The Mating System, Genetic Variability and Genetic Relationships Between Centrosema Species
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Euphytica DOI 10.1007/s10681-011-0415-0 Genetic studies in Centrosema pubescens benth, a tropical forage legume: the mating system, genetic variability and genetic relationships between Centrosema species A. C. B. Sousa • M. A. Carvalho • A. K. B. Ramos • T. Campos • D. A. Sforc¸a • M. I. Zucchi • L. Jank • A. P. Souza Received: 11 December 2010 / Accepted: 16 March 2011 Ó Springer Science+Business Media B.V. 2011 Abstract In this study, we used microsatellite loci to occurs in related individuals. A paternity correlation of estimate the outcrossing rate of Centrosema pubescens 14% suggests that there is a low probability of finding in open-pollinated populations of 10 progenies that full sibs in the progeny. Cross-amplification of the 26 each contained 20 genotypes. The multilocus outcross- microsatellite loci available for C. pubescens was ing rate was 27%, which suggested a mixed mating evaluated across 11 different Centrosema species. system with a predominance of autogamy. The single Nineteen of the 26 tested microsatellites were success- locus outcrossing rate was 13%. The difference was fully transferable across the Centrosema species. The 0.040, which indicated that only 4% of outcrossing polymorphism information content and discriminating power evaluated had averages of 0.64 and 0.77, respectively. A total of three clusters were assembled A. C. B. Sousa Á T. Campos Á D. A. Sforc¸a Á to demonstrate the genetic relationships between A. P. Souza (&) Centrosema species. The transferable microsatellite Genetic Engineering and Molecular Biology Center loci should be useful for exploiting the genetic (CBMEG), University of Campinas (UNICAMP), CP 6010, Campinas, SP CEP 13083-970, Brazil resources of the Centrosema species and determining e-mail: [email protected] the outcrossing rate, which are essential for proposing effective approaches for conservation and for estab- M. A. Carvalho Á A. K. B. Ramos lishing strategies for the selection and improvement of Brazilian Agricultural Research Corporation, EMBRAPA Cerrados, BR 020, Km 18, Planaltina, Centrosema spp. DF CEP 73310-970, Brazil Keywords Tropical legume Á Cross-amplification Á M. I. Zucchi Mating system Á Autogamy Agronomic Institute of Campinas, Po´lo Apta Centro Sul, Rod. SP 127 Km 30, CP 28, Piracicaba, SP CEP 13400-970, Brazil Introduction L. Jank Forage Breeding Department, Brazilian Agricultural Research Corporation, EMBRAPA Beef Cattle, The genus Centrosema (DC.) Benth belongs to the CP 154, Campo Grande, MS CEP 79002-970, Brazil family Fabaceae (alt. Leguminosae), the subfamily Faboideae and the tribe Phaseoleae. The genus is A. P. Souza composed of 34 species that are native to Central and Plant Biology Department (DBV), Biology Institute, University of Campinas (UNICAMP), CP 6109, South America (Williams and Clements 1990). Campinas, SP CEP 13083-970, Brazil Several Centrosema species occur naturally in Brazil 123 Euphytica where wide genetic diversity is found (Schultze-Kraft that microsatellites can be transferred from one genera/ and Clements 1990). Centrosema includes species species to another (Eujayl et al. 2004; Gutierrez et al. that can adapt to diverse habitats such as the dry 2005). In addition to being applicable to genetic tropics, high-altitude tropics, subtropics, poorly diversity, genetic mapping, and marker-assisted selec- drained and seasonally flooded areas and acidic, tion, microsatellites are useful for estimating the low-fertility soils (Keller-Grein et al. 2000). Because mating systems in plants (Varshney et al. 2005). of this adaptability, Centrosema has been agronom- Centrosema is assumed to be a predominantly self- ically evaluated in Brazil (Borges 2006), Nigeria pollinating genus, although insect cross-pollination has (Odeyinka et al. 2008), Columbia (Keller-Grein et al. been reported in C. brasilianum with outcrossing rates 2000) Australia (Schultze-Kraft et al. 1997), Asia ranging from 31.2 to 53.5% (Maass and Torres 1992). (Humphreys et al. 1990) and Peru (Rea`tegui et al. However, the reproductive system of other Centrosema 1985). Of the promising fodder crop species of species has not been clearly elucidated. This informa- Centrosema, three are of interest in the tropical and tion is essential for proposing effective approaches for subtropical areas of America: C. pubescens (Centro- conservation and for establishing strategies for selec- sema molle Mart. Ex Benth), C. acutifolium and tion and improvement. In this study, we used C. C. brasilianum. The chromosome number for these pubescens-specific microsatellite markers to assess three species is 2n = 2x = 22 (Novaes and Penteado cross-transferability in 11 different Centrosema species 1993), whereas numbers of 2n = 2x = 18 and 20 and to estimate the outcrossing rate in C. pubescens. have been reported in other species of this genus (Battistin and Vargas 1989; Miles et al. 1990). In addition to the currently cultivated species, the Material and methods genus Centrosema includes other promising species that may be used as pasture crops. However, genetic Plant material knowledge in Centrosema is still limited and has restricted their domestication and exploitation in Twelve Centrosema species: C. pubescens, C. pascuo- breeding programs. Advancing the knowledge in this rum, C. brachypodum, C. brasilianum, C. rotundifoli- area will require the application of genomic tools such um, C. acutifolium, C. terezae, C. arenarium, as molecular markers. In C. pubescens, 26 polymorphic C. tetragonolobum, C. macrocarpum, C. plumieri, microsatellite loci have been reported (Sousa et al. and C. sagittatum were used in this study (Table 1). 2009). These accessions were obtained from the Cerrados Microsatellite loci are short (1–6 bp) tandem repeat Research Center Germplasm Bank of the Brazilian DNA sequences that are dispersed randomly through- Agricultural Research Corporation—EMBRAPA, out the genome. Replication slippage or unequal Distrito Federal—Brası´lia, Brazil and the Instituto de crossing over events produce variation in the number Zootecnia, Sa˜o Paulo—Nova Odessa, Brazil. of repeat motifs, and these loci thus represent hyper- variable regions of the genome. These regions are Microsatellite loci highly polymorphic, codominant and can result in high rates of transferability across species (Gaita´n-Solı´s Twenty-six microsatellite loci were selected for this et al. 2002). study based on the primer sequences used previously in The cross-amplification of microsatellite loci C. pubescens (Sousa et al. 2009). The lengths of these among closely related species depends on the extent microsatellites varied from 18 to 22 nucleotides, and of homology and sequence conservation in regions the product lengths varied from 165 to 298 bp. Primers flanking the simple sequence repeats. A high rate of were synthesized by Invitrogen, CA, USA. transferability has already been documented in plant species. Cross-amplification of microsatellites in DNA extraction, polymerase chain reaction closely related Oryza species has been reported (Wu (PCR) amplification and genotyping and Tanksley 1993). Choumane et al. (2000) reported the conservation of microsatellite loci in different taxa Genomic DNA was extracted from freeze-dried leaf of Fabaceae. In legumes, several reports have shown samples using the cetyltrimethylammonium bromide 123 Euphytica Table 1 Centrosema species and their respective accession Taq DNA Polymerase (Invitrogen, CA, USA). All numbers PCR amplifications were performed in a PTC-200 Name species Accession no. thermal cycler (MJ Research, Waltham, MA/USA) using the touchdown PCR parameters: 94°C for a 1 Centrosema pubescens CPAC 4205 2 min; 2x [15 cycles of 94°C for 1 min, 60°C(-1°C/ a 2 Centrosema pubescens CPAC 4247 cycle) for 1 min and 72°C for 2 min]; 30 cycles of a 3 Centrosema pubescens CPAC 4250 94°C for 1 min, 48°C for 1 min and 72°C for 2 min; a Centrosema pubescens CPAC 4251 and 72°C for 5 min (Don et al. 1991). Amplification a Centrosema pubescens CPAC 4252 products were genotyped by electrophoresis on 6% a Centrosema pubescens CPAC 4253 denaturing polyacrylamide gels in 19 TBE buffer Centrosema pubescensa CPAC 4254 using a 10 bp ladder (Invitrogen, CA, USA) as a size Centrosema pubescensa CPAC 4255 standard. The DNA fragments were visualized by Centrosema pubescensa CPAC 4256 silver staining according to Creste et al. (2001). Centrosema pubescensa CPAC 4257 4 Centrosema pascuorum CPAC 2945 5 Centrosema pascuorum CPAC 2955 Allele scoring and data analysis 6 Centrosema. brachypodum CIAT 5833 7 Centrosema. brachypodum CIAT 5850 Polymorphism information content