

0.6354

3

179–213

3

181–205

EC601277.1

HBE193

55

218

3

196–220

0.5382

2

185–204

3

160–284

EC601217.1

HBE196

55

231

3

190–235

0.6215

3

180–220

3

121–220

EC600725.1

HBE200

55

187

2

180–220

0.4965

3

200–225

3

180–233

EC600478.1

HBE206

55

165

2

154–189

0.375

3

168–196

1 (2.3)

167

 

Mean

  

2.47

 

0.3833

2.40

   

aNumber of species showing amplification; Number in the braces (see Table 1) indicate species that failed to amplify

Analysis of genetic diversity

A total of 272 alleles were detected by 87 validated primer pairs from all samples of the 16 plant materials, with an average of 3.12 alleles per polymorphic SSR, slightly higher than that of previous 30 primer pairs. All the polymorphic SSRs (87) were applied to genetic diversity analysis where the results are shown in Fig. 2.
https://static-content.springer.com/image/art%3A10.1007%2Fs11032-008-9216-0/MediaObjects/11032_2008_9216_Fig2_HTML.gif
Fig. 2

Dendrogram of rubber tree varieties based on 87 EST-SSRs primer pairs

As shown in Fig. 2, all 16 materials (12 varieties of cultivated species and four related species) were divided into three groups on the level of similarity coefficient 0.77, H. spruceana, H. Nitida, H. benthamiana and H. pauciflora were in group I, PR107, Haiken2, Reyan 8-79, Reyan88-13, Reken523, Yunyan77-2, Wenchang217 and RRIM600 in group II. IAN873, Reken525, GT1 and Reyan7-33-97 in group III.

Group II can be divided into three subgroups on the level of similarity coefficient 0.84. They are, PR107, Reyan8-79, Reken523 and Haiken2 in subgroup I, Reyan88-13 and RRIM600 in subgroup II, Yunyan77-2 and Wenchang217 in subgroup III.

On the level of similarity coefficient 0.96, all the materials can be distinguished from each other except Reken525 and GT1.

Cross-species/genera transferability

To investigate cross-species and genera transferability, a total of 52 PCR bands of five selected EST-SSRs amplified from some cultivated varieties, related species and intergenera materials as well were cloned and sequenced, and the results are shown in Fig. 3. The expected SSR loci were amplified in all material investigated with variation of the repeated number of SSRs and their flanking sequences on occasion. The homology products in Ricinus communis L. and Manihot utilissima amplified by primer pairs HBE114 and HBE140 validated the cross-species/genera transferability of the developed EST-SSRs in this study.
https://static-content.springer.com/image/art%3A10.1007%2Fs11032-008-9216-0/MediaObjects/11032_2008_9216_Fig3_HTML.gif
Fig. 3

Sequences obtained using five EST-SSRs markers amplifying across intraspecies, interspecies and intergenera. (ae) Represent the marker HBE014, HBE117, HBE140, HBE156, HBE206, respectively; RefESTs in (ae) represent the accession numbers: EC609118.1, EC605512.1, EC604443.1, EC603602.1 and EC600478 in NCBI database, respectively. The suffix ‘a1’ and ‘a2’ represent the allele numbers. H.spr., H.nit., H.ben., H.pau., R.com., M.uti., Ry88-13, Ry8-79 represent Hevea spruceana, Heveanitida, Hevea benthamiana, Hevea pauciflora, Ricinus communis L., Manihot utilissima, Reyan88-13, Reyan8-79, respectively

Discussion

SSR frequency and distribution

EST-SSR frequency was ~1/2.25 kb for rubber tree transcriptome, which was higher than that of wheat (15.6 kb) (Kantety et al. 2002), barley (~1/6.3 kb) (Thiel et al. 2003), and Arabidopsis thaliana (~1/13.83 kb), tomato (~1/11.1 kb), cotton (~1/20.0 kb), soybean (~1/7.4 kb) and poplar (~1/14.0 kb), etc. (Cardle et al. 2000). However, it was lower than coffee (~1/1.56 kb) (Aggarwal et al. 2007). The distribution, frequency and abundance level of EST-SSR may fluctuate due to SSR search criteria, the size of the database and SSR development tools (Varshney et al. 2005). For example, Rota et al. (2005) found that, in rice, the frequency of EST-SSRs was reduced from 50 to 1% when SSR search criteria changed from 12 to 30 bp. In this study, we have also shown that changing the SSR search criteria also resulted in frequency fluctuation of EST-SSRs in rubber-tree, i.e., if the criteria was: MNR ≥ 10, DNR ≥ 6, TNR ≥ 5, TTNR to HNR ≥ 4, and the EST-SSR frequency would be ~1/1.82 kb.

Up to now, DNRs and TNRs were mostly reported in plants, but the dominant repeat motifs were different. For example, TNRs were the leading repeat motif in Arabidopsis thaliana, wheat, rice, corn, soybean (Cardle et al. 2000), sugar cane, grapes (Cordeiro et al. 2001), barley (Thiel et al. 2003) and citrus (Chen et al. 2006) while DNRs were dominant in Kiwi (Fraser et al. 2004), apricot and peach (Jung et al. 2005), coffee (Aggarwal et al. 2007). In rubber tree, based on our results, DNRs (42.2%) were the leading repeat motif, followed by TNRs (11.9%), if not considering the MNRs. In addition, AG/TC were the predominant DNRs, which was consistent with the report of Roy et al. (2004). Observation of AAG/TTC as the leading repeat motif of TNRs in the rubber tree, is similar to that in Arabidopsis thaliana (Cardle et al. 2000), soybean (Gao et al. 2003), and citrus (Chen et al. 2006). No PNRs in our study was possibly related to the stringent searching criteria. In fact, PNRs could be obtained when the searching criteria was reduced (data not shown). This also verified an earlier viewpoint that EST-SSR distribution and frequency were related to the search criteria (Varshney et al. 2005).

The GC repeat motif is rare in most plants. There were no GC repeat motifs found in rice, corn, soybean (Gao et al. 2003), wheat (Nicot et al. 2004), Arabidopsis thaliana, apricot and peach (Jung et al. 2005). However, Aggarwal et al. (2007) observed that the GC/CG repeat motif was at a very low frequency in coffee EST-SSRs. The GC/CG repeat motif was not found in rubber tree EST-SSRs either in our study, which is consistent with the results in other plants.

Novel EST-SSR molecular markers

Hitherto, little work has been done on the development and application of SSR markers in rubber tree genetic and breeding studies. Previously, rubber tree SSR markers were developed by screening genomic library with probe containing microsatellite sequence. In doing so, only 23 SSR primer pairs were successfully designed among 39 SSR (Lespinasse et al. 2000). Although SSR marker development strategy was reported in other plant species (Ramakrishna et al. 1998; Davierwala et al. 2001; Varshney et al. 2005), no SSR markers of rubber tree have been developed based on their cross-species/genera transferability i.e. the homology of DNA sequence among species and/or genera. Among 184 primer pairs designed based on SSR-ESTs in rubber tree, 59.3% resulted in clear bands from not only cultivated rubber tree species but also related species and 47.3% produced polymorphic bands. This level of polymorphism was lower compared to that of genome-developed SSR marker in other crops (Eujayl et al. 2002; Ferguson et al. 2004; Gonzalo et al. 2005; Pinto et al. 2006) which might be related to the EST conservatism (Dreisigacker et al. 2004; Varshney et al. 2005). If not the unavoidable inadequate primer design, then the possible reason that 74 primer pairs failed to be amplified might be introns existing between the two primers (Varshney et al. 2005), for the primer design criterion of the expected product size was less than 300 bp, this could not completely eliminate the probability of pre-amplified region inserting introns.

Polymorphism analysis and cross-species/genera transferability

The PIC value amplified by 30 primer pairs across the cultivated species was 0.383, which represented the medium polymorphism of our developed EST-SSRs. These results indicated that the 30 markers would be useful for genetic and breeding studies in rubber tree. Only three of the 30 primer pairs in related species and five in cultivated species amplified monomorphic bands. Among all the polymorphic primer pairs, 9 of 25 (36.0%) in cultivated species and 9 of 27 (33.3%) in related species could amplify 12–20 bp of SSR repeat motif, respectively, while 16 of 25 (64.0%) in cultivated species and 18 of 27 (66.7%) in related species could amplify 20 bp of SSR repeat motif or more, respectively. The results showed that the polymorphism of SSRs with repeat motif of 20 bp or more was higher than that of SSRs with repeat motif of 12–20 bp, confirmed the conclusion in rice research by Temnykh et al. (2001). Dreisigacker et al. (2004) found that higher-order repeat motifs (higher-order repeat motif refers to repeat motif more than three bases as well as the compound repeat) have lower polymorphism than lower-order repeat ones. In our study, only one higher-order repeat motifs (4%) in the cultivated species and two (7.4%) in related species were detected by the above-mentioned polymorphic primer pairs, and the lower-order repeat motifs were predominant among all the polymorphic SSR loci. One possible explanation that the higher-order repeat motifs were less polymorphic was their lower slippage possibilities during the process of DNA replication.

Recent studies showed that the transfer efficiency of EST-SSRs was higher than that of genomic SSRs in cross-species/genera. Liewlaksaneeyanawin et al. (2004) compared the transferability of 14 Pinus taeda (loblolly pine) EST-SSRs from public domain EST databases and 99 traditional microsatellite markers (including seven genomic SSRs) and found that EST-SSRs had higher transfer rates than the traditional microsatellite markers. Peakall et al. (1998) used the SSR markers developed from soybean genome amplifying across Glycine max and the results showed that 65% of the markers can be amplified in cross-species but only 3–13% in cross-genera. Similar results were obtained in coffee (Baruah et al. 2003; Aggarwal et al. 2007). Transferability of EST-SSRs among closely related genera has been reported in many more crop species such as Actinidia chinesis, Oryza, Arabidopsis, apricot and grape (Huang et al. 1998; Chen et al. 2002; Clauss et al. 2002; Decroocq et al. 2003). In the study by Gao et al. (2005), the transfer rate decreased slightly in the more closely related species and became lower in the most distantly related species in the genus. According to Liewlaksaneeyanawin et al. (2004), the transferability success decreased as the evolutionary distance between the source and target species increased. In our study, all of the randomly selected 30 EST-SSR markers developed from the rubber tree can be amplified in both cultivated species and related species while 73.3% of them can be amplified in cross-genera completely. Our results show that the transfer rate decreased from 93.3% of Manihot utilissima, 90% of Ricinus communis L., to 80% of Phyllanthus emblica and seemed to indicate that the Phyllanthus emblica had a more distantly relationship with Hevea than Ricinus communis L. and Manihot utilissima.

Evaluation of genetic diversity using the developed EST-SSR markers

Hevea brasiliensis has had a long history of cultivation since its introduction to China in 1904. Due to limitation in China climate conditions, they are distributed in areas south of latitude 24° no. Among the 12 varieties of the cultivated species investigated in this study, eight varieties were bred by Rubber Research Institute of China. As for the other four varieties, RRIM600 was bred in Malaysia, PR107 and GT1 in Indonesia and IAN873 in Brazil. Based on 272 alleles detected by 87 EST-SSR markers reported in this study, the high-yielding Reyan8-79 clone (descendant from a RRIM600 × PilB84 cross) and the high-yielding and wind-resistant Ry88-13 clone (descendant from Reyan8-79 × Reyan217 cross) were clustered in the same category. This clustering result coincided with the pedigree analysis. Our analysis also showed that the Yeyan77-2, Wenchang217, Haiken2 varieties were clustered in the same category as PR107. Yunyan77-2 is a descendant of a GT1 × PR107 cross and is a high-yielding, fast-growing and cold-tolerance variety. Wenchang217 is a high-yielding and wind-resistant variety from a Haken1 × PR107 cross while Haiken2 is a wind-resistant and high-yielding variety from a PB86 × PR107 cross. Clearly, the three varieties (Yunyan77-2, Wenchang217, Haiken2) which have been clustered in the same category share a common parent, PR107. As PR107 is known for its characteristic high dry rubber yield, thick leaves and fast growth, this clustering result coincided with the pedigree analysis as well.

The narrow genetic basis crops is the bottleneck of genetic improvement especially for the traits of resistance and adaptability (Callow et al. 1997). Utilization of wild species, local varieties and mutants would be an effective way to change this situation (Swanson 1996). Therefore, it is important for plant breeders to evaluate genetic diversity comprehensively not only in various cultivated plant varieties but also in related wild species and multifarious mutants of plants. Previous studies have indicated that the genetic basis of H. brasiliensis is very narrow (Varghese 1992). The related species had about 77% similarity with the cultivated species: it seemed to represent the potential value of genetic diversity on future genetic improvement of cultivated H. brasiliensis. The results of the dendrogram of rubber tree varieties could provide a reference of selection of parents in genetic breeding.

Validation of cross-species/genera amplicon

Cross-species/genera transferability of SSR markers has been previously reported (Ramakrishna et al. 1998; Davierwala et al. 2001). The results of amplification of expected products by five EST-SSRs in 10 materials of cultivated species, related species and intergenera, respectively, together with their homology to the original EST sequences (the RefEST) provided clear evidence for the conservatism and transferability of the EST-SSR markers in H. brasiliensis. It also supported the fact that SSR markers can be obtained through the transfer amplification (Varshney et al. 2005).

However, compared to the RefEST, insertions and deletions were detected in the regions of SSR motifs in intraspecies, interspecies and intergenera while point mutations or deletions in flanking regions were found more often in intergenera and less in interspecies. Gutierrez et al. (2005) found that the variations in the sequences are mainly due to the diversification of the number of repeat motifs in the SSR region combined with insertions and base substitutions in Medicago truncatula. Xie et al. (2006) had identified that allelic size variation in almond resulted exclusively from differences in the structures of repeat motifs which involved interruptions or occurrences of new motif repeats in addition to varying number of AG/CT repeats. Guo et al. (2006) reported that no correlation was found between the repeat motif type and cross-species amplification. No affirmative pattern of allelic variation and transferability and their certain relationship to the repeat motif type for the number of repeat was confirmed in our study were six or seven with trinucleotide repeat motif.

Acknowledgements

Project support was provided by the National Non-profit Institute Research Grant. Prof. Jun Zhang of Shandong Academy of Agricultural Science and Prof. Xing-Quan Zhu of South China Agricultural University are thanked for their constructive comments on the content of the manuscript. Dr. Keng-See Chow of the Rubber Research Institute of Malaysia is thanked for the critical reading of the manuscript. Prof. John M. Woodruff, University of Georgia, is thanked for the English usage.

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© Springer Science+Business Media B.V. 2008