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BMC Research Notes

Open Access

Microsatellite markers for Urochloa humidicola (Poaceae) and their transferability to other Urochloa species

  • Jean CS Santos1,
  • Mariana A Barreto1,
  • Fernanda A Oliveira1,
  • Bianca BZ Vigna2 and
  • Anete P Souza1, 3Email author
BMC Research Notes20158:83

https://doi.org/10.1186/s13104-015-1044-9

Received: 18 August 2014

Accepted: 3 March 2015

Published: 15 March 2015

Abstract

Background

Urochloa humidicola is a warm-season grass commonly used as forage in the tropics and is recognized for its tolerance to seasonal flooding. This grass is an important forage species for the Cerrado and Amazon regions of Brazil. U. humidicola is a polyploid species with variable ploidy (6X–9X) and facultative apomixis with high phenotypic plasticity. However, this apomixis and ploidy, as well as the limited knowledge of the genetic basis of the germplasm collection, have constrained genetic breeding activities, yet microsatellite markers may enable a better understanding of the species’ genetic composition. This study aimed to develop and characterize new polymorphic microsatellite molecular markers in U. humidicola and to evaluate their transferability to other Urochloa species.

Findings

A set of microsatellite markers for U. humidicola was identified from two new enriched genomic DNA libraries: the first library was constructed from a single sexual genotype and the second from a pool of eight apomictic genotypes selected on the basis of previous results. Of the 114 loci developed, 72 primer pairs presented a good amplification product, and 64 were polymorphic among the 34 genotypes tested. The number of bands per simple sequence repeat (SSR) locus ranged from 1 to 29, with a mean of 9.6 bands per locus. The mean polymorphism information content (PIC) of all loci was 0.77, and the mean discrimination power (DP) was 0.87. STRUCTURE analysis revealed differences among U. humidicola accessions, hybrids, and other Urochloa accessions. The transferability of these microsatellites was evaluated in four species of the genus, U. brizantha, U. decumbens, U. ruziziensis, and U. dictyoneura, and the percentage of transferability ranged from 58.33% to 69.44% depending on the species.

Conclusions

This work reports new polymorphic microsatellite markers for U. humidicola that can be used for breeding programs of this and other Urochloa species, including genetic linkage mapping, quantitative trait loci identification, and marker-assisted selection.

Keywords

MicrosatelliteGenomic librarySSR transferabilityForageGrass

Findings

Background

Urochloa humidicola (Rendle) Morrone & Zuloaga (syn. Brachiaria humidicola (Rendle) Schweick.), commonly known as koronivia grass, is a perennial tropical grass native to eastern Africa that was introduced to Brazil in the 1950s [1,2]. U. humidicola is an apomictic polyploid species with variable levels of ploidy (6X–9X) [3-7].

In Brazil, the grasses of the genus Urochloa occupy 85% of the cultivated pasture areas [8]. U. humidicola is cultivated as forage in several tropical regions worldwide and is particularly recognized for its tolerance to poorly draining soils, seasonal flooding, and infertile acidic soils [9]. For this reason, this species has been largely exploited in the tropics as a forage option over other Urochloa grasses, mostly in the African savannas and similar environments, such as the Brazilian Cerrado [7].

The development and adoption of new U. humidicola cultivars with a broad genetic base are crucial for the diversification of forage pastures in the tropics, primarily because there are few cultivars of this species in Brazil (Tully, Llanero, and BRS Tupi). However, the development of new cultivars must be a dynamic process, providing cultivars with high nutritional value, increased biotic and abiotic resistance, and economic competitiveness.

Molecular markers are important tools to the progress of breeding programs, and their utilization would favor a more dynamic development of new cultivars of this species. However, there is a lack of information about the U. humidicola genome. Indeed, little or nothing is known about the number of genes, distribution of gene families, abundance and diversity of retro-elements, QTL localization of traits of economic importance, genome colinearity with model species, or abundance of repetitive sequences. Molecular markers are widely used in the fingerprinting of cultivars, the detection of genetic diversity in evaluating population structure in the mapping genes of interest, and in the selection of elite genotypes in breeding programs. SSR markers, in particular, are often used due to their codominant and multi-allelic characteristics [10]; moreover, they are highly site specific and transferable to related species [11].

Some microsatellite markers have already been developed for U. humidicola [12,13] and have been used for germplasm diversity studies [7,13], with all of them from the same microsatellite-enriched library constructed from genotype H016. Moreover, our research group identified four different gene pools among U. humidicola accessions; genotype H031 was found to be completely different from all other accessions, which was verified by a population structure analysis and by the fact that 18.5% of the tested markers did not amplify in this accession [7]. As a large number of markers are necessary for molecular breeding programs, our goal was to isolate and characterize new polymorphic microsatellite markers for U. humidicola genotype H031 (accession 12) to ensure that its genome was well represented by the new set of markers and also different accessions that belong to different gene pools and to test the transferability of these markers to four other Urochloa species (U. brizantha, U. decumbens, U. ruziziensis, and U. dictyoneura). The results were compared with previously reported data [12,13].

Methods

The plant material for library construction and marker validation was obtained from young leaves from several Urochloa genotypes. For the first library (Lb-1) construction, a single sexual genotype (H031) was used. For the second library (Lb-2) construction, a pool of eight apomictic genotypes (H010, H013, H015, H034, H041, H043, H101, and H108) was used. For marker validation, 34 genotypes were selected, consisting of 20 U. humidicola germplasm accessions, six intra-specific hybrids, and eight Urochloa accessions, as represented by two different accessions from each of the following species: U. brizantha, U. decumbens, U. ruziziensis, and U. dictyoneura. These genotypes were selected based on the four gene pools found by a previous study [7], from which two genotypes were selected from each gene pool. All of the accessions used are from the Urochloa germplasm collection maintained at Embrapa Beef Cattle, Campo Grande, MS, Brazil. They have been personally identified by S. A. Renvoize, from the Royal Botanic Gardens, Kew, UK and their identity have been confirmed by C. B. do Valle when transferred to Brazil [9]. The annotation numbers, accession numbers (as recorded in Embrapa Beef Cattle (EBC) and Center for Tropical Agriculture (CIAT)), genotypes, and species identifications are shown in Table 1. Genomic DNA was extracted from freeze-dried leaf samples using the CTAB method [14]. The DNA samples were evaluated on a 1% agarose gel and quantified by comparison to known quantities of uncut λ phage DNA (Invitrogen, Carlsbad, CA, USA).
Table 1

Genotypes of U. humidicola and four species of the genus Urochloa used for the characterization and transferability analyses of new microsatellite markers

AN

CIAT

BRA

EBC

Genotype

Species

1

16181

4821

H004

germplasm accession

U. humidicola

2

16182

4839

H005

germplasm accession

U. humidicola

3

16867

4863

H006

germplasm accession

U. humidicola

4

16871

4901

H008

germplasm accession

U. humidicola

5

16880

4952

H010

germplasm accession

U. humidicola

6

16882

4979

H012

germplasm accession

U. humidicola

7

16886

5011

H013

germplasm accession

U. humidicola

8

26141

5088

H015

germplasm accession

U. humidicola

9

26149

5118

H016

germplasm accession

U. humidicola

10

16877

4928

H023

germplasm accession

U. humidicola

11

16894

5070

H030

germplasm accession

U. humidicola

12

26146

5100

H031

germplasm accession

U. humidicola

13

26413

6131

H035

germplasm accession

U. humidicola

14

26432

6203

H041

germplasm accession

U. humidicola

15

16884

4995

H044

germplasm accession

U. humidicola

16

NA

NA

H048

germplasm accession

U. humidicola

17

NA

1929

H107

germplasm accession

U. humidicola

18

6705

2208

H112

germplasm accession

U. humidicola

19

6133

1449

H125

germplasm accession

U. humidicola

20

6369

0370

H126

germplasm accession

U. humidicola

21

-

-

20

hybrid

U. humidicola

22

-

-

45

hybrid

U. humidicola

23

-

-

184

hybrid

U. humidicola

24

-

-

215

hybrid

U. humidicola

25

-

-

264

hybrid

U. humidicola

26

-

-

320

hybrid

U. humidicola

27

16162

-

B057

germplasm accession

U. brizantha

28

16467

-

B166

germplasm accession

U. brizantha

29

16499

004481

D009

germplasm accession

U. decumbens

30

26300

004707

D028

germplasm accession

U. decumbens

31

26163

005568

R102

germplasm accession

U. ruziziensis

32

26174

005614

R104

germplasm accession

U. ruziziensis

33

16186

007889

DT157

germplasm accession

U. dictyoneura

34

16188

007901

DT159

germplasm accession

U. dictyoneura

NA: not available, AN: annotation number, CIAT: Center for Tropical Agriculture, BRA: codes from EMBRAPA, EBC: codes from EMBRAPA Beef Cattle.

Genomic DNA was restriction digested with Afa I (Invitrogen), enriched in microsatellite fragments using (CT)8 and (GT)8 probes, and then used to construct a microsatellite-enriched library following the protocol of Billotte et al. [15]. The enriched microsatellite fragments were cloned into pGEM-T (Promega, Madison, WI), and the ligation products were used to transform Escherichia coli XL1-Blue competent cells. All 94 clones from both libraries were sequenced with an ABI 377 automated sequencer (Applied Biosystems, Foster City, CA) using the BigDye terminator cycle sequencing kit (Applied Biosystems, Foster City, CA).

The microsatellites were identified using MISA software [16]. Only mono-nucleotides with twelve or more repeats, di-nucleotides with six or more repeats, tri-nucleotides with four or more repeats, and tetra-, penta-, and hexa-nucleotides with three or more repeats were considered. Primer pairs were designed using the Primer Select 5.01 software (DNASTAR Inc.) and the Primer3Plus software [17]. Polymerase chain reactions (PCRs) were carried out as previously described [12]. The amplification products were resolved by electrophoresis through 3% agarose gels prior to vertical electrophoresis through 6% denaturing polyacrylamide gels. The gels were then silver stained [18], and the product sizes were determined by comparison to a 10-bp DNA ladder (Invitrogen, Carlsbad, CA).

Polyploid microsatellite genotyping is difficult due to the closeness of fragment sizes, stutter peaks observed and allele overlap due to multiple alleles of the same size. Few methods have been developed to overcome allele overlapping and estimate the allele frequencies, such as the estimation of alleles based on the electropherogram peak ratios [19] or the statistical estimation of allele frequencies [20]. However, for the present study work, we restricted the project to describe the new SSR markers, which were visually scored based on the presence (1) or absence (0) of a band in the polyacrilamide gels for each of the Urochloa genotypes. PIC (Polymorphic Information Content) [21] and DP (Discriminatory Power) [22] values were calculated to estimate polymorphisms at each locus.

The microsatellite scores for the 34 individuals were evaluated using a model-based method with Bayesian clustering approach in STRUCTURE software version 2.2 [23-25]. The admixture model was tested with 200,000 replicates for burn-in and 100,000 replicates for Markov Chain Monte Carlo (MCMC) processes through ten iterations (runs). The numbers of clusters (K) were tested from 2 to 20. The optimal number of clusters was estimated using the ΔK value, as previously described [26], and the final graphs were visualized using the STRUCTURE HARVESTER software [27]. The individuals were grouped into clusters according to the association coefficient (Q) proportion of each allelic pool in an individual.

A joint analysis (Lb-c) was performed with the data from the polymorphic loci derived from the new libraries Lb-1 and Lb-2. Data from a previous study [12] that used SSRs developed from accession 9 (H016) were used to compare the three libraries. The data were reanalyzed under the same parameters as those used for the new libraries, resulting in Lb-3. Another joint analysis (Lb-ct) was performed with data from the three libraries together (Lb-1, Lb-2, and Lb-3). The results obtained by STRUCTURE software were permuted by CLUMPP software [28], and the figures were generated using DISTRUCT software [29].

Results

Microsatellite enrichment success for the U. humidicola DNA libraries was 79.0% for Lb-1 and 61.2% for Lb-2. From all of the sequenced clones, 183 microsatellites were identified. Di-nucleotide repeats were the most abundant class of microsatellites detected, representing 76.4% and 72.7% of the loci for Lb-1 and Lb-2, respectively, followed by mono-nucleotide and tetra-nucleotide repeats. Perfect microsatellites were the most abundant (Table 2).
Table 2

Characterization of new microsatellite-enriched libraries from U. humidicola

Library

 

Lb-1

Lb-2

Total clones sequenced

 

86.0

80.0

Sequences containing microsatellites (%)

 

79.0

61.2

Total number of SSRs identified

 

106.0

77.0

Type of repeat (%)

By nucleotide string

Mono-nucleotides

12.7

6.5

Di-nucleotides

76.4

72.7

Tri-nucleotides

1.9

5.2

Tetra-nucleotides

5.6

11.6

Penta-nucleotides

2.8

3.9

Hexa-nucleotides

0.9

0.0

By form

Perfect

79.1

80.6

Imperfect

9.3

1.6

Perfect Compound

5.8

9.7

Imperfect Compound

5.8

8.1

Of the 114 SSR primer pairs designed and tested, 72 were successfully amplified in U. humidicola genotypes, and 64 SSRs were polymorphic. A description of the number of alleles per locus and PIC and DP values for both the U. humidicola accessions and Urochloa accessions is presented in Table 3. The loci BhUNICAMP68 to BhUNICAMP108 are derived from Lb-1, and the loci BhUNICAMP109 to BhUNICAMP139 are derived from Lb-2. Based on the allelic frequencies estimated by STRUCTURE software, 36.43% of the alleles are rare (frequency < 0.05), 60.06% are intermediate alleles (0.05 < frequency < 0.30), and 3.50% are abundant alleles (frequency > 0.30).
Table 3

Characterization of the 72 polymorphic SSR markers developed for U. humidicola

SSR locus

GenBank accession number

Repeat motif

Ta (°C) a

Primer sequences (5′-3′)

Urochloa species accessions*

U. humidicola accessions**

Size range (bp)

A b

PIC c

A b

PIC c

DP d

BhUNICAMP068

KM068303

(CACACC)4(CA)17

58.5

F_CCACAAACGTGAACACATACA R_AGGGACGGAAACACCCTTAG

226-261

10

0.87

10

0.87

0.95

BhUNICAMP069

KM068304

(TC)25

64.5

F_GAGGAACTCCTTTGGGTAGA R_TTCAGAGAGAGGATGGTATAGAG

285-300

2

0.36

2

0.36

0.58

BhUNICAMP070

KM068305

(GT)9

65

F_CCCCGGTCTCGACCTATC R_GAGGCTGCCCCCTTACTC

174-214

12

0.84

6

0.78

0.54

BhUNICAMP071

KM068306

(AC)11

65

F_CGCAACGAAGCTCCAATAG R_CGATCGCAAGCGTGTATCTA

160-228

11

0.86

11

0.86

0.94

BhUNICAMP072

KM068307

(GT)7

56.5

F_CCCCATGTAAACAACCGTAGA R_CCATGGTTGACCGCTAGAA

174-186

3

0.56

3

0.56

0.85

BhUNICAMP073

KM068308

(TG)10

60

F_TGAACATGTGAATGCCCACT R_ATTGCAGGATGCGGACTCTA

240-304

10

0.85

10

0.85

0.94

BhUNICAMP074

KM068309

(CT)6

58.5

F_ACGAACGATCCGACCAACTA R_TGCTTACGAGACGGCATAGA

231-255

7

0.81

7

0.81

0.92

BhUNICAMP075

KM068310

(TC)22

50

F_TGAATGCTTTTGTCCTGGTATC R_ACGTGCAGCAGCAACAGTA

148-236

28

0.95

24

0.95

0.98

BhUNICAMP076

KM068311

(AC)18

51.5

F_CCGATGGTCAAAGGTCAGTT R_GGTGGGCATATACCATGTTT

206-234

10

0.84

10

0.84

0.66

BhUNICAMP077

KM068312

(AC)7

65

F_CGGGAAGTCCTACTCCGTAA R_GGAGCTCAAGGTAGGGATTG

212-230

8

0.83

8

0.83

0.93

BhUNICAMP078

KM068313

(GT)7

58.5

F_ACCAGTGCACGTCTGAAAGA R_CGATCACTGCTGCGTCATA

216-218

2

0.35

2

0.35

0.52

BhUNICAMP079

KM068314

(AG)12G(GA)17

62.5

F_GGATTGAAAGTTGGAGCACA R_GCATGCTGTGAAGGAGGTTA

180-222

17

0.92

17

0.92

0.96

BhUNICAMP080

KM068315

(GA)26

50

F_CAAGCCTCTTCATGCAAGTAAC R_TGTCATACCCCCATGATTAAGA

176-230

22

0.93

21

0.93

0.93

BhUNICAMP081

KM068316

(AGC)5ACAAT(CA)11

55

F_CTGGCATGGGTCCCTTTAC R_TCTTCTTCCTCCAGCCACAT

160-179

5

0.75

5

0.75

0.95

BhUNICAMP082

KM068317

(CA)23

60

F_TTGCCGGGAACAGTTATACA R_GAAGCTCTATCAAACAGCCCT

157-192

9

0.82

9

0.82

0.92

BhUNICAMP083

KM068318

(AG)22

56.5

F_AAACATGCACCGTCATAACT R_GGGCTTGATTCATTTGTTA

152-190

6

0.68

4

0.68

0.77

BhUNICAMP084

KM068319

(TG)15

65

F_GGCGAAGACCATACCCTGTA R_TGCTGGTGGAAGAAGATGAA

159-182

9

0.80

9

0.80

0.96

BhUNICAMP085

KM068320

(GT)9

60

F_CGATTTATCGACGACCGAGT R_CCTTACTCGCAGGTCTGTCC

158-171

5

0.76

5

0.76

0.64

BhUNICAMP086

KM068321

(TC)19

65

F_AGTTGAATGGGCTGAACCAT R_TGCACTTCCAGGATCAGACA

238-326

10

0.82

10

0.82

0.93

BhUNICAMP087

KM068322

(GT)10

50

F_GGCCATTTCTAGCCAAACAA R_CCTTACTCGCAGGTCTGTCC

240

1

0.00

1

0.00

0.00

BhUNICAMP088

KM068323

(TG)12

65

F_AGAGGTTCCATGGACATTGC R_CTCATCAACAGACGCCTGAA

178

1

0.00

1

0.00

0.00

BhUNICAMP089

KM068324

(AC)7

65

F_CCGGATAGAAGGTCTGAACG R_AGTCGTCGAAGCGAGCTCTA

175

1

0.00

1

0.00

0.00

BhUNICAMP090

KM068325

(CA)10

65

F_CAGAGTAAGCTTCCGGGACA R_CGATTTATCGACGACCGAGT

200-300

12

0.85

11

0.85

0.91

BhUNICAMP091

KM068326

(AC)8

65

F_CTTGTGCCACTTCCACCTTT R_TCGTGTGGACACTTCCTCTG

120-150

9

0.83

9

0.83

0.95

BhUNICAMP092

KM068327

(TG)6

65

F_ATGCCTTGCTCCCACTAACA R_TAAATGCTCCAGCGACCTTC

135-168

11

0.85

11

0.85

0.91

BhUNICAMP093

KM068328

(AAG)4

65

F_GGAGCGCTAATTTCGTTCAG R_CCTCCGTTCTCGCTAATGAC

230

1

0.00

1

0.00

0.00

BhUNICAMP094

KM068329

(TG)7

65

F_TTGGAGCTTTCCCTAGCTCA R_GAACAAGAAGGGAGGAAGCA

272-290

4

0.31

4

0.31

0.39

BhUNICAMP095

KM068330

(TC)16(TG)14

65

F_GGGTTGGCCTACACACTGAT R_CGCACGACATTGATACCTTG

268-320

6

0.75

6

0.75

0.92

BhUNICAMP096

KM068331

(TC)8TT(TC)40

65

F_TGTTCTGCTCACTGGTTTGG R_TCAGCTCTCTACGGCTGGAT

157-255

11

0.87

11

0.87

0.95

BhUNICAMP097

KM068332

(GT)6

65

F_GCGAGCTACCGAGGTATTTG R_ACGTCAATGTCGAGCTTCCT

129-148

5

0.69

5

0.69

0.80

BhUNICAMP098

KM068333

(GT)10(G)18

65

F_GGACTGGTCGTCTTTCCATC R_GCTTTCTGCAAGCGGTAGAT

250-312

9

0.85

9

0.85

0.95

BhUNICAMP099

KM068334

(CA)10TG(GA)10

65

F_TTTGTGGCACCTGCAGAATA R_CGCTTCGTGCTGACAGATTA

124-174

16

0.91

16

0.91

0.99

BhUNICAMP100

KM068335

(TG)12

65

F_GCGCCATGGTTTCATCTATT R_GGTGGTTCCTCGTGTGAGAT

178-219

7

0.79

7

0.79

0.98

BhUNICAMP101

KM068336

(TG)28

65

F_GGTAAAGAAGGGCCGGACT R_GCATGGCATGTTCCTACTGA

128-184

14

0.89

12

0.89

0.97

BhUNICAMP102

KM068337

(GCGA)4

65

F_TGGTGGGCTCCACTATCTCT R_TCCGCCATCTCTCCTCTCT

224-260

12

0.89

12

0.89

0.94

BhUNICAMP103

KM068338

(CT)22

65

F_AGCTCTCCCGCCTCTCTCT R_CATCCACACCGTCTCTCTCA

100-156

14

0.91

14

0.91

0.96

BhUNICAMP104

KM068339

(TG)26

60

F_ACGACGACCTAATGGGTGAA R_ACCCAGCAACAAATCTCGTC

190-274

15

0.87

13

0.87

0.96

BhUNICAMP105

KM068340

(AC)10ATACACACACAC(AG)53

50

F_CTCCATCACGTGCTTGCTAA R_GTGTGATCGGCTGGAGATTT

100-176

30

0.93

29

0.93

0.98

BhUNICAMP106

KM068341

(TTTGT)3

50

F_GCTGTTCGGAGAGGAATCTG R_ATGAGAGGAGGGAAGGAAGG

135-155

8

0.79

7

0.79

0.91

BhUNICAMP107

KM068342

(GA)18

50

F_GGGTCAGTGTCGTCTCAGTTT R_CAGATTCCTCTCCGAACAGC

118-190

26

0.94

26

0.94

0.98

BhUNICAMP108

KM068343

(CT)16

65

F_TTGCCATTACTGGATCTGGA R_GCGCCACCCATAACTTAAA

112-160

14

0.85

13

0.85

0.94

BhUNICAMP109

KM068344

(GT)9

60

F_AGCGAGTCAAGCACAAGGAT R_GGGTCCAATCTCCCTCTCTC

186-226

9

0.82

9

0.82

0.93

BhUNICAMP110

KM068345

(TG)8

65

F_TCTGCATCCACTAGGCTCAG R_TCCTCCACCTTCTTTCCGTA

148-164

4

0.39

4

0.39

0.46

BhUNICAMP111

KM068346

(TG)27

65

F_AACTCCGACTATCTTCCAGTTGA R_AATGCATGGGTAGGATCTGC

250-330

15

0.89

15

0.89

0.96

BhUNICAMP112

KM068347

(AC)26

65

F_GACCAAACCCTCCGAAGTTA R_GGTTGCAACTACACGACCAG

246-300

10

0.81

10

0.81

0.94

BhUNICAMP113

KM068348

(CGTG)3

63

F_AACTTCGAGAGGTTCGTCCA R_ACCGGCAATCTATCCGTGT

144-179

3

0.45

3

0.45

0.51

BhUNICAMP114

KM068349

(CT)21

63

F_TATACAAGGCGCATCCACAA R_GCTCTTTCCTCACGCTGTTC

200-266

15

0.89

15

0.89

0.96

BhUNICAMP115

KM068350

(AC)27(AT)7

60

F_CTTCCTGCCAATAAGCGAAG R_CGAGCTTCCAGATTCTTTGG

240

1

0.00

1

0.00

0.00

BhUNICAMP116

KM068351

(TG)8

65

F_CTCCGCACCGCTTAAATTAG R_GTTGGAAATGGTGCTTCCAC

288-306

3

0.52

3

0.52

0.62

BhUNICAMP117

KM068352

(TGA)7

65

F_CCAACTGAACGGCCATACTT R_CCCACAAAGGAACCCTGAT

290-300

4

0.61

4

0.61

0.77

BhUNICAMP118

KM068353

(AG)9

50

F_CTGCATAACTTTCAGCCATCTC R_TTGGCACAACTGGAACGTAG

149

1

0.00

1

0.00

0.00

BhUNICAMP119

KM068354

(AAG)7

65

F_AAGGGCGTGATGTTCTGAAG R_AGGCCAAACGAATTTCTCAA

189-204

4

0.66

4

0.66

0.82

BhUNICAMP120

KM068355

(AT)8ACACACACACG(CA)9

65

F_TCCAGCAGTGTGTTCCTCAG R_ACCAGGAGTGCATAGCCAAG

190-200

6

0.71

6

0.71

0.75

BhUNICAMP121

KM068356

(TC)12

65

F_CGCTACTGCTGCACACAAAT R_CTGAGTGCGCCGTATGTTTA

170-195

6

0.71

6

0.71

0.92

BhUNICAMP122

KM068357

(GT)15

65

F_AGGAAGGCTCGCACTCACTA R_CCAAAGGCGGTGGTTAGATA

200-315

14

0.90

14

0.90

0.95

BhUNICAMP123

KM068358

(TTA)4

65

F_CCAAACTCTAGCTTTCACAGCA R_TTGGATCCACGTCAAACAAG

280

1

0.00

1

0.00

0.00

BhUNICAMP124

KM068359

(AG)23

65

F_TTGGAGTTGCTGGGCTATTT R_GAACCAAGCATAAGGCAACA

218-320

12

0.85

10

0.85

0.95

BhUNICAMP125

KM068360

(GT)8GAATGTGTGT(GA)7

65

F_TGTTATCAGTGCAGGTGTTGG R_GAGGCTGACGAAAGCTCAAC

258-280

7

0.81

7

0.81

0.93

BhUNICAMP126

KM068361

(AC)10

65

F_GGGAACCCAGGGTATCGTAT R_CTCTCCCAGCGTCTTTCCTT

210

1

0.00

1

0.00

0.00

BhUNICAMP127

KM068362

(GT)6

65

F_CCACCATTGCTTCCAGAGTAA R_ATTCGCCTCTCCTAGCACAA

272-320

7

0.69

7

0.69

0.91

BhUNICAMP128

KM068363

(GA)37

65

F_TGCCTGGAGACTGAGAAAGG R_CCTGCAGCAGACCTTCACAT

150-240

17

0.91

17

0.91

0.98

BhUNICAMP129

KM068364

(AC)7ATGAA(CATG)3(CA)22

63

F_TGTGTTTAGACCGCCAACAA R_TTATCGGCTCCCATTCACTC

207-310

11

0.84

10

0.84

0.95

BhUNICAMP130

KM068365

(AC)7

63

F_ACGCAGGAGAACTGCGTATC R_ATGGGATCCAACCGAACATA

236-300

12

0.79

11

0.79

0.87

BhUNICAMP131

KM068366

(AC)7(A)16

60

F_CATCAGATGCCTCAAACAGC R_GCAGGTGTGCAGCAAATAGA

184-238

14

0.87

14

0.87

0.93

BhUNICAMP132

KM068367

(TG)7(T)29

50

F_TCACTAGTGCGTCTGCTGCT R_GCACTCCATTGCAGACCTAAG

184-196

4

0.53

3

0.53

0.63

BhUNICAMP133

KM068368

(TG)10

50

F_CATGACTTATGTCCTTGGTGGA R_TCGACAGTGGAGCCACAA

114-162

19

0.89

16

0.89

0.97

BhUNICAMP134

KM068369

(CCGG)3

60

F_CAAACGGAGGAAGAGAGACG R_GGTGTCAATGCAGCCAAGTA

114-135

9

0.75

5

0.75

0.83

BhUNICAMP135

KM068370

(AG)27

65

F_CATGAGCCATCTCGTTGTTG R_TGCATTGACTTGACGTCTCC

176-260

14

0.90

9

0.90

0.91

BhUNICAMP136

KM068371

(AC)9(ACAA)3

50

F_TCCTGGTAAAGTTCCTCGTCA R_ACAACAATGCACGTCGAGAA

225-290

7

0.75

6

0.75

0.93

BhUNICAMP137

KM068372

(GA)23

65

F_TAGGTTTGGGTGGCACTAGG R_CTCCATGCTGCGTTGCTAT

258-320

11

0.85

9

0.85

0.91

BhUNICAMP138

KM068373

(T)12

60

F_TGCTCATGTGGGTCACATTT R_TGTGTGCCTGTGTGATGCTA

270-288

5

0.70

5

0.70

0.95

BhUNICAMP139

KM068374

(AAAAG)3

65

F_TCCTTTCTTTGAGCCGAGAG R_GCTGATGCTGACATCAAGGA

248-294

6

0.67

5

0.67

0.97

Total average

     

10.26

0.77

9.60

0.77

0.87

Lb-1 average

     

11.05

0.79

10.48

0.79

0.87

Lb-2 average

     

9.18

0.75

8.40

0.75

0.86

*Species evaluated: Urochloa humidicola (Rendle) Morrone & Zuloaga, Urochloa brizantha (Hochst. ex A. Rich.) R.D. Webster, Urochloa decumbens (Stapf) R.D. Webster, Urochloa dictyoneura (Figure & De Not.) Veldkamp, Urochloa ruziziensis (R. Germ. & C.M. Evrard) Crins.

**Hybrids included.

a Amplification temperature (°C).

b Maximum number of alleles observed.

c Polymorphism Information Content.

d Discrimination Power.

A survey of the potential transferability of the microsatellite markers from U. humidicola to other Urochloa species identified that 61.11% of the 72 markers resulted in amplified PCR products in at least one U. brizantha genotype, 58.33% were amplified in U. decumbens, 59.72% were amplified in U. ruziziensis, and 69.44% were amplified in U. dictyoneura. The number of successfully amplified genotypes per number of genotypes tested per species is shown in Table 4.
Table 4

Cross-amplification of the 72 SSR markers among other Urochloa species

Transferability a,b

SSR locus

U. brizantha

U. decumben

U. ruziziensis

U. dictyoneura

BhUNICAMP068

0/2

0/2

0/2

0/2

BhUNICAMP069

0/2

0/2

0/2

0/2

BhUNICAMP070

2/2

2/2

2/2

2/2

BhUNICAMP071

0/2

0/2

0/2

2/2

BhUNICAMP072

1/2

1/2

0/2

1/2

BhUNICAMP073

0/2

0/2

0/2

2/2

BhUNICAMP074

0/2

0/2

0/2

0/2

BhUNICAMP075

2/2

2/2

2/2

2/2

BhUNICAMP076

2/2

2/2

2/2

1/2

BhUNICAMP077

2/2

2/2

2/2

2/2

BhUNICAMP078

1/2

0/2

1/2

1/2

BhUNICAMP079

2/2

2/2

2/2

1/2

BhUNICAMP080

2/2

1/2

2/2

1/2

BhUNICAMP081

0/2

0/2

0/2

0/2

BhUNICAMP082

2/2

0/2

1/2

1/2

BhUNICAMP083

1/2

1/2

1/2

2/2

BhUNICAMP084

2/2

2/2

2/2

2/2

BhUNICAMP085

2/2

2/2

2/2

2/2

BhUNICAMP086

2/2

2/2

2/2

2/2

BhUNICAMP087

2/2

2/2

2/2

2/2

BhUNICAMP088

2/2

2/2

2/2

2/2

BhUNICAMP089

0/2

0/2

0/2

0/2

BhUNICAMP090

2/2

2/2

2/2

2/2

BhUNICAMP091

0/2

0/2

0/2

2/2

BhUNICAMP092

0/2

0/2

0/2

2/2

BhUNICAMP093

0/2

0/2

0/2

0/2

BhUNICAMP094

2/2

2/2

1/2

2/2

BhUNICAMP095

0/2

0/2

0/2

0/2

BhUNICAMP096

2/2

2/2

1/2

2/2

BhUNICAMP097

2/2

2/2

2/2

2/2

BhUNICAMP098

0/2

0/2

0/2

0/2

BhUNICAMP099

0/2

0/2

0/2

0/2

BhUNICAMP100

0/2

0/2

0/2

0/2

BhUNICAMP101

2/2

2/2

2/2

2/2

BhUNICAMP102

0/2

0/2

0/2

0/2

BhUNICAMP103

2/2

1/2

2/2

2/2

BhUNICAMP104

2/2

2/2

2/2

2/2

BhUNICAMP105

2/2

2/2

2/2

2/2

BhUNICAMP106

2/2

2/2

2/2

2/2

BhUNICAMP107

2/2

2/2

2/2

1/2

BhUNICAMP108

2/2

2/2

2/2

1/2

BhUNICAMP109

2/2

2/2

2/2

2/2

BhUNICAMP110

2/2

2/2

2/2

2/2

BhUNICAMP111

2/2

2/2

2/2

2/2

BhUNICAMP112

2/2

2/2

2/2

2/2

BhUNICAMP113

0/2

0/2

0/2

0/2

BhUNICAMP114

0/2

0/2

0/2

0/2

BhUNICAMP115

0/2

0/2

0/2

0/2

BhUNICAMP116

2/2

2/2

2/2

2/2

BhUNICAMP117

0/2

0/2

0/2

2/2

BhUNICAMP118

0/2

0/2

0/2

0/2

BhUNICAMP119

0/2

0/2

0/2

0/2

BhUNICAMP120

2/2

2/2

2/2

2/2

BhUNICAMP121

2/2

2/2

2/2

2/2

BhUNICAMP122

0/2

0/2

0/2

0/2

BhUNICAMP123

0/2

0/2

0/2

2/2

BhUNICAMP124

2/2

2/2

2/2

0/2

BhUNICAMP125

0/2

0/2

0/2

0/2

BhUNICAMP126

2/2

2/2

2/2

2/2

BhUNICAMP127

0/2

0/2

0/2

0/2

BhUNICAMP128

0/2

0/2

0/2

0/2

BhUNICAMP129

2/2

2/2

2/2

0/2

BhUNICAMP130

2/2

2/2

2/2

2/2

BhUNICAMP131

2/2

2/2

2/2

2/2

BhUNICAMP132

0/2

0/2

0/2

2/2

BhUNICAMP133

2/2

2/2

2/2

2/2

BhUNICAMP134

2/2

2/2

2/2

2/2

BhUNICAMP135

2/2

2/2

2/2

2/2

BhUNICAMP136

2/2

2/2

1/2

2/2

BhUNICAMP137

2/2

2/2

2/2

2/2

BhUNICAMP138

0/2

0/2

0/2

2/2

BhUNICAMP139

2/2

2/2

2/2

2/2

Total

44

42

43

50

Amplification %

61,11

58,33

59,72

69,44

a Number of successfully amplified genotypes/Number of tested genotypes.

b Nomenclatural classification: Urochloa humidicola (Rendle) Morrone & Zuloaga, Urochloa brizantha (Hochst. ex A. Rich.) R.D. Webster, Urochloa decumbens (Stapf) R.D. Webster, Urochloa dictyoneura (Figure & De Not.) Veldkamp, Urochloa ruziziensis (R. Germ. & C.M. Evrard) Crins.

The population structure analysis based on SSR allelic data showed differentiation among the U. humidicola accessions, hybrids, and other Urochloa species. The STRUCTURE analysis for Lb-1 and Lb-2 and the joint analysis of data from both libraries (Lb-c) showed K = 18, K = 17, and K = 17 allelic pools, respectively, with each one represented by a different color in Figure 1. Clusters I to V were composed of U. humidicola accessions. Cluster VI was composed of two U. humidicola accessions (accessions 9 and 12) and six hybrids derived from a controlled cross between these two accessions. The other Urochloa species were grouped into Clusters VII and VIII for Lb-1 and Lb-c and in Cluster VII for Lb-2.
Figure 1

Analysis performed with STRUCTURE software. Lb-1: Library constructed from a sexual accession (H031), Lb-2: Library constructed from a pool of eight apomictic accessions, Lb-3: Library constructed from an apomictic accession (H016) [12], Lb-c: Joint analysis of Lb-1 and Lb-2, Lb-ct: Joint analysis of Lb-1, Lb-2, and Lb-3. Each of the 34 genotypes is represented by a single column divided into colored segments with lengths proportional to each of the allelic pools inferred by K through Evanno method [24]. Each K is represented by a different color and Lb-1 presented K = 18, Lb-2 K = 17, Lb-c K = 17, Lb-3 K = 15, and Lb-ct K = 18. The individuals were grouped into clusters according to the Q proportion of each allelic pool in an individual. Eight clusters were identified for Lb-1, Lb-c, Lb-3, and Lb-ct (I to VIII) and seven clusters for Lb-2 (I to VII). The left scale indicates the association coefficient (Q) for the assignment of genotypes into groups. The genotypes are named according to the annotated numbers listed in Table 1.

The STRUCTURE analysis for Lb-3 and Lb-ct showed K = 15 and K = 18 allelic pools, respectively (Figure 1), classified in the same clusters as for Lb-1 and Lb-c.

Discussion

In the present study, we described 72 new SSRs for U. humidicola, 64 of which are polymorphic. Along with the 67 previous developed SSRs [12,13], these markers contribute to the genetic breeding of the species and other species of the genus Urochloa in efforts to obtain new cultivars and better understanding of the species genetic, through genetic mapping, marker-assisted selection, genome sequencing and synteny.

The increased occurrence of di-nucleotide motifs for Lb-1 and Lb-2 is in accordance with the enrichment of both libraries with (CT)8 and (GT)8 probes. In addition, Morgante et al. [30] reported a higher occurrence of microsatellites with di-nucleotide motifs in plants, which may have been a contributing factor in our observation.

Among the microsatellites analyzed, 88% had a polymorphism among the evaluated genotypes. The most informative loci in this panel of SSRs were those with the highest PIC and DP values (BhUNICAMP075 and BhUNICAMP107). Locus BhUNICAMP094 showed the lowest values for PIC and DP, at 0.3165 and 0.3969, respectively, even though it was amplified in all the Urochloa species evaluated. This also occurred with the BhUNICAMP030 locus [12]. Both loci may be useful markers for studies in Urochloa because it may be the result of a conserved region among the species studied herein. Monomorphic loci may be useful in other studies.

The transferability rates of the loci from U. humidicola to four other species were very similar. Although these results were not highly variable, U. dictyoneura presented the highest transferability, corroborating the genetic closeness between U. dictyoneura and U. humidicola, as has been previously described [2,31] and the results obtained in another study [13].

For the population structure analysis, different numbers of allelic pools [K] were observed for all analyses. However, the individual composition presented in each cluster was maintained into Lb-1, Lb-c, Lb-3, and Lb-ct analyses, but for Lb-2 analysis, the Clusters VII and VIII were grouped into Cluster VII.

The genotypes of the species U. brizantha, U. decumbens, and U. ruziziensis were grouped into the same cluster in all the analyses. However, the U. dictyoneura genotypes were grouped separately from the other species for all the analyses, except for Lb-2, with the four species grouping into Cluster VII.

In all analyses, Cluster VI included accessions 9 and 12, and six hybrids derived from crosses between these two accessions grouped together. However, in a previous study, the progenitors did not group together with the hybrids [13], as only runs from K = 1 to K = 10 were performed. These hybrids are part of an F1 population that is being mapped with the SSRs described in this study and previously published [12,13].

Although some discrepancies were found among the three libraries (Lb-1, Lb-2, and Lb-3), the set of loci belonging to each was able to satisfactorily differentiate the accessions. Comparing the three libraries developed, Lb-1 presented the highest number of allelic pools, which may be correlated to the usage of the accession H031, a highly diverse genotype, as described by [7]. The genotype used for the enriched library construction directly influences the results. The joint analysis of the three libraries (Lb-ct) would be the most recommended way to differentiate among accessions, because it uses loci derived from many different genotypes, conferring a greater reliability of the observed results.

These markers are immediately useful for U. humidicola breeding programs, aiding in areas such as the construction of linkage and QTL maps, gene flow and mating system evaluation, and marker-assisted selection.

Availability of supporting data

The datasets supporting the results of this article are included in the article.

Abbreviations

A: 

Maximum number of alleles observed

AN: 

Annotation number

BRA: 

Codes of the accessions from EMBRAPA

CAPES: 

Coordination of Improvement of Higher Education Personnel

CIAT: 

Center for Tropical Agriculture

CTAB: 

Cetyltrimethyl ammonium bromide

DNA: 

Deoxyribonucleic acid

DP: 

Discrimination power

EBC: 

Embrapa beef cattle

EMBRAPA: 

Brazilian Agricultural Research Corporation

FAPESP: 

Foundation for Research Support of the State of Sao Paulo

K: 

Number of clusters

Lb-1: 

Library construction from a sexual accession (H031)

Lb-2: 

Library construction from a pool of eight apomictic accessions

Lb-3: 

Library construction from an apomictic accession (H016) [12]

Lb-c: 

Joint analysis of Lb-1 and Lb-2

Lb-ct: 

Joint analysis of Lb-1, Lb-2, and Lb-3

MCMC: 

Markov Chain Monte Carlo

NA: 

Not available

bp: 

Base pairs

PCR: 

Polymerase chain reaction

PIC: 

Polymorphism information content

Q: 

Association coefficient from STRUCTURE analysis

QTL: 

Quantitative trait locus

SSR: 

Simple sequence repeat

Ta (°C): 

Annealing temperature

Declarations

Acknowledgements

The authors thank Dr. Cacilda Borges do Valle, Dr. Leticia Jungmann and the Brazilian Agricultural Research Corporation (EMBRAPA Beef Cattle) for providing the Urochloa accessions used. This work was supported by grants from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, 2008/52197-4) and a scholarship to FAO (2013/14903-2). JCSS is a recipient of a scholarship from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES-EMBRAPA Program), and MAB is a recipient of a scholarship from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). APS is a recipient of a research fellowship from CNPq.

Authors’ Affiliations

(1)
Centro de Biologia Molecular e Engenharia Genética (CBMEG), Universidade Estadual de Campinas (UNICAMP), Cidade Universitária Zeferino Vaz
(2)
EMBRAPA Southeast Livestock, Brazilian Agricultural Research Corporation
(3)
Departamento de Biologia Vegetal, Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP), Cidade Universitária Zeferino Vaz

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© Santos et al.; licensee BioMed Central. 2015

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

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