Cross-amplification and characterization of microsatellite markers in species of Manihot Mill. (Euphorbiaceae) endemic to the Brazilian Cerrado

Kássia Marques Corrêa Miranda Rejane Araújo Guimarães Marcos José da Silva Patrícia Rasteiro Ordiale Oliveira Thania Gonçalves Ribeiro Thainara Policarpo Mendes Mariana Pires de Campos Telles Thannya Nascimento Soares About the authors

ABSTRACT

The genus Manihot Mill. contains about 120 species of which about 104 occur in Brazil. We tested the cross-amplification of ten microsatellite markers developed for Manihot esculenta in 15 species of Manihot endemic to the Brazilian Cerrado. We also evaluated the genetic diversity of Manihot irwinii, M. orbicularis, and M. purpureocostata. Ten pairs of primers were amplified among 14 species of Manihot. The percentage of polymorphic loci per species varied from 70 to 100 %. Nine markers showed amplification and polymorphism when evaluated on polyacrylamide gel. The markers were combined to form three sets for multiplex genotyping for genetic diversity analysis, and showed 51, 75, and 75 alleles in M. irwinii, M. orbicularis, and M. purpureocostata, respectively. The levels of genetic diversity for the transferred markers were high for the three species and proved to be useful for population genetics studies of species of Manihot endemic to the Cerrado. The results of this study will help to better understand the genetic diversity, taxonomy and relationships among species Manihot, and to develop conservation programs for the genus.

Keywords:
Genetic diversity; polymorphism; SSR; transferability wild cassava

Introduction

The genus Manihot has a Mesoamerican origin with its center of diversity in Brazil (Silva 2014Silva MJ. 2014. Manihot veadeirensis (Euphorbiaceae s. s.): a New species from the Brazilian Cerrado. Systematic Botany 39: 1161-1165.). The Cerrado of Central Brazil presents about 104 documented species, so the main center of diversity for the genus within the country, (Silva & Amaral 2020Silva MJ, Amaral US. 2020. Novelties in wild cassava (Manihot, Euphorbiaceae) from Brazil. Brittonia 72: 164-176.).

Floristic and taxonomic studies have shown that species of this genus have problems of taxonomic delimitation due to the lack of taxonomic studies in Brazil. Nonetheless, several new species have been described in recent years (Silva et al. 2016Silva MJ, Alonso AA, Sodré RC. 2016. Manihot pachycaulis sp. nov. (Euphorbiaceae) from the Brazilian Cerrado. Nordic Journal of Botany 34: 60-65.; Silva 2016Silva MJ. 2016. Manihot gratiosa and M. lourdesii spp. nov. (Manihoteae, Euphorbiaceae) from the Brazilian Cerrado. Nordic Journal of Botany 34: 66-74.; Silva et al. 2017Silva MJ, Soares TN, Oliveira PRO. 2017. Morphological characteristics and genetic evidence reveals a new species of Manihot (Euphorbiaceae, Crotonoideae) from Goiás, Brazil. PhytoKeys 77: 99-111.; Mendoza et al. 2018Mendoza M, Simon MF, Arquelão TKM, Cavalcanti TB. 2018. Novas espécies de Manihot (Euphorbiaceae) do Brasil Central. Rodriguésia 69: 915-932.), showing significant advances in knowledge of the diversity of the genus. However, little is known about the genetic diversity of these species, especially in the Central-West Region of Brazil.

The development of microsatellites or simple sequence repeats (SSRs) provides an ideal tool for investigating patterns of genetic variation due to their codominant inheritance and multiallelic and highly polymorphic properties, as well as being abundant and well distributed throughout the genome (Li et al. 2002Li Y-C, Korol AB, Fahima T, Beiles A, Nevo E. 2002. Microsatellites: genomic distribution, putative functions and mutational mechanisms: a review. Molecular Ecology 11: 2453-2465.; Ellegren 2004Ellegren H. 2004. Microsatellites: simple sequences with complex evolution. Nature Reviews Genetics 5: 435-45.). However, given the time-consuming and expensive process of isolating SSRs, it is advantageous to test available microsatellite markers for phylogenetically close species by cross-amplification before investing in the development of species-specific markers.

The success of cross-species amplification depends on the conservation of primer sites within flanking sequences and on the maintenance of sequences that promote polymorphisms (FitzSimmons et al. 1995FitzSimmons NN, Moritz C, Moore SS. 1995. Conservation and dynamics of microsatellite loci over 300 million years of marine turtle evolution. Molecular Biology and Evolution 12: 432-40.). Several studies have demonstrated the utility of using primer pairs designed from one species for others of the same genus (Bernardes et al. 2014Bernardes V, Anjos DE, Gondim SGCA, Murakami DM, Bizão N, Telles MPC. 2014. Isolation and characterization of microsatellite loci in Byrsonima cydoniifolia (Malpighiaceae) and cross-amplification in B. crassifolia. Applications in Plant Sciences 2: 1400016. doi: 10.3732/apps.1400016
https://doi.org/10.3732/apps.1400016...
; Buzatti et al. 2016Buzatti RSO, Chicata FSL, Lovato MB. 2016. Transferability of microsatellite markers across six Dalbergia (Fabaceae) species and their characterization for Dalbergia miscolobium. Biochemical Systematics and Ecology 69: 161-165.) and even for species of other genera (Barbará et al. 2007Barbará T, Palma-Silva C, Paggi GM, Bered F, Fay MF, Lexer C. 2007. Cross-species transfer of nuclear microsatellite markers: potential and limitations. Molecular Ecology 16: 3759-3767.; Santos et al. 2015Santos J, Barreto MA, Oliveira FA, Vigna B, Souza AP. 2015. Microsatellite markers for Urochloa humidicola (Poaceae) and their transferability to other Urochloa species. BMC Research Notes 8: 83. doi: 10.1186/s13104-015-1044-9
https://doi.org/10.1186/s13104-015-1044-...
; Fagundes et al. 2016Fagundes BS, Silva LF, Giacomin RM, Secco D, Díaz-Cruz JA, Da-Silva PR. 2016. Transferability of microsatellite markers among Myrtaceae species and their use to obtain population genetics data to help the conservation of the Brazilian Atlantic Forest. Tropical Conservation Science 9: 408-422.; Miranda et al. 2016Miranda EAGC, Boaventura-Novaes CRD, Braga RS, Reis EF, Pinto JFN, Telles MPC. 2016. Validation of EST-derived microsatellite markers for two cerrado-endemic Campomanesia (Myrtaceae) species. Genetics and Molecular Research 15: 15017658. doi: 10.4238/gmr.15017658
https://doi.org/10.4238/gmr.15017658...
).

Considering the insufficient knowledge available regarding the diversity of wild species of the genus Manihot, along with the taxonomic and phylogenetic complexity of the genus and the lack of SSR and molecular information, we tested microsatellite markers developed for M. esculenta (cultivated cassava) (Chavarriaga-Aguirre et al. 1998Chavarriaga-Aguirre P, Maya MM, Bonierbale MW, et al. 1998. Microsatellites in cassava (Manihot esculenta Crantz); discovery, inheritance and variability. Theoretical and Applied Genetics 97: 493-501.; Mba et al. 2001Mba REC, Stephenson P, Edwards K, et al. 2001. Simple sequence repeat (SSR) markers survey of the cassava (Manihot esculenta Crantz) genome: Towards an SSR-based molecular genetic map of Cassava. Theoretical and Applied Genetics 102: 21-31.) by cross-amplification in 15 congeners endemic to the Cerrado. Moreover, we evaluated the genetic diversity of the markers in three of these species (M. irwinii, M. orbicularis, and M. purpureocostata), to provide tools for population genetics studies on species of Manihot endemic to the Cerrado.

Materials and methods

Plant material and cross-species amplification

Amplification tests used leaves sampled from three individuals from each of the know 15 species of Manihot of the Cerrado, Goiás State, Central-West Brazil (Tab. 1). Marker polymorphism evaluation used eight individuals per species. A standard protocol of 2 % CTAB was used for DNA extraction (Doyle & Doyle 1987Doyle JJ, Doyle JL. 1987. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochemical Bulletin 19: 11-15.). Ten nuclear microsatellite markers previously developed for M. esculenta were tested in the 15 Manihot species: GA 134, GA 136, AG 126, AG 21, AG 12, AG 131, AG 16, GAGG 5 (Chavarriaga-Aguirre et al. 1998), and SSRY 12 and SSRY 82 (Mba et al. 2001Mba REC, Stephenson P, Edwards K, et al. 2001. Simple sequence repeat (SSR) markers survey of the cassava (Manihot esculenta Crantz) genome: Towards an SSR-based molecular genetic map of Cassava. Theoretical and Applied Genetics 102: 21-31.).

Table 1
Sampling locations for the Manihot species used in the present study and the vouchers deposited in the herbarium of the Universidade Federal de Goiás.

PCR amplifications were performed in a final volume of 10 μl using 4.5 ng template DNA, 0.18 μM primers (forward + reverse), 0.15 μM dNTP, 2.16 mg bovine serum albumin (BSA), 1x reaction buffer (10 mM Tris), HCl [pH 8.3], 50 mM KCl, 1.5 mM MgCl 2, and 0.75 units of Taq DNA polymerase (5U; Phoneutria, Belo Horizonte, Brazil) under the following conditions: 30 cycles of 94 °C for 1 min, 54-65 °C (depending on the initiator, Tab. 2) for 1 min, and of 72° C for 1 min, and a final extension at 72 °C for 45 min.

Table 2
Cross amplification of ten microsatellite loci of Manihot esculenta Crantz for 15 wild species of Manihot endemic to the Cerrado.

The annealing temperature of the primers was adjusted for each marker until an acceptable amplification pattern was found on 3 % agarose gel (Tab. 2). Polymorphism of the markers in the 15 species of Manihot was determined through standard 6 % acrylamide gel electrophoresis visualized by silver staining procedures (Creste et al. 2001Creste S, Neto Tulmann A, Figueira A. 2001. Detection of single sequence repeat polymorphisms in denaturing polyacrylamide sequencing gels by silver staining. Plant Molecular Biology Reporter 19: 299-306.). Allele size was determined by reference to 10 bp and 50 bp DNA standards (Invitrogen ™).

Genetic variability of polymorphic loci

The markers that showed the best polyacrylamide gel amplification profiles, associated with verified polymorphism in the 15 species, were selected for characterizing the genetic diversity of three Manihot species: M. orbicularis Pohl, M. purpureocostata Pohl, and M. irwinii DJ Rogers & Appan. A sample of 24 individuals, representing known occurrence, was used for each species, for a total of 72 individuals. DNA extraction and amplification followed the same protocols as described above.

Forward sequences of selected primer pairs were labeled with one of four fluorescent dyes: VIC, NED, 6-FAM, or PET. The sizes of amplification products were determined using a GeneScan 600 LIZ internal marker (Applied Biosystems) in an ABI PRISM® 3500 DNA Genetic Analyzer (Applied Biosystems). Microsatellite loci with greater clarity in their amplification detected by capillary electrophoresis were arranged in multiplex panels for analysis of the three Manihot species.

Allele calling was performed using GeneMapper 5.0 software (Applied Biosystems). Genotypes were confirmed using an allelic ladder constructed with all alleles found for each locus in this study. Micro-Checker software (Oosterhout et al. 2004Oosterhout CV, Hutchinson WF, Wills DPM, Shipley P. 2004. MICRO- CHECKER: software for identifying and correcting genotyping errors in microsatellite data. Molecular Ecology Notes 4: 535-538.) was then used to detect errors due to stuttering, allele dropout, and null alleles.

The power of individual discrimination with the total loci set and with each locus was evaluated by estimates of probability of genetic identity (I) (Paetkau et al. 1995Paetkau D, Calvert W, Stirling I, Strobeck C. 1995. Microsatellite analysis of population structure in Canadian polars bears. Molecular Ecology 4: 347-354.) and the probability of paternity exclusion (Q) (Weir 1996Weir BS. 1996. Genetic data analysis II: methods for discrete population genetic data. Sunderland, MA, Sinauer Associates .), using Identity 1.0 software (Wagner & Sefc 1999Wagner HW, Sefc KM. 1999. IDENTITY 1.0 Centre for Applied Genetics. University of Agricultural Sciences, Vienna. http:/boku. ac. at/zag/forsgh/identity. htm. 10 Oct. 2020.
http:/boku. ac. at/zag/forsgh/identity. ...
). Genetic variability analysis, including allelic richness, observed heterozygosity (Ho), and expected heterozygosity under the Hardy-Weinberg equilibrium (He ), were estimated using Genetic Data Analysis 1.0 software (GDA) (Lewis & Zaykin 2001Lewis PO, Zaykin D. 2001. GDA (Genetic Data Analysis): Computer Program for the Analysis of Allelic Data (version 1.1). http://phylogeny.uconn.edu/software/. 10 Oct. 2020.
http://phylogeny.uconn.edu/software/...
). Linkage disequilibrium was evaluated using Bonferroni correction in FSTAT 2.9.3.2 software (Goudet 2002Goudet J. 2002. FSTAT: a program to estimate and test gene diversities and fixation indices (version 2.9.3.2). http://www2. unil. ch/popgen/softwares/fstat. htm. 10 Oct. 2020.
http://www2. unil. ch/popgen/softwares/f...
).

Results and discussion

The markers amplified in all 15 species of Manihot were dinucleotides, most with the GA motif. The least conserved marker was GAGG5 (tetranucleotide), which did not amplify for one of the 15 species (M. irwinii).

All ten primer pairs tested in the wild Manihot species were polymorphic (100 %) in nine of them (M. gabrielensis, M. violacea, M. orbicularis, M. purpureocostata, M. saxatilis, M. alutacea, M. attenuata, M. divergens, and M. irwinii), while nine (90 %) were polymorphic for M. mossamedensis, M. confertiflora, and M. pentaphylla; eight (80 %) for M. paviifolia; and seven (70 %) for M. peltata and M. tripartita (Tab. 2). The results obtained regarding the percentage of polymorphic loci among the three species of Manihot are similar to those reported in the literature for other species of the same genus and characterized by a set of similar primers. Among these, we have included the cultivated species M. esculenta and six other different species (all wild), M. esculenta subsp. flabellifolia, M. esculenta subsp. peruviana, M. aesculifolia, M. brachyloba, M. carthaginensis, and M. tristis (Roa et al. 2000Roa AC, Chavarriaga-Aguirre P, Duque MC, et al. 2000. Cross-species amplification of cassava (Manihot esculenta) (Euphorbiaceae) microsatellites: allelic polymorphism and degree of relationship. American Journal of Botany 87: 1647-1655.). Thus, in the present study, the percentage of polymorphic loci obtained from cross-amplification among species within the genus Manihot tends to maintain close to 100 %, reflecting high conservation of genomic regions among species. This conservation may be evidence of recent diversification of the genus Manihot, which would explain the low taxonomic resolution for some sets of Manihot species (Duputié et al. 2011Duputié A, Salick J, Mckey D. 2011. Evolutionary biogeography of Manihot (Euphorbiaceae), a rapidly radiating Neotropical genus restricted to dry environments. Journal of Biogeography 38: 1033-1043.).

The high microsatellite cross-amplification observed among species of Manihot reveal their genetic proximity with M. esculenta. This finding corroborates the results of Raji et al. (2009Raji AA, Anderson J V, Kolade OA, Dixon AG, Ingelbrecht IL. 2009. Gene-based microsatellites for cassava (Manihot esculenta Crantz): Prevalence, polymorphisms, and cross-taxa utility. BMC Plant Biology 9: 118. doi: 10.1186/1471-2229-9-118
https://doi.org/10.1186/1471-2229-9-118...
), who showed that 92 % of the markers were transferable to wild relatives of M. esculenta belonging to the same genus (M. epruinosa, M. glaziovii, M. brachyandra, and M. tripartita), while only a small fraction was transferable to species of other genera.

Cross-species amplification has been reported for six wild species of the genus Manihot (M. aesculifolia, M. brachyloba, M. carthaginensis, M. esculenta subsp. flabellifolia, M. esculenta subsp. peruviana, and M. tristis) (Roa et al. 2000Roa AC, Chavarriaga-Aguirre P, Duque MC, et al. 2000. Cross-species amplification of cassava (Manihot esculenta) (Euphorbiaceae) microsatellites: allelic polymorphism and degree of relationship. American Journal of Botany 87: 1647-1655.), showing that, in general, microsatellite primers work throughout the genus. However, as phylogenetic distance increases, successful amplification of loci tends to decrease. This relationship with phylogenetic distance was also observed by Bressan et al. (2012Bressan EDA, Scotton DC, Ferreira RR, et al. 2012. Development of microsatellite primers for Jatropha curcas (Euphorbiaceae) and transferability to congeners. American Journal of Botany 99: 237-239.) when reporting cross-amplification with the species Jatropha curca, which belongs to the same family as Manihot (Euphorbiaceae). They found amplification of alleles in species of the same genus, but not in other genera of Euphorbiaceae, such as Hevea brasiliensis, Manihot esculenta, and Ricinus communis. Successful transferability of microsatellite markers among closely related species has also been verified for other Cerrado species, such as Anacardium humile (Soares et al. 2013Soares TN, Sant’Ana LL, Oliveira LK, Telles MPC, Collevatti RG. 2013. Transferability and characterization of microssatellite loci in Anacardium humile A. St. Hil. (Anacardiaceae). Genetics and Molecular Research 12: 3146-3149.), Byrsonima cydoniifolia (Bernardes et al. 2014Bernardes V, Anjos DE, Gondim SGCA, Murakami DM, Bizão N, Telles MPC. 2014. Isolation and characterization of microsatellite loci in Byrsonima cydoniifolia (Malpighiaceae) and cross-amplification in B. crassifolia. Applications in Plant Sciences 2: 1400016. doi: 10.3732/apps.1400016
https://doi.org/10.3732/apps.1400016...
), and Campomanesia adamantium and C. pubescens (Miranda et al. 2016Miranda EAGC, Boaventura-Novaes CRD, Braga RS, Reis EF, Pinto JFN, Telles MPC. 2016. Validation of EST-derived microsatellite markers for two cerrado-endemic Campomanesia (Myrtaceae) species. Genetics and Molecular Research 15: 15017658. doi: 10.4238/gmr.15017658
https://doi.org/10.4238/gmr.15017658...
). This success can be explained by the conservation of microsatellite flanking regions in closely related species (FitzSimmons et al. 1995FitzSimmons NN, Moritz C, Moore SS. 1995. Conservation and dynamics of microsatellite loci over 300 million years of marine turtle evolution. Molecular Biology and Evolution 12: 432-40.; Barbará et al. 2007Barbará T, Palma-Silva C, Paggi GM, Bered F, Fay MF, Lexer C. 2007. Cross-species transfer of nuclear microsatellite markers: potential and limitations. Molecular Ecology 16: 3759-3767.).

The markers were polymorphic for most species (eight individuals), varying among 12 to 15 species. The most polymorphic markers were GA 136, GA 134, GA131, and SSRY12, which were polymorphic in all 15 species (Tab. 2). Such high levels of polymorphism were expected according to data in the literature for wild species of the genus Manihot (Roa et al. 2000Roa AC, Chavarriaga-Aguirre P, Duque MC, et al. 2000. Cross-species amplification of cassava (Manihot esculenta) (Euphorbiaceae) microsatellites: allelic polymorphism and degree of relationship. American Journal of Botany 87: 1647-1655.; Raji et al. 2009Raji AA, Anderson J V, Kolade OA, Dixon AG, Ingelbrecht IL. 2009. Gene-based microsatellites for cassava (Manihot esculenta Crantz): Prevalence, polymorphisms, and cross-taxa utility. BMC Plant Biology 9: 118. doi: 10.1186/1471-2229-9-118
https://doi.org/10.1186/1471-2229-9-118...
).

Out of the ten markers, nine exhibited better amplification and polymorphism patterns and so were combined into three sets for multiplex genotyping and characterization of the loci (Tab. 3). Out of the nine markers with clear amplicons tested on the three wild species of Manihot, four loci had null alleles, the locus GA136 for M. orbicularis and M. irwinii, GA126 for M. orbicularis and M. purpureocostata, GA16 for M. irwinii, and SSRY12 for M. orbicularis. Null alleles have been commonly found in studies of transferability of microsatellite markers in wild species of Manihot (Roa et al. 2000Roa AC, Chavarriaga-Aguirre P, Duque MC, et al. 2000. Cross-species amplification of cassava (Manihot esculenta) (Euphorbiaceae) microsatellites: allelic polymorphism and degree of relationship. American Journal of Botany 87: 1647-1655.; Raji et al. 2009Raji AA, Anderson J V, Kolade OA, Dixon AG, Ingelbrecht IL. 2009. Gene-based microsatellites for cassava (Manihot esculenta Crantz): Prevalence, polymorphisms, and cross-taxa utility. BMC Plant Biology 9: 118. doi: 10.1186/1471-2229-9-118
https://doi.org/10.1186/1471-2229-9-118...
). However, no null alleles have been observed in M. esculenta (Roa et al. 2000Roa AC, Chavarriaga-Aguirre P, Duque MC, et al. 2000. Cross-species amplification of cassava (Manihot esculenta) (Euphorbiaceae) microsatellites: allelic polymorphism and degree of relationship. American Journal of Botany 87: 1647-1655.). In this sense, the the occurrence de null alleles may be an artifact in the sequences flanking the microsatellite, caused by transferability (Dabrowski et al. 2015Dabrowski MJ, Bornelöv S, Kruczyk M, Baltzer N, Komorowski J. 2015. “True” null allele detection in microsatellite loci: A comparison of methods, assessment of difficulties and survey of possible improvements. Molecular Ecology Resources 15: 477-488.).

No significant changes in linkage disequilibrium (P> 0.05) were found for any pair of loci in any species. Deviations from HWE (P <0.05) were observed at locus GA21 for all species, GA136 for M. irwinii and M. orbicularis, GA16 and SSRY82 for M. irwinii, GA126 for M. orbicularis and M. purpureocostata, and GA131 for M. orbicularis. Among the loci that deviated from HWE, some showed null alleles, which may explain the deviation. Analysis with more populations and a larger number of individuals per population may confirm this result.

Table 3
Three sets of microsatellite markers for multiplex genotyping for the species Manihot irwinii, M. orbicularis and M. purpureocostata.

The present study detected 51 alleles in M. irwinii, 75 alleles in M. orbicularis and 75 alleles in M. purpureocostata, which demonstrates a high level of polymorphism. These results are similar to those reported in the literature for wild species of the genus Manihot, which found 79 alleles (Roa et al. 2000Roa AC, Chavarriaga-Aguirre P, Duque MC, et al. 2000. Cross-species amplification of cassava (Manihot esculenta) (Euphorbiaceae) microsatellites: allelic polymorphism and degree of relationship. American Journal of Botany 87: 1647-1655.) and 50 alleles (Silva et al. 2017Silva MJ, Soares TN, Oliveira PRO. 2017. Morphological characteristics and genetic evidence reveals a new species of Manihot (Euphorbiaceae, Crotonoideae) from Goiás, Brazil. PhytoKeys 77: 99-111.). Studies of M. esculenta have found 46 alleles (Siqueira et al. 2009Siqueira MVBM, Queiroz-silva JR, Bressan EA, et al. 2009. Genetic characterization of cassava (Manihot esculenta) landraces in Brazil assessed with simple sequence repeats. Genetics and Molecular Biology 32: 104-110.), 45 alleles (Roa et al. 2000Roa AC, Chavarriaga-Aguirre P, Duque MC, et al. 2000. Cross-species amplification of cassava (Manihot esculenta) (Euphorbiaceae) microsatellites: allelic polymorphism and degree of relationship. American Journal of Botany 87: 1647-1655.), and 47 alleles (Aragon et al. 2012Aragon E, Aguilar V, Arguello J. 2012. Diversidad genética en cultivares de Manihot esculenta Crantz en Nicarágua determinada mediante microsatélites (SSR). Biotecnologia Vegetal 12: 211-218.). These results follow Roa et al. (2000),Roa AC, Chavarriaga-Aguirre P, Duque MC, et al. 2000. Cross-species amplification of cassava (Manihot esculenta) (Euphorbiaceae) microsatellites: allelic polymorphism and degree of relationship. American Journal of Botany 87: 1647-1655. who state that the wild species of the genus Manihot have a larger pool of SSR alleles than M. esculenta.

The number of alleles per locus found for the three species of Manihot varied ​​from three to 16, with averages of 5.7, 8.3, and 8.3 alleles per locus for M. irwinii, M. orbicularis, and M. purpureocostata, respectively (Tab. 4). Thus, the high average number of alleles per locus found in the present study suggests that the set of markers used substantially represents the polymorphism of the loci. This can be confirmed by the high polymorphism along with the low probability of identity (1.672 x 10-7, 9.13 x 10-10, and 2.61 x 10-9) and high power of paternity exclusion (0.999, 0.999, and 0.998), observed for the species M. irwinii, M. orbicularis, and M. purpureocostata, respectively. These results show that the nine markers are suitable for discriminating individuals at each locus under analysis (Paetkau et al. 1995Paetkau D, Calvert W, Stirling I, Strobeck C. 1995. Microsatellite analysis of population structure in Canadian polars bears. Molecular Ecology 4: 347-354.) and demonstrated a high power of paternity exclusion (Weir & Evett 1998Weir BS, Evett I. W. 1998. Interpreting DNA evidence: statistical genetics for forensic scientists. Sunderland, MA, Sinauer Associates.), allowing an efficient characterization of the genetic variability existing in populations of wild Manihot species (Tab. 4).

Table 4
Genetic characterization of nine microsatellite loci of Manihot esculenta in three wild species of Manihot. Species (number of individuals); A: number of alleles per loco; He: heterozygosity expected by the Hardy-Weinberg equilibrium; Ho: observed heterozygosity; Q: probability of paternity exclusion; I: probability of identity.

The average genetic diversity (He ) of the markers was high for M. irwinii (74 %), M. orbicularis (82 %), and M. purpureocosta (78 %). These results are equivalent to the maximum genetic diversity expected (0.833, 0.879, and 0.879), according to Hennink & Zeven (1991Hennink S, Zeven AC. 1991. The interpretation of Nei and Shannon-Weaver within population variation indices. Euphytica 51: 235-240.), considering the number of alleles found per locus.

The average observed heterozygosity (Ho= 0.517, 0.615, and 0.669) was lower than the expected (He =0.618, 0.724, and 0.689; Tab. 4) for M. irwinii, M. orbicularis, and M. purpureocosta, respectively. This result suggests that the heterozygote deficiency may be due to several factors, such as inbreeding (Halsey et al. 2008Halsey ME, Olsen KM, Taylor NJ, Chavarriaga-Aguirre P. 2008. Reproductive biology of cassava (Manihot esculenta Crantz) and isolation of experimental field trials. Crop Science 48: 49-58.), limited sample size, and presence of null alleles, with the latter being a common factor in transferability studies with wild species of Manihot (Roa et al. 2000Roa AC, Chavarriaga-Aguirre P, Duque MC, et al. 2000. Cross-species amplification of cassava (Manihot esculenta) (Euphorbiaceae) microsatellites: allelic polymorphism and degree of relationship. American Journal of Botany 87: 1647-1655.; Raji et al. 2009Raji AA, Anderson J V, Kolade OA, Dixon AG, Ingelbrecht IL. 2009. Gene-based microsatellites for cassava (Manihot esculenta Crantz): Prevalence, polymorphisms, and cross-taxa utility. BMC Plant Biology 9: 118. doi: 10.1186/1471-2229-9-118
https://doi.org/10.1186/1471-2229-9-118...
), and with other species of the Cerrado (Ciampi et al. 2008Ciampi AY, Azevedo VCR, Gaiotto FA, Ramos ACS, Lovato MB. 2008. Isolation and characterization of microsatellite loci for Hymenaea courbaril and transferability to Hymenaea stigonocarpa, two tropical timber species. Molecular Ecology Resources 8: 1074-1077.; Feres et al. 2009Feres JM, Martinez MLL, Martinez C, Mestriner MA, Alzate-Marin AL. 2009. Transferability and characterization of nine microsatellite markers for the tropical tree species Tabebuia roseo-alba. Molecular Ecology Resources 9: 434-437.; Soares et al. 2013Soares TN, Sant’Ana LL, Oliveira LK, Telles MPC, Collevatti RG. 2013. Transferability and characterization of microssatellite loci in Anacardium humile A. St. Hil. (Anacardiaceae). Genetics and Molecular Research 12: 3146-3149.; Fagundes et al. 2016Fagundes BS, Silva LF, Giacomin RM, Secco D, Díaz-Cruz JA, Da-Silva PR. 2016. Transferability of microsatellite markers among Myrtaceae species and their use to obtain population genetics data to help the conservation of the Brazilian Atlantic Forest. Tropical Conservation Science 9: 408-422.; Miranda et al. 2016Miranda EAGC, Boaventura-Novaes CRD, Braga RS, Reis EF, Pinto JFN, Telles MPC. 2016. Validation of EST-derived microsatellite markers for two cerrado-endemic Campomanesia (Myrtaceae) species. Genetics and Molecular Research 15: 15017658. doi: 10.4238/gmr.15017658
https://doi.org/10.4238/gmr.15017658...
).

The present study documented ten polymorphic microsatellite markers for the 15 studied species of Manihot. Nine of these markers were indicated as having high potential to detect genetic variation in the three analyzed wild species of Manihot. Thus, the results of this study are promising and valuable for developing further studies of genetic variability of these species and for studies aiming to properly understand the relationships among the species of the genus Manihot, as well as their genetic diversity, taxonomy, and conservation.

Acknowledgements

We acknowledge support: projects Universal 14/2014, PRO-CENTRO-OESTE 31/2010 and Genpac 2; CAPES for scholarships to KM Corrêa Miranda, RA Guimarães and PRO Oliveira, and the technical scholarship to TP Mendes; FAPEG for the scholarship to TG Ribeiro; and the CNPq productivity grant to MPC Telles, MJ Silva, and TN Soares. This research was developed in the context of Instituto Nacional de Ciência e Tecnologia (INCT) in Ecologia, Evolução e Conservação da Biodiversidade, supported by MCTIC/CNPq and FAPEG.

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Publication Dates

  • Publication in this collection
    22 Mar 2021
  • Date of issue
    Oct-Dec 2020

History

  • Received
    20 Nov 2019
  • Accepted
    12 Oct 2020
Sociedade Botânica do Brasil SCLN 307 - Bloco B - Sala 218 - Ed. Constrol Center Asa Norte CEP: 70746-520 Brasília/DF - Alta Floresta - MT - Brazil
E-mail: acta@botanica.org.br