Development of new genomic microsatellite markers from robusta coffee (<it>Coffea canephora </it>Pierre ex A. Froehner) showing broad cross-species transferability and utility in genetic studies

1471-2229-8-51 1471-2229 Research article Development of new genomic microsatellite markers from robusta coffee (<it>Coffea canephora </it>Pierre ex A. Froehner) showing broad cross-species transferability and utility in genetic studies Hendre Suresh Prasad prasadhendre@gmail.com Phanindranath Regur phanindra@ccmb.res.in Annapurna V purnavneni@yahoo.com Lalremruata Albert albert.ccmb@gmail.com Aggarwal K Ramesh rameshka@ccmb.res.in

Centre for Cellular and Molecular Biology (CCMB), Uppal Road, Tarnaka, Hyderabad- 500 007, Andhra Pradesh, India

BMC Plant Biology 1471-2229 2008 8 1 51 http://www.biomedcentral.com/1471-2229/8/51 18447947 10.1186/1471-2229-8-51 27 9 2007 30 4 2008 30 4 2008 2008 Hendre et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background

Species-specific microsatellite markers are desirable for genetic studies and to harness the potential of MAS-based breeding for genetic improvement. Limited availability of such markers for coffee, one of the most important beverage tree crops, warrants newer efforts to develop additional microsatellite markers that can be effectively deployed in genetic analysis and coffee improvement programs. The present study aimed to develop new coffee-specific SSR markers and validate their utility in analysis of genetic diversity, individualization, linkage mapping, and transferability for use in other related taxa.

Results

A small-insert partial genomic library of Coffea canephora, was probed for various SSR motifs following conventional approach of Southern hybridisation. Characterization of repeat positive clones revealed a very high abundance of DNRs (1/15 Kb) over TNRs (1/406 kb). The relative frequencies of different DNRs were found as AT >> AG > AC, whereas among TNRs, AGC was the most abundant repeat. The SSR positive sequences were used to design 58 primer pairs of which 44 pairs could be validated as single locus markers using a panel of arabica and robusta genotypes. The analysis revealed an average of 3.3 and 3.78 alleles and 0.49 and 0.62 PIC per marker for the tested arabicas and robustas, respectively. It also revealed a high cumulative PI over all the markers using both sib-based (10^-6and 10^-12for arabicas and robustas respectively) and unbiased corrected estimates (10^-20and 10^-43for arabicas and robustas respectively). The markers were tested for Hardy-Weinberg equilibrium, linkage dis-equilibrium, and were successfully used to ascertain generic diversity/affinities in the tested germplasm (cultivated as well as species). Nine markers could be mapped on robusta linkage map. Importantly, the markers showed ~92% transferability across related species/genera of coffee.

Conclusion

The conventional approach of genomic library was successfully employed although with low efficiency to develop a set of 44 new genomic microsatellite markers of coffee. The characterization/validation of new markers demonstrated them to be highly informative, and useful for genetic studies namely, genetic diversity in coffee germplasm, individualization/bar-coding for germplasm protection, linkage mapping, taxonomic studies, and use as conserved orthologous sets across secondary genepool of coffee. Further, the relative frequency and distribution of different SSR motifs in coffee genome indicated coffee genome to be relatively poor in microsatellites compared to other plant species.

Background

Coffee tree, a member of the family Rubiaceae, belongs to the genus Coffea that comprises > 100 species. Of these two species, the tetraploid Coffea arabica L. (i.e. arabica coffee; 2n = 4x = 44) and the diploid C. canephora Pierre ex A. Froehner (i.e. robusta coffee; 2n = 2x = 22), are cultivated commercially. Coffee, one of the most popular non-alcoholic beverages, is consumed regularly by 40% of the world population mostly in the developed world 1, and thus occupies a strategic position in the world socio-economy.

Efforts undertaken globally to improve coffee, though successful, have proven to be too slow and severely constrained owing to various factors. The latter includes: genetic and physiological makeup (low genetic diversity and ploidy barrier in arabicas, and self incompatibility/easy cross-species fertilization in robustas), long generation cycle, requirement of huge land resources, and equally the dearth of easily accessible and assayable genetic tools/techniques for screening/selection. The situation warrants recourse to newer, easy, practical technologies that can provide acceleration, reliability and directionality to the breeding efforts, and allow characterization of cultivated/secondary genepool for proper utilization of the available germplasm in genetic improvement programs. In this context, development of DNA marker tools and availability of markers-based molecular linkage maps becomes imperative for MAS-based accelerated breeding of improved coffee genotypes.

Among the different types of DNA markers, the Short Sequence Repeats (SSR) based microsatellite markers promise to be the most ideal ones due to their multi-allelic nature, high polymorphism content, locus specificity, reproducibility, inter-lab transferability and ease for automation 2. Microsatellite markers have been developed for a large number of plant species and are increasingly being used for ascertaining germplasm diversity, linkage analysis and molecular breeding 3. Despite these advantages, only ~180 microsatellite markers have been reported till to date for coffee 456789101112, signifying the need for expanding the repertoire of these genetically highly informative markers for efficient management and improvement of coffee germplasm resources. Here we report, a set of 44 novel microsatellite markers developed by radioactive screening of a small-insert partial genomic library of C. canephora (robusta coffee). Interestingly, all these markers exhibit broad cross-species transferability. We also demonstrate their utility as genetic markers for ascertaining the germplasm diversity, genotype individualization, linkage mapping and taxonomic affinities.

Results

The present study aimed to isolate new coffee-specific informative SSRs useful as genetic markers for characterizing coffee genome and linkage mapping studies. For the purpose, a partial small-insert genomic library was constructed from a commercially cultivated robusta variety 'Sln-274'. The library was screened using radioactive SSR oligo probes to isolate SSR-containing DNA fragments, which were sequenced and used for designing primer pairs from the flanking regions and subsequent conversion to PCR-based SSR markers. The designed primer pairs were standardized for PCR amplification, and then validated for utility as genetic markers using panels of elite coffee genotypes, a mapping population for linkage studies, and related taxa of coffee for cross-species transferability. In addition, sequence data of the screened and putative SSR-positive selected clones were used to assess the relative abundance of different SSR motifs in robusta coffee genome. In total 44 new highly informative SSR markers are developed.

Screening/Identification of SSR positive genomic sequences from the small insert partial genomic library of Sln-274

The small-insert partial genomic library constructed from robusta variety Sln-274 comprised 15,744 clones. Radioactive screening of the arrayed and blotted clones indicated 446 putative positives of which good quality sequence data could be obtained for 199 clones. The average insert size of the sequenced clones was 773.5 bp. Considering the latter, and that the sequenced clones represented a random sample of the genomic library with respect to the size, the total size of the cloned genome amounted to 12.2 Mb which equaled to ca. 1.5 % of the robusta coffee genome 13 (Table 1). SSR search of the clone sequences using the MISA search module, detected 76 genuine SSR-positive clones (0.48% of the total library) containing both targeted and non-targeted SSR motifs. Overall, these clones contained 92 SSRs comprising DNRs (48.3%), TNRs (25.9%), and HO-NRs (4.8%), and 24 SSRs comprising only MNRs (20.7%) (Table 1, 2). Among the targeted repeat motifs (screened SSR-oligo nucleotides), AG was the most abundant repeat (26.7%), followed by AC (12.9%) and AGC (7.8%), whereas CCG (0.9%) was the least abundant and ACT was not detected at all (Table 2). Similarly, among the non-targeted SSR motifs other than MNRs, AT was the most abundant repeat (8.6%, Table 2).

Table 1

Summary statistics of screening of the small-insert partial genomic library of robusta coffee for putative SSR positive clones/sequences and SSRs.

Summary of Screening/sequencing

Total Number of clones screened (X)

15,744

Number of clones selected and sequenced after screening

446 (2.83% of X)

Number of good quality sequences obtained

199 (1.27% of X)

Total number of SSR containing clones (Y)

76 (0.48% of X)

Number of sequences containing more than 1 SSR core

26 (34.21% of Y)

Number of sequences containing compound SSRs

15 (12.93% of Y)

Number of SSR+ sequences used for primer design/synthesis

58 (0.37% of X)

Number of working primer pairs

53 (0.34% of X)

Average size of the cloned/sequenced insert

773.5 bp

Haploid genome size of C. canephora [13]

809 Mb

Estimated genome screened (number of library clones x. average insert size)

12.2 Mb (1.5 % genome equivalent)

C. canephora genome sequenced (good quality sequences × average insert size)

0.15 Mb (0.01 % of robusta genome)

Summary of SSRs identified in the library

Number of non-targeted MNRs of minimum 12-mer length (a)

24 (0.15% of X)

Number of targeted DNRs having a minimum of 6 repeats (b)

46 (0.29% of X)

Number of non-targeted DNRs having a minimum of 6 repeats (c)

10 (0.06% of X)

Total number of DNRs (b+c)

56 (0.36% of X)

Number of targeted TNRs having a minimum of 5 repeats (d)

30 (0.19% of X)

Total number of DNRs and TNRs (b+c+d)

86 (0.55% of X)

Total Number of non-targeted HO-NRs having a minimum of 5 repeats (e)

6 (0.04% of X)

Total Number of DNRs, TNRs and HO-NRs (b+c+d+e)

92 (0.58% of X)

Total Number of SSRs (a+b+c+d+e)

116 (0.73% of X)

Table 2

Summary statistics of distribution and abundance of detected SSRs in the tested genomic library and SSR frequency estimates for robusta coffee genome

SSR motif

SSRs detected in the library (% of total SSRs)

Mean no. of repeats/SSR (Range of repeat iterations in the SSR core)

Estimated number/distance of SSRs in the robusta coffee genome

Total SSRs/genome (X = n.a/b)*

SSRs/Mb genome (Y = X/a)

SSR spacing in the genome^@(Z = 1000/Y)

Targeted SSRs (DNRs^T+ TNRs^T)

31(26.7)

10.0 (6 to 29)

2057

2.5

393

15 (12.9)

8.4 (6 to 14)

995

1.2

812

DNRs^T

46 (39.7)

9.6 (6 to 29)

3053

3.8

265

AGC

9 (7.8)

6.8 (5 to 10)

597

0.7

1354

ATC

4 (3.5)

5.0 (5)

265

0.3

3048

ACG

3 (2.6)

6.7 (5 to 9)

199

0.3

4063

ACC

3 (2.6)

5.7 (5 to 7)

199

0.3

4063

AAT

3 (2.6)

5.3 (5 to 6)

199

0.3

4063

AAC

3 (2.6)

5.0 (5)

199

0.3

4063

AGG

2 (1.7)

6.0 (5 to 7)

133

0.7

6095

AAG

2 (1.7)

5.5 (5 to 6)

133

0.7

6095

CCG

1 (0.9)

6.0 (6)

0.1

12190

ACT

TNRs^T

30 (25.9)

5.9 (5 to 10)

1991

2.5

406

SSRs^T

76 (65.5)

8.3 (5 to 29)

5044

6.2

160

Non-targeted DNRs (DNRs^NT)

AT/AT

10 (8.6)

10.3 (6 to 23)

50563^#

62.50

Miscellaneous non-targted SSRs

A/T

21 (18.1)

C/G

3 (2.6)

Note: Three of these MNRs were detected as part of the compound SSR motifs

AAAT

2 (1.7)

AAGTGG

2 (1.7)

AATT

1 (0.7)

AAAAAT

1 (0.7)

DNRs^T+NT

56 (48.3)

11.5 (6 to 29)

53616

66.3

DNRs^T+NT& TNRs^T

86 (74.1)

9.5 (5 to 29)

55607

68.7

nc: Not calculated

*: X = estimated number of SSRs in genome; n = No. of detected SSRs in the library; a = 809 Mb -size of the haploid robusta genome [13]; b = 12.19 Mb- size of the screened robusta genome (see table 1)

^#: b = 0.16 Mb -size of genome sequenced

^@: Distance (in Kb) between two consecutive SSRs

^T: Targeted SSRs; ^NT: Non-targeted SSRs

Frequency and distribution of SSRs in coffee genome

A total of 76 targeted SSRs (DNRs and TNRs) and 10 non-targeted DNRs were assessed for their lengths, distribution in the present library, and their relative abundance in the robusta genome (Table 2). Average length (in terms of repeat units) for the DNRs and TNRs was 9.6 and 5.9, respectively. Among DNRs, AT and AG were comparable and longer than AC, whereas ACG and AGC were the longest of the TNRs (Table 2). The size of cloned/screened genomic library and the observed data for identified SSRs were considered along with the earlier predicted size of the robusta genome 13 to derive relative estimates for frequency/distribution of different SSR motifs in the robusta genome. The analysis revealed coffee genome to be enriched in AT type DNRs (AT-DNR), which were estimated to be many fold more than any other SSR motifs (targeted and/or non-targeted). The results indicated one AT-DNR per 16 Kb (1/16 Kb) of robusta genome; this was almost 20-fold higher than the next most abundant DNR i.e. AG (ca. 1/393 Kb). The DNRs as a single class were estimated to be 1/15 Kb genome when AT (comprising 94% of the total DNRs) was included, and 1/265 Kb coffee genome for the remaining ones. In comparison, the overall frequency of TNRs was calculated to be 1/406 Kb with AGC being the most predominant (ca. 1/1300 Kb) and CCG the least (ca. 1/12200 Kb). In addition, a few other higher order SSRs (mainly the AT-rich) were also detected but these were not used for estimate calculations, as their numbers were very low. Thus, the present study indicated an abundance of one SSR (either DNR or TNR) per 15 Kb of robusta coffee genome, wherein the DNRs were ~27 times more abundant than the TNRs.

Development of microsatellite markers

All the identified SSR-positive sequences were tried to design primer pairs for conversion to microsat markers using 'SSR motif length' (of ≥ 7 and 5 repeats for DNRs and higher order SSRs, respectively) as one major criterion. As a result, only 56 of the total 92 identified SSRs (all except MNRs) were found suitable for primer design indicating 60.9% primer suitability. These comprised 42.2% DNRs, 40.7% compound SSRs, 6.8% TNRs, 5.1% TtNRs and 1.7% HNRs. In addition, primers were also designed for 2 of the randomly chosen 14 MNRs to test their potential for conversion to SSR markers. Among the SSRs found unsuitable for primer design, 70.6% had shorter motif length and 29.4% had flanking regions unsuitable for primer modeling. Of the 58 potential primer pairs designed, 52 could be successfully amplified and 44 of these could further be validated (Table 3, 4) as useful markers indicating ~76% primer to marker conversion ratio.

Table 3

Details of the newly developed SSR primers

Sl. No.

Primer Id

Primer sequence (F: Forward; R: reverse)

Repeat unit

Ta (°C)

Amplicon (bp)

GenBank accession No.

Linkage group

CaM02

F: CGCCAGCCACAGCCACTTGC

(AGG)7

224

EU526557

R: GCGGGGGTAAGAAAGAGGCGAG

CaM03

F: CGCGCTTGCTCCCTCTGTCTCT

(AC)11

173

EU526558

CLG03

R: TGGGGGAGGGGCGGTGTT

CaM06

F: ACCCGATATTCAACCGACATGC

(CT)7

278

EU526559

R: CATGACTTGAGCGCTAATATTTGAT

CaM08

F: CAGCTGAAGTGGTGAAAAACAAGAG

(TC)8

202

EU526560

R: CGCTTTCTTGTTTTCTCCATTTCAG

CaM09

F: CAGGAAGAGAAGAAAGTGAAATTGAC

(TC)8

137

EU526560

R: CGCTTTCTTGTTTTCTCCATTTC

CaM11

F: GTCCCCGCTTAAATAATATACACACA

(AC)8–15 bp-AC(6)(AT)6

285

EU526561

R: ATAGGACGGAGGGAGTAATAGAATAAA

CaM12

F: TTCGGGCTCACCTGGCAG

(CAG)10

155

EU526562

R: CGCGGAAGCAGGACATGGATT

CaM13

F: CCTCGCCCTCAATCACCTCCTAG

(AAAT)5

287

EU526563

R: GGCTCCCCAAGAATCCTCAACTC

CaM15

F: AGCCCTAGACGAGATGGATTCC

(CAG)5

170

EU526564

R: CGGCTCCTTCTGCACTCCCATTT

CaM16

F: AAGGCAGCTGAAGCGGGACAAA

(TC)11

199

EU526565

CLG11

R: TGGGGAGAGCTGCAGTTGGAGG

CaM17

F: CGGGCGTTTCTTCTTTTGAGTTGC

(GTC)6

212

EU526566

R: TCACGGTTTCTCAAGTCGGGGATTTA

CaM18

F: CCGACTTGGACTGATGCGAAATTGA

(TC)9

181

EU526567

R: AAAGCAAAAAACCAGAAAACACGAAGA

CaM20

F: GAAACCGCTGAAATTCGGTA

(TATGGG)3

217

EU526568

CLG16

R: CCCTCTGATTTCTCCTTTCATC

CaM21

F: GGGCTTACCGACCGCTCACAG

(TC)8

161

EU526569

R: CCGCTATTGTTGCTGCTATGGAGTTG

CaM22

F: CCCCTCCTCCTCCTACTAGATGGTGGT

(AT)15

113

EU526570

CLG02

R: GGTCCAGGGTCCATCCATTCTTGA

CaM23

F: TGCTTGTAAGGGAATTTCTGGTCAG

(AATT)5

154

EU526571

R: GTGCGAATGTGGAACCTTTTAAGTCA

CaM24

F: GGATTCGACAAGGTTGGCAGAGC

(CCT)5–87 bp-(CTG)6

193

EU526572

R: TGCCGAAGAAGAGGGAGATAGTGATG

CaM25

F: TCCATCTTCCTTCATTTCTGCTGCTAA

(GA)9

186

EU526573

R: CCTTCACCCCCTTTGCACTTCCTTA

CaM26

F: CGTTGCCATTTCTTCCCTTCTTTCTTC

(TG)7–21 bp-(GA)9

236

EU526574

R: ACACCTTACCCCCTTATCGTTTAGAA

CaM27

F: AAGAGTGTTTGGGATTGCATTTTTAT

(TA)7(GT)14

178

EU526575

R: CCGCGTAGGCTTTGTTTGG

CaM30

F: TTGCCTTCCGGATTTTTGATTCA

(CA)6(TA)5

222

EU526576

R: AGTTCTAAGGCTGAGGCGGCTAAAG

CaM31

F: ATCCACTGCTGTCACCTTTTGTTA

(TAA)5

261

EU526577

R: AGCAGTGTGTGTGTTAAAGAGGAGTT

CaM32

F: CAGACAGACCAGAGAGAGACACCTAAC

(TA)12

204

EU526577

CLG12

R: CCCCCTCCAAAATAATTCAGAAAA

CaM33

F: GCGCATTAGGCGTGGGAGAA

(A)13–5 bp-(AG)18

240

EU526578

R: CAGAGGTTGTCGGTCAGGTGGAGAA

CaM34

F: CTCCAAATTATTAAGCACAACAAACAA

(GA)10

202

EU526579

R: ATCCGCCTCCAGGTCTTATCC

CaM35

F: CGAGCTAGAATGGATGACTTGGTTGG

(TGGAAG)5

203

EU526580

CLG04

R: GTTGCTCGCACCCGCTTCC

CaM36

F: TGGTTTTAGTTTGTTTATTTTGATGTGAT

(TTA)7

185

EU526581

R: CGAGCCCTCCCCTTGCA

CaM38

F: GAAGCTGAAGCGGGAGGGTAGTAATT

(G)13(GA)7

228

EU526582

R: CCCATCCACCCAACCTTCATTTC

CaM39

F: GAGCAGAGGGAGACGGTGTGGT

(GA)12

196

EU526583

R: CGCGCAACTCTTCGAACTCTAACC

CaM40

F: TTGACACGAAACAGGAAATAAATATAG

(CGA)8

238

EU526584

R: CCCTTCCCCTCATAGCCCTTT

CaM41

F: CATCGTCTCCATCGTTGCTCTATC

(TAAA)5

242

EU526585

R: CCCTCCCCCTCTTTCCTATCTAAT

CaM42

F: TGGGTCAAGGATCCGTGTAAGAAAGA

(CT)8

191

EU526586

CLG01

R: CCCTCACCAGTTCCCGATGTCAG

CaM43

F: CCTGACCGTGAACCTGACCGTGAC

(CT)8

202

EU526587

R: TCGGGACTTGTTTTGGTTTTTGGGT

CaM44

F: TGCTCTTGCCCTCTTTCATCC

222

EU526588

CLG09

R: TCCCGAAAAAGAAAATAAGATAAAGAG

(CT)9

CaM45

F: CGCGGCCAGTGAATTCGAGCTC

(GT)8(GA)5

218

EU526589

R: TCGCCATTTGGAGCTGCTGATTCA

CaM46

F: TGGTGCGGTGTTTTTCAGTTTGGAGA

(AT)9 (AC)12

222

EU526590

CLG11

R: AACCACCCACGCCCACCAATTAAAT

CaM49

F: CCGGTTAATACATTGGTCTTT

(A)33

200

EU526591

R: ATGACATTGTTGACTTTGCTATAA

CaM52

F: TGCCACTCGGAGCTCACTTCA

(CCG)6

160

EU526592

R: GGCTGCCGAGGTTCCAATT

CaM53

F: TTAGGTGTGAGGAGGGATGGGACTG

(GGC)9

172

EU526593

R: CCACAGACTCCTCGTTCGGCAATC

CaM54

F: ACGGGTGAGTCGAAGGGGGAGCAGT

(GGCAGA)4–22 bp-(GCA)9

185

EU526593

R: CACGCCGGCCCACATCTCGAAA

CaM55

F: ATGGGGGGTGTCGGTCTATGTGA

(GA)4(G)4 (A)27

183

EU526594

R: CGCAATTCGCTGTCACCTCCG

CaM57

F: CGAACTCGAACTCAAGCTCAGA

(TA)23

190

EU526595

R: AAGGATATATACGGTAATTTTA

CaM58

F: ACCCCCTCTCCCTCTCCATTTTTAC

CAGA(CA)7

192

EU526596

R: GCACGAGGATGGAGCAGAGCACT

CaM59

F: AAGTGAGTGGTTGTGGCATTAAAT

GATA(GA)8

229

EU526591

R: TTCTTACAAAATCTCATCCCCTCAT

CaM: Canephora Microsatellite marker; '--': Unmapped; these were not polymorphic among parents of the tested mapping population; CLG: Combined Linkage Group (as per [13]). The amplicon size is based on the original clone of Sln-274 genomic library from which the marker was designed.

Table 4

Allelic diversity attributes of new SSR markers as revealed across elite genotypes of arabica and robusta, and related coffee taxa

Primer Id

C. arabica (n = 8)

C. canephora (n = 8)

Coffea spp. (n = 12)

Psilanthus spp. (n = 2)

N_A

PA^$

Allele range

H_o

H_e

PIC

N_A

PA^$

Allele range

H_o

H_e

PIC

N_A

PA^$

Allele range

N_A

PA^$

Allele range

CaM02

252–262

Duplicated loci

256–268

0.71

0.69

0.67

4^a,c,k,l

252–278

262–272

CaM03

1⁶

164–184

0.38

0.74*

0.70

171–194

0.63

0.88

0.83

5^c,e,f,j,l

165–201