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

Keywords: Microsatellite, Genomic library, SSR transferability, Forage, Grass

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. 

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][4][5][6][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). 
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, trinucleotides 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][24][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 DIS-TRUCT 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). 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). 
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. 
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. 
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 F 1 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. 
(See figure on previous page.) 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. 

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. 


Figure 1 (

 1 Figure 1 (See legend on next page.) 


Table 1

 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		4821	H004	germplasm accession U. humidicola
	2		4839	H005	germplasm accession U. humidicola
	3		4863	H006	germplasm accession U. humidicola
	4		4901	H008	germplasm accession U. humidicola
	5		4952	H010	germplasm accession U. humidicola
	6		4979	H012	germplasm accession U. humidicola
	7		5011	H013	germplasm accession U. humidicola
	8		5088	H015	germplasm accession U. humidicola
	9		5118	H016	germplasm accession U. humidicola
	10		4928	H023	germplasm accession U. humidicola
	11		5070	H030	germplasm accession U. humidicola
	12		5100	H031	germplasm accession U. humidicola
	13		6131	H035	germplasm accession U. humidicola
	14		6203	H041	germplasm accession U. humidicola
	15		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		-	B057	germplasm accession U. brizantha
	28		-	B166	germplasm accession U. brizantha
	29		004481 D009	germplasm accession U. decumbens
	30		004707 D028	germplasm accession U. decumbens
	31		005568 R102	germplasm accession U. ruziziensis
	32		005614 R104	germplasm accession U. ruziziensis
	33		007889 DT157 germplasm accession U. dictyoneura
	34		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.

Table 2

 2 Characterization of new microsatellite-enriched libraries from U. humidicola 


	Library		Lb-1	Lb-2
	Total clones sequenced		86.0	80.0
	Sequences containing		79.0	61.2
	microsatellites (%)			
	Total number of SSRs		106.0	77.0
	identified			
	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

Table 3

 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* Size range (bp) A b	PIC c	U. humidicola accessions** A b PIC c	DP d
	BhUNICAMP068	KM068303	(CACACC) 4	58.5	F_CCACAAACGTGAACACATACA	226-261	10	0.87	10	0.87	0.95
			(CA) 17		R_AGGGACGGAAACACCCTTAG						
	BhUNICAMP069	KM068304	(TC) 25	64.5	F_GAGGAACTCCTTTGGGTAGA	285-300	2	0.36	2	0.36	0.58
					R_TTCAGAGAGAGGATGGTATAGAG						
	BhUNICAMP070	KM068305	(GT) 9	65	F_CCCCGGTCTCGACCTATC	174-214	12	0.84	6	0.78	0.54
					R_GAGGCTGCCCCCTTACTC						
	BhUNICAMP071	KM068306	(AC) 11	65	F_CGCAACGAAGCTCCAATAG	160-228	11	0.86	11	0.86	0.94
					R_CGATCGCAAGCGTGTATCTA						
	BhUNICAMP072	KM068307	(GT) 7	56.5	F_CCCCATGTAAACAACCGTAGA	174-186	3	0.56	3	0.56	0.85
					R_CCATGGTTGACCGCTAGAA						
	BhUNICAMP073	KM068308	(TG) 10	60	F_TGAACATGTGAATGCCCACT	240-304	10	0.85	10	0.85	0.94
					R_ATTGCAGGATGCGGACTCTA						
	BhUNICAMP074	KM068309	(CT) 6	58.5	F_ACGAACGATCCGACCAACTA	231-255	7	0.81	7	0.81	0.92
					R_TGCTTACGAGACGGCATAGA						
	BhUNICAMP075	KM068310	(TC) 22	50	F_TGAATGCTTTTGTCCTGGTATC	148-236	28	0.95	24	0.95	0.98
					R_ACGTGCAGCAGCAACAGTA						
	BhUNICAMP076	KM068311	(AC) 18	51.5	F_CCGATGGTCAAAGGTCAGTT	206-234	10	0.84	10	0.84	0.66
					R_GGTGGGCATATACCATGTTT						
	BhUNICAMP077	KM068312	(AC) 7	65	F_CGGGAAGTCCTACTCCGTAA	212-230	8	0.83	8	0.83	0.93
					R_GGAGCTCAAGGTAGGGATTG						
	BhUNICAMP078	KM068313	(GT) 7	58.5	F_ACCAGTGCACGTCTGAAAGA	216-218	2	0.35	2	0.35	0.52
					R_CGATCACTGCTGCGTCATA						
	BhUNICAMP079	KM068314	(AG) 12 G(GA) 17	62.5	F_GGATTGAAAGTTGGAGCACA	180-222	17	0.92	17	0.92	0.96
					R_GCATGCTGTGAAGGAGGTTA						
	BhUNICAMP080	KM068315	(GA) 26	50	F_CAAGCCTCTTCATGCAAGTAAC	176-230	22	0.93	21	0.93	0.93
					R_TGTCATACCCCCATGATTAAGA						
	BhUNICAMP081	KM068316	(AGC) 5 ACAA	55	F_CTGGCATGGGTCCCTTTAC	160-179	5	0.75	5	0.75	0.95
			T(CA) 11		R_TCTTCTTCCTCCAGCCACAT						
	BhUNICAMP082	KM068317	(CA) 23	60	F_TTGCCGGGAACAGTTATACA	157-192	9	0.82	9	0.82	0.92
					R_GAAGCTCTATCAAACAGCCCT						
	BhUNICAMP083	KM068318	(AG) 22	56.5	F_AAACATGCACCGTCATAACT	152-190	6	0.68	4	0.68	0.77
					R_GGGCTTGATTCATTTGTTA						
	BhUNICAMP084	KM068319	(TG) 15	65	F_GGCGAAGACCATACCCTGTA	159-182	9	0.80	9	0.80	0.96
					R_TGCTGGTGGAAGAAGATGAA						
	BhUNICAMP085	KM068320	(GT) 9	60	F_CGATTTATCGACGACCGAGT	158-171	5	0.76	5	0.76	0.64
					R_CCTTACTCGCAGGTCTGTCC						
	BhUNICAMP086	KM068321	(TC) 19	65	F_AGTTGAATGGGCTGAACCAT	238-326	10	0.82	10	0.82	0.93
					R_TGCACTTCCAGGATCAGACA						

Table 3

 3 Characterization of the 72 polymorphic SSR markers developed for U. humidicola (Continued) 


	BhUNICAMP087	KM068322	(GT) 10	50	F_GGCCATTTCTAGCCAAACAA	240	1	0.00	1	0.00	0.00
					R_CCTTACTCGCAGGTCTGTCC						
	BhUNICAMP088	KM068323	(TG) 12	65	F_AGAGGTTCCATGGACATTGC	178	1	0.00	1	0.00	0.00
					R_CTCATCAACAGACGCCTGAA						
	BhUNICAMP089	KM068324	(AC) 7	65	F_CCGGATAGAAGGTCTGAACG	175	1	0.00	1	0.00	0.00
					R_AGTCGTCGAAGCGAGCTCTA						
	BhUNICAMP090	KM068325	(CA) 10	65	F_CAGAGTAAGCTTCCGGGACA	200-300	12	0.85	11	0.85	0.91
					R_CGATTTATCGACGACCGAGT						
	BhUNICAMP091	KM068326	(AC) 8	65	F_CTTGTGCCACTTCCACCTTT	120-150	9	0.83	9	0.83	0.95
					R_TCGTGTGGACACTTCCTCTG						
	BhUNICAMP092	KM068327	(TG) 6	65	F_ATGCCTTGCTCCCACTAACA	135-168	11	0.85	11	0.85	0.91
					R_TAAATGCTCCAGCGACCTTC						
	BhUNICAMP093	KM068328	(AAG) 4	65	F_GGAGCGCTAATTTCGTTCAG	230	1	0.00	1	0.00	0.00
					R_CCTCCGTTCTCGCTAATGAC						
	BhUNICAMP094	KM068329	(TG) 7	65	F_TTGGAGCTTTCCCTAGCTCA	272-290	4	0.31	4	0.31	0.39
					R_GAACAAGAAGGGAGGAAGCA						
	BhUNICAMP095	KM068330	(TC) 16 (TG) 14	65	F_GGGTTGGCCTACACACTGAT	268-320	6	0.75	6	0.75	0.92
					R_CGCACGACATTGATACCTTG						
	BhUNICAMP096	KM068331	(TC) 8 TT(TC) 40	65	F_TGTTCTGCTCACTGGTTTGG	157-255	11	0.87	11	0.87	0.95
					R_TCAGCTCTCTACGGCTGGAT						
	BhUNICAMP097	KM068332	(GT) 6	65	F_GCGAGCTACCGAGGTATTTG	129-148	5	0.69	5	0.69	0.80
					R_ACGTCAATGTCGAGCTTCCT						
	BhUNICAMP098	KM068333	(GT) 10 (G) 18	65	F_GGACTGGTCGTCTTTCCATC	250-312	9	0.85	9	0.85	0.95
					R_GCTTTCTGCAAGCGGTAGAT						
	BhUNICAMP099	KM068334	(CA) 10 TG(GA) 10	65	F_TTTGTGGCACCTGCAGAATA	124-174	16	0.91	16	0.91	0.99
					R_CGCTTCGTGCTGACAGATTA						
	BhUNICAMP100	KM068335	(TG) 12	65	F_GCGCCATGGTTTCATCTATT	178-219	7	0.79	7	0.79	0.98
					R_GGTGGTTCCTCGTGTGAGAT						
	BhUNICAMP101	KM068336	(TG) 28	65	F_GGTAAAGAAGGGCCGGACT	128-184	14	0.89	12	0.89	0.97
					R_GCATGGCATGTTCCTACTGA						
	BhUNICAMP102	KM068337	(GCGA) 4	65	F_TGGTGGGCTCCACTATCTCT	224-260	12	0.89	12	0.89	0.94
					R_TCCGCCATCTCTCCTCTCT						
	BhUNICAMP103	KM068338	(CT) 22	65	F_AGCTCTCCCGCCTCTCTCT	100-156	14	0.91	14	0.91	0.96
					R_CATCCACACCGTCTCTCTCA						
	BhUNICAMP104	KM068339	(TG) 26	60	F_ACGACGACCTAATGGGTGAA	190-274	15	0.87	13	0.87	0.96
					R_ACCCAGCAACAAATCTCGTC						
	BhUNICAMP105	KM068340	(AC) 10 ATACAC	50	F_CTCCATCACGTGCTTGCTAA	100-176	30	0.93	29	0.93	0.98
			ACACAC(AG) 53		R_GTGTGATCGGCTGGAGATTT						
	BhUNICAMP106	KM068341	(TTTGT) 3	50	F_GCTGTTCGGAGAGGAATCTG	135-155	8	0.79	7	0.79	0.91
					R_ATGAGAGGAGGGAAGGAAGG						

Table 3

 3 Characterization of the 72 polymorphic SSR markers developed for U. humidicola (Continued) 


	BhUNICAMP107	KM068342	(GA) 18	50	F_GGGTCAGTGTCGTCTCAGTTT	118-190	26	0.94	26	0.94	0.98
					R_CAGATTCCTCTCCGAACAGC						
	BhUNICAMP108	KM068343	(CT) 16	65	F_TTGCCATTACTGGATCTGGA	112-160	14	0.85	13	0.85	0.94
					R_GCGCCACCCATAACTTAAA						
	BhUNICAMP109	KM068344	(GT) 9	60	F_AGCGAGTCAAGCACAAGGAT	186-226	9	0.82	9	0.82	0.93
					R_GGGTCCAATCTCCCTCTCTC						
	BhUNICAMP110	KM068345	(TG) 8	65	F_TCTGCATCCACTAGGCTCAG	148-164	4	0.39	4	0.39	0.46
					R_TCCTCCACCTTCTTTCCGTA						
	BhUNICAMP111	KM068346	(TG) 27	65	F_AACTCCGACTATCTTCCAGTTGA	250-330	15	0.89	15	0.89	0.96
					R_AATGCATGGGTAGGATCTGC						
	BhUNICAMP112	KM068347	(AC) 26	65	F_GACCAAACCCTCCGAAGTTA	246-300	10	0.81	10	0.81	0.94
					R_GGTTGCAACTACACGACCAG						
	BhUNICAMP113	KM068348	(CGTG) 3	63	F_AACTTCGAGAGGTTCGTCCA	144-179	3	0.45	3	0.45	0.51
					R_ACCGGCAATCTATCCGTGT						
	BhUNICAMP114	KM068349	(CT) 21	63	F_TATACAAGGCGCATCCACAA	200-266	15	0.89	15	0.89	0.96
					R_GCTCTTTCCTCACGCTGTTC						
	BhUNICAMP115	KM068350	(AC) 27 (AT) 7	60	F_CTTCCTGCCAATAAGCGAAG	240	1	0.00	1	0.00	0.00
					R_CGAGCTTCCAGATTCTTTGG						
	BhUNICAMP116	KM068351	(TG) 8	65	F_CTCCGCACCGCTTAAATTAG	288-306	3	0.52	3	0.52	0.62
					R_GTTGGAAATGGTGCTTCCAC						
	BhUNICAMP117	KM068352	(TGA) 7	65	F_CCAACTGAACGGCCATACTT	290-300	4	0.61	4	0.61	0.77
					R_CCCACAAAGGAACCCTGAT						
	BhUNICAMP118	KM068353	(AG) 9	50	F_CTGCATAACTTTCAGCCATCTC	149	1	0.00	1	0.00	0.00
					R_TTGGCACAACTGGAACGTAG						
	BhUNICAMP119	KM068354	(AAG) 7	65	F_AAGGGCGTGATGTTCTGAAG	189-204	4	0.66	4	0.66	0.82
					R_AGGCCAAACGAATTTCTCAA						
	BhUNICAMP120	KM068355	(AT) 8 ACACACA	65	F_TCCAGCAGTGTGTTCCTCAG	190-200	6	0.71	6	0.71	0.75
			CACG(CA) 9		R_ACCAGGAGTGCATAGCCAAG						
	BhUNICAMP121	KM068356	(TC) 12	65	F_CGCTACTGCTGCACACAAAT	170-195	6	0.71	6	0.71	0.92
					R_CTGAGTGCGCCGTATGTTTA						
	BhUNICAMP122	KM068357	(GT) 15	65	F_AGGAAGGCTCGCACTCACTA	200-315	14	0.90	14	0.90	0.95
					R_CCAAAGGCGGTGGTTAGATA						
	BhUNICAMP123	KM068358	(TTA) 4	65	F_CCAAACTCTAGCTTTCACAGCA	280	1	0.00	1	0.00	0.00
					R_TTGGATCCACGTCAAACAAG						
	BhUNICAMP124	KM068359	(AG) 23	65	F_TTGGAGTTGCTGGGCTATTT	218-320	12	0.85	10	0.85	0.95
					R_GAACCAAGCATAAGGCAACA						
	BhUNICAMP125	KM068360	(GT) 8 GAATGT	65	F_TGTTATCAGTGCAGGTGTTGG	258-280	7	0.81	7	0.81	0.93
			GTGT(GA) 7		R_GAGGCTGACGAAAGCTCAAC						
	BhUNICAMP126	KM068361	(AC) 10	65	F_GGGAACCCAGGGTATCGTAT	210	1	0.00	1	0.00	0.00
					R_CTCTCCCAGCGTCTTTCCTT						

Table 3

 3 Characterization of the 72 polymorphic SSR markers developed for U. humidicola (Continued) 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. 


	BhUNICAMP127	KM068362	(GT) 6	65	F_CCACCATTGCTTCCAGAGTAA	272-320	7	0.69	7	0.69	0.91
					R_ATTCGCCTCTCCTAGCACAA						
	BhUNICAMP128	KM068363	(GA)37	65	F_TGCCTGGAGACTGAGAAAGG	150-240	17	0.91	17	0.91	0.98
					R_CCTGCAGCAGACCTTCACAT						
	BhUNICAMP129	KM068364	(AC) 7 ATGAA	63	F_TGTGTTTAGACCGCCAACAA	207-310	11	0.84	10	0.84	0.95
			(CATG) 3 (CA) 22		R_TTATCGGCTCCCATTCACTC						
	BhUNICAMP130	KM068365	(AC) 7	63	F_ACGCAGGAGAACTGCGTATC	236-300	12	0.79	11	0.79	0.87
					R_ATGGGATCCAACCGAACATA						
	BhUNICAMP131	KM068366	(AC) 7 (A) 16	60	F_CATCAGATGCCTCAAACAGC	184-238	14	0.87	14	0.87	0.93
					R_GCAGGTGTGCAGCAAATAGA						
	BhUNICAMP132	KM068367	(TG) 7 (T) 29	50	F_TCACTAGTGCGTCTGCTGCT	184-196	4	0.53	3	0.53	0.63
					R_GCACTCCATTGCAGACCTAAG						
	BhUNICAMP133	KM068368	(TG) 10	50	F_CATGACTTATGTCCTTGGTGGA	114-162	19	0.89	16	0.89	0.97
					R_TCGACAGTGGAGCCACAA						
	BhUNICAMP134	KM068369	(CCGG) 3	60	F_CAAACGGAGGAAGAGAGACG	114-135	9	0.75	5	0.75	0.83
					R_GGTGTCAATGCAGCCAAGTA						
	BhUNICAMP135	KM068370	(AG) 27	65	F_CATGAGCCATCTCGTTGTTG	176-260	14	0.90	9	0.90	0.91
					R_TGCATTGACTTGACGTCTCC						
	BhUNICAMP136	KM068371	(AC) 9 (ACAA) 3	50	F_TCCTGGTAAAGTTCCTCGTCA	225-290	7	0.75	6	0.75	0.93
					R_ACAACAATGCACGTCGAGAA						
	BhUNICAMP137	KM068372	(GA) 23	65	F_TAGGTTTGGGTGGCACTAGG	258-320	11	0.85	9	0.85	0.91
					R_CTCCATGCTGCGTTGCTAT						
	BhUNICAMP138	KM068373	(T) 12	60	F_TGCTCATGTGGGTCACATTT	270-288	5	0.70	5	0.70	0.95
					R_TGTGTGCCTGTGTGATGCTA						
	BhUNICAMP139	KM068374	(AAAAG) 3	65	F_TCCTTTCTTTGAGCCGAGAG	248-294	6	0.67	5	0.67	0.97
					R_GCTGATGCTGACATCAAGGA						
	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: **Hybrids included. a Amplification temperature (°C). b Maximum number of alleles observed. c Polymorphism Information Content. d Discrimination Power. 
Table 4

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


	Transferability a,b			
	SSR locus	U.	U.	U.	U.
		brizantha	decumben	ruziziensis	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

Table 4

 4 Cross-amplification of the 72 SSR markers among other Urochloa species (Continued) 


	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. 
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. 


Availability of supporting data

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


Competing interests

The authors declare that they have no competing interests. 
Authors' contributions JCSS and MAB developed the microsatellite-enriched libraries, participated in the microsatellite marker validation, performed the statistical analysis, and drafted the manuscript. JCSS and FAO carried out computational searches for microsatellite identification and drafted the manuscript. BBZV participated in the design and implementation of the study and helped to draft the manuscript. APS conceived of and supervised the study and helped to draft the manuscript. All authors read and approved the final manuscript.
Jean Cs Santos, Mariana A Barreto, Fernanda A Oliveira, Bianca Bz Vigna, Anete P Souza
Microsatellite markers for Urochloa humidicola (Poaceae) and their transferability to other Urochloa species