Genetic diversity analysis among pigeonpea genotypes adapted to South American regions based on microsatellite markers

The pigeonpea [Cajanus cajan (L) Millspaugh] is one of the most important perennial legume crops utilized in the food, fodder, soil conservation, crop-livestock integrated systems, reclaiming of degraded pastures and symbiotic nitrogen fixation. Microsatellite markers were used to estimate the genetic diversity of 77 pigeonpea genotypes selected from the germplasm collections at Embrapa Cattle-Southeast and, to evaluate their transferability to Phaseolus vulgaris and Vigna unguiculata species. The number of alleles per locus ranged from 2 to12, with an average of 5.1 alleles. The PIC values ranged from 0.11 to 0.80 (average 0.49) and the D values from 0.23 to 0.91 (average 0.58). The averages of observed and expected heterozygosity were 0.25 and 0.47, respectively, showing a deficit in heterozygosity. A model-based Bayesian approach implemented in the software STRUCTURE was used to assign genotypes into clusters. A dendrogram was constructed based on the modified Roger’s genetic distances using a neighbor-joining method (NJ). A total of four clusters were assembled by STRUCTURE and a strong tendency of correspondence between the Bayesian clusters in the NJ tree was observed. The genetic distance ranged from 0.09 to 0.62 (average 0.37), showing a low genetic diversity in the pigeonpea genotypes. Transferability of pigeonpea-specific microsatellites revealed a cross-amplification and the presence of polymorphic alleles in P. vulgaris and V. unguiculata.


Introduction
The pigeonpea [Cajanus cajan (L.) Millspaugh] is one of the most important perennial legume crops in the tropic and subtropic regions of the world.Because of its multiple usages in food, fodder, soil conservation, crop-livestock integrated systems, reclaiming of degraded pastures and symbiotic nitrogen fixation, the pigeonpea plays an important role in subsistence agriculture (Reddy et al., 2005).
Because of the potential of the pigeonpea as a forage legume, the Brazilian Agricultural Research Corporation (Embrapa Cattle-Southeast, state of São Paulo-SP) has germplasm collections of selected genotypes with desirable agronomic traits such as high yield, quality of forage and lowest tannin content (Godoy et al., 1995).Over time, the selected genotypes showed phenotypic segregation in subsequent generations.Therefore, these genotypes were self-fertilized and subsequently selected in order to obtain inbred lines (Godoy et al., 1994(Godoy et al., , 1997)).Several studies have been conducted to characterize genotypes and inbred lines of the pigeonpea and provide basic information for breeding.The genetic variability of a partial set of accessions from this collection was assessed using Random Amplification of Polymorphic DNA (RAPD) molecular markers.Results showed low genetic variability and the need to broaden the genetic base for use in crop-livestock integrated systems and reclaiming degraded pastures (Godoy et al., 2003) The knowledge of the genetic variability is very important in for pigeonpea germplasm collections and pigeonpea breeding programs.Microsatellite markers are quite effective for estimating genetic diversity and genetic relationships and in predicting the genetic value of selected genotypes derived from intraspecific crosses and the performance of their hybrid progenies (Gaitán-Solís et et al., 2002;Varshney et al., 2005).In this study, we used 43 microsatellite markers to evaluate the genetic diversity of 77 pigeonpea selected genotypes from the Embrapa collection.In addition, we studied cross-species amplification in Phaseolus vulgaris L. and Vigna unguiculata L. Walp.

Materials and Methods
We have selected 43 microsatellite markers described in the literature (Burns et al., 2001;Odeny et al., 2007) to analyze 77 pigeonpea genotypes (  Tropics (ICRISAT) (Table 2).The inbred lines have distinct morphological characteristics such as color of the stem, flowers, seeds and pods.These inbred lines were obtained from selfing of genotypes introduced from ICRISAT and have been incorporated to the breeding programs at Embrapa.In addition, cross-amplification evaluations were made using two other legume species: Phaseolus vulgaris (CAL-143, IAC-UNA, BAT-93 and JALO-EEP558 varieties) and Vigna unguiculata ("Fradinho" cultivar), both from the germplasm collection of the Agronomic Institute of Campinas (IAC) (Campinas, SP, Brazil).Genomic DNA was extracted from freeze-dried leaf samples using the cetyltrimethyl ammonium bromide (CTAB) method with modifications (Faleiro et al., 2003).DNA samples were quantified by comparison with known quantities of λ phage DNA on a 1% agarose gel.
The PCR was carried out in a total reaction volume of 25 μL containing 0.5 ng of DNA template, 0.8 μM of each forward and reverse primers, 100 μM of each dNTP (MBI Fermentas), 1.5 mM MgCl 2 , 10 mM Tris-HCl, 50 mM KCl and 0.5 U Taq DNA Polymerase (Invitrogen).All PCR amplifications were performed in a PTC-200 thermal cycler (MJ Research, Waltham, MA/USA) using the following conditions: 94ºC for 1 min followed by 30 cycles of 94°C for 1 min, specific annealing temperature for 1 min, 72°C for 1 min, and a final extension of 72°C for 5 min.Amplification products were genotyped by electrophoresis on 6% denaturing polyacrylamide gels in 1X TBE buffer using a 10 bp ladder (Invitrogen) as a standard size.The DNA fragments were visualized by silver staining according to Creste et al. (2001).
The polymorphism information content (PIC) values were calculated for estimates of marker informativeness according to the equation of Botstein et al. (1980), where f i is the frequency of the i th allele, f j is the frequency of the j th allele and the summation extends over n alleles.In order to compare marker efficiencies in varietal identification, the discriminating power (D) was estimated for each primer based on the formula, where N is the number of individuals and p j is the frequency of the j th pattern (Tessier et al., 1999).The observed heterozygosity (H O ) and the expected heterozygosity (H E ) were analyzed using the GDA software (Lewis and Zaykin, 2002).Genetic distance was calculated from microsatellite marker data using modified Roger's genetic distances.A genetic distance matrix was estimated using tools for genetic population analysis (TFPGA v 1.3) (Miller, 1997).Cluster analysis was performed using the neighbor-joining (NJ) method with the DARwin v. 5.0.157software (Perried and Jacquemound-Collet, 2006).The reliability of the generated dendrogram was also tested by bootstrap analysis using the BooD program with 1000 iterations (Coelho, 2002).The software STRUCTURE version 2.2 (Pritchard et al., 2000) was used to generate a Bayesian inference of the structure of the populations.By this method, a model of K populations is assumed and samples are grouped in order to minimize linkage disequilibrium and to maximize conformity to Hardy-Weinberg equilibrium across all analyzed loci.As a preliminary step, analysis was performed a single time for each K value ranging from 2 to 20.Each run was performed using the admixture model and
Knowledge of the genetic diversity in germplasm collections is fundamental for further breeding programs to fully exploit existing diversity by genotypes selection.As evident from the clustering of genotypes, it is clear that these microsatellite markers are efficacious.The pigeonpea is an important crop of the Phaseoleae tribe, which has limited genomic resources.As microsatellite markers are highly polymorphic, reproducible, co-dominant in nature and distributed throughout the genome, they have become the ideal marker system for genetic analysis and breeding applications.

Conclusions
The microsatellite markers revealed low genetic diversity among genotypes of pigeonpea, especially between the Brazilian inbred lines selected for use in crop-livestock integrated systems and reclaiming degraded pastures.The modified Roger's genetic distances revealed the presence of genetically close genotypes.
Pigeonpea-specific microsatellite markers were transferable to P. vulgaris and V. unguiculata.The transferable loci exhibited polymorphism among some genotypes.Transferability studies of microsatellite loci from other cultures can be highly advantageous.
Sci. Agric.(Piracicaba, Braz.), v.68, n.4, p.431-439, July/August 2011 1000 replicates for burn-in and 10,000 replicates during analysis.The most probable number of K was calculated based onEvanno et al. (2005) using an ad hoc statistic ΔK, which represents the rate of change in log probability of the data between successive K values rather than the log probability of the data.

Figure 1 -
Figure 1 -Genetic diversity of pigeonpea genotypes and cross-species amplification between Phaseolus vulgaris and Vigna unguiculata.a Population structure analysis.Each genotype is represented by a thin vertical segment, which can be partitioned into K colored segments that represent the individual estimated membership to the K cluster.Membership coefficients obtained at the optimal K value (K = 4 clusters).b Neighbor-joining tree analysis.The numbers at the tip of tree branches indicate the accession number.The colors of the bar and the tree branch indicate the 4 groups identified through the STRUCTURE program (C1 = red, C2 = green, C3 = blue and C4 = yellow).

Table 1
) of the Brazilian Agricultural Research Corporation (Embrapa Cattle-Southeast) germplasm collection, in São Carlos, SP, Brazil.Thirty-nine of them are Brazilian inbred lines, three are commercial cultivars and thirty-five came from the International Crops Research Institute for the Semi-Arid

Table 3 -
Characteristics of pigeonpea microsatellite loci, including number of alleles, PIC, D, H O and H E values.Number of alleles in pigeonpea. 2PIC -Polymorphism information content. 3D -Discriminating power. 4H O -Observed heterozygosity. 5H E -Expected heterozygosity. 1

Table 4 -
Characteristics of pigeonpea-specific microsatellite markers transferable to Phaseolus vulgaris and Vigna unguiculata.