SSR Based Genetic Diversity Analysis in Diploid Algaroba ( Prosopis spp . ) Population

Algaroba (Prosopis palida and Prosopis juliflora species) provides important environmental and economic benefits for semi-arid regions of the world. These are resistant to drought, and its fruits are used in the manufacture of flour and algarobina syrup. In the present study, the chromosome number, the ploidy level, and the genetic diversity based on 40 microsatellite loci of Prosopis spp. were determined in samples of a Brazilian algaroba population. The cytogenetic analysis in the metaphase showed only diploid individuals (2n = 28), with multiple cells featuring two CMA/DAPI heterochromatic blocks suggesting diploid level. However, polysomatism was found by the presence of some cells with four CMA/DAPI blocks, showing the tetraploid level just in some somatic cells. Among all of the primers tested for cross-amplification in algaroba, 22 were selected to characterize the samples. Thirteen loci revealed allele polymorphisms in the population and are recommended for future population studies and genetic improvement. The mean values of the analyzes showed low genetic diversity (two alleles per locus and HE = 0.181), reflecting the history of the introduction of algaroba in this sampled locality, and suggesting the genetic bottleneck and probable events of founders, as well as the characteristics of the species of this genera. However, amplified loci indicated low inbreeding (allelic fixation index of -0.007), although heterozygosis was higher than expected by the Hardy-Weinberg equilibrium. Therefore, this algaroba population is formed by diploid individuals and adjusts to the tendency of low number of alleles per locus SSR commonly observed in different species of Prosopis.


Introduction
Prosopis juliflora (Sw.)DC. is known as algaroba in Brazil, and as algarroba or algarrobo in some Spanish-speaking countries, as well as mesquite in African, Asian countries and others.Prosopis juliflora and P. pallida (H. & B. ex. Wild).H.B.K. are legumes, and they form a complex due to taxonomic issues not yet solved (Burkart, 1976;Pasiecznik et al., 2001).The species presents a basic chromosome number of x = 14, but the variable ploidy level present in most of the sample studies has caused cytotypes to be recognized as tetraploid (Hunziker et al., 1975;Pasiecznik et al., 2001, Nogueira et al., 2007;Trenchard et al., 2008).In this case, variation from 2n = 2x = 28 to 2n = 4x = 56 was principally in the genus Prosopis (Trenchard et al., 2008).
The species complex are distributed in Central and South America, Africa, Asia, and Oceania.Algaroba is widely found in the dry forests of Peru and was introduced in Brazil in 1940 (Azevedo, 1955;Burkart, 1976;Pasiecznik et al., 2001).Algaroba is a tropical species and has spread throughout the world for economic reasons and due to its ease of adaptation in arid and semi-arid regions, thus guaranteeing its survival for many centuries.The species is well adapted to regions that suffer periods of severe drought and unproductivity worldwide (Burkart, 1976).The algaroba has the potential to provide a wide range of products in the northeastern part of Brazil, where few useful species are found.It is one of the few economic resources for farmers and inhabitants of the region.Algaroba is a food source for animals, an ingredient of breads, flour, sweets, and used as wood, firewood, and coal (Cruz, 1990;Silva, 1996;Felker, 1984;Figueiredo, 2000;Ribaski, 2009).
Considering the value of algaroba, the molecular diversity studies of Prosopis spp.are crucial for understanding the genetics of these invasive plants, their response to adverse conditions and events of genetic drift, and their use in studies on crop's genetic improvement.
Molecular methods for genetic detection of polymorphic microsatellite loci (SSR) can be performed by cross-amplification of markers from related species (Landeras et al., 2006).There are very limited molecular studies for Prosopis spp., and no polymorphic microsatellites were identified to evaluate genetic diversity in Brazilian algaroba populations.Cross-amplification of microsatellite markers was successful between distant taxa (Yasodha et al., 2005).Additionally, the algaroba chromosome number, the existence of species with variation in ploidy, and marker analysis of species with known ploidy will help on determination and evaluation of useful parameters.This phenomenon is commonly observed in plants with high adaptation.
SSR loci were originally developed for P. chilensis and P. flexuosa (Mottura et al., 2005), P. alba (Torales et al., 2013), P. alba and P. chilensis (Bessega et al., 2013), P. rubriflora and P. ruscifolia (Alves, 2014).Cross-species amplification of six microsatellites markers developed for P. chilensis have been tested in seven Prosopis species from Argentina: P. alba, P. caldenia, P. ferox, P. hassleri, P. nigra, P. ruscifolia, P. torquata and P. brasiliensis (Mottura et al., 2005).These six SRR loci were also used in cross-application of eight additional Prosopis species: P. tamarugo hybrid, P. pallida, P. juliflora, P. laevigata, P. glandulosa var.torreyana, P. velutina, P. articulata, P. caldenia (Sherry et al., 2011).So, cross-amplification with the six microsatellite loci in 15 different Prosopis species indicates that there must be a high level of similarity of flanking sequences from repetitive sites in Prosopis spp.However, these primers were not genetically characterized at the level of populations of these species; this is a necessary information to plan the use of the loci in population studies.In addition, the number of loci existing for each species is not sufficient for more comprehensive genetic studies in populations.Thus, the cross-amplification of SSR loci available in Prosopis is an alternative to characterize primers aiming studies of algaroba populations.
The genetic aspects of representative algaroba population in the state of Bahia, Brazil, was studied with the following objectives: i) To test the chromosomal number, ploidy level of this selected population to determine the use of chosen molecular markers; ii) To test the cross-amplification of selected simple sequence repeat/microsatellite loci in Prosopis juliflora and P. pallida; iii) To evaluate the polymorphism of the number of alleles per locus, heterozygosity and coefficient of inbreeding; iv) To determine the genetic diversity of the Brazilian algaroba population and to compare this diversity with previous studies of different populations.

Study Site and Plant Material
The samples were collected in a farm located in the municipality of Manoel Vitorino, Bahia, Brazil (Figure 1), in a semi-arid region characterized by the predominance of the Caatinga biome and bordered by the De Contas River.It is an area of the occurrence and cultivation of the Prosopis species for animal feed.
Leaves were collected from 20 representative genotypes of the population, and they were identified, stored, and taken to the Laboratory of the Center for Biotechnology and Genetics (CBG) at the State University of Santa Cruz (UESC) for the development of the molecular marker research.Among the 40 primers tested, 34 (85%) were cross-amplified.However, only 22 primers were selected as a functional with ideal minimum size of 100 base pair (bp) fragment for genotyping, excluding the possibility of false allele interpretations in reading the peaks (Table 1).Of these amplified primers, at least one allele per locus was verified.Amplicons in the 176 bp and 371 bp range were detected.The amplification temperature of most of the primers ( 16) was at 56 °C, and the others required specific temperatures of 52 °C to 58 °C.

Genetic Characterization of SSR Loci
The genetic data analysis in 20 algaroba genotypes for polymorphic loci is described (Table 2).Among 22 primers, 13 amplified more than one allele, four of which were the maximum number of alleles found per locus, and a total of 46 alleles were distributed among the 20 accessions of the algaroba.The loci showed a mean H O that was greater than the H E , and the mean of the PIC was 0.167, having values lower than or similar to those of the species to which the loci were developed.The mean for the combined exclusion of the loci (Q) was 0.96, and the coefficient of identity (I) estimate was 0.00022963.
In the analysis of linkage disequilibrium, no non-random association was found for the great majority of pairs of microsatellite loci, and loci with only one allele were not computed.The associated pairs were (Mo07, Prsc5), (Prsc5, Prsc10), and (Prsc1, Prsc11).

Grouping of Individuals in the Population
In the analysis of the main components, three groups were observed: one group with eight individuals, one group with 11 individuals, and another with just one individual (Figure 3).

Characteristics of Markers
In the analysis of the genetic diversity of different populations of the genus of Prosopis characterized by the microsatellite loci of several studies previously established in the literature, it can be observed that the mean values of H O and H E of all populations were intermediate or low (Table 3).(PCA) based o study et al., 2015 et al., 2015 et al., 2015 et al., 2015 et al., 2015 et al., 2015 , 2014 , 2014 , 2014 , 2014 al., 2013 al., 2013 al., 2005 al., 2005 .FSM  and DAPI, which facilitated the visualization and definition of the chromosome due to its small size, in comparing to Giemsa staining in our previous studies.
In the genus Prosopis the most common chromosome number found has been 2n = 28, based on the basic number x = 14 we can consider these diploid species.However, the P. juliflora taxon, found naturally in both North and South America, has been shown to be tetraploid with 2n = 4x = 56 (Trenchard et al., 2008).However, in Brazil, algaroba can present both diploid and polyploid cytotypes (Hunziker et al., 1975).It also corroborates the findings of Nogueira et al. (2007), which analyzed a population of P. juliflora from three municipalities of different Brazilian states.Only one of the accessions sampled in one of the populations was found in the tetraploid form while the others were diploids.Therefore, although the algaroba population analyzed in the present study is considered locally as P. juliflora, this population should be P. pallida.
The occurrence of polysomatism in the species consisting of cells or organs of the same individual with levels of ploidy was verified.Barow (2006) reports that polysomatism is common in well-adapted plants and is important for accelerated plant growth, as well as to support certain physiological cellular functions.This event differs from polyploidy, which consists of the entirety of the individual's cells having extra chromosome sets.The erroneous interpretations of polyploidy for P. juliflora have been explained by the polysomatism that commonly occurs in root tissues that are used in analyses, or even by non-accurate counting errors (Burkart, 1976).Additionally, the low frequency with which tetraploid cells are reported in the literature could be understood as possible polysomatism and not necessarily the appearance of polyploid individuals.Specifically, in relation to the results obtained in the present study, it has been proven to be a case of polysomatism.
Prosopis juliflora polyploidy origin is still unknown (Pasiecznik, 2001).Trenchard et al. (2008) found only tetraploid individuals for this species from different source, indicating that P. juliflora would be the only tetraploid specie related, whereas P. pallida and several other species of Prosopis would be diploid, since 32 species from the genera has been cytological analyzed.However, the algaroba population evaluated in the present study, known among those who cultivate P. juliflora, is a true diploid, with rare polysomatism.Introductions of P. pallida were made in Brazil, but there were no records or information on the places of these introductions (Burkart, 1976).This divergence proves the taxonomic complexity of the group, and even though there were errors of identification in the native populations of Peru and the Pacific coast.These data suggest the need for future taxonomic, cytogenetic, and phylogenetic studies from their native scale that can clarify both the history and the identification of consensus for these Prosopis materials in Brazil.
Our analyses using molecular markers revealed that loci I-P03211 and Prsc4 showed a deviation to the proportions expected by the Hardy-Weinberg equilibrium considering the 95% and 99% confidence interval, respectively.This result is due to the number of heterozygotes observed to be much higher than expected.
Despite the low allelic diversity, the average inbreeding coefficient or loss of heterozygotes (F) was considered to be optimal because the population presented a low level of heterozygosity (-0.007).On the other hand, the means for the combined exclusion of the loci (Q) and the estimation of the coefficient of identity (I) were low.
The 13 amplified loci with more than one allele are useful tools for future population and genetic diversity studies of algaroba, although they are not recommended for studies aimed at plant identification and protection due to the low values of the "Q" and "I" indices.
The microsatellite loci "Mo05" developed by Mottura et al. (2005) for P. chilensis and P. flexuosa detected more alleles in the algaroba, to which it was cross-amplified, than in the species to which it was developed.In addition, the same series of loci (Mo05, Mo07, Mo08, Mo09, Mo13, and Mo16) developed by Mottura et al. (2005) were cross-amplified in six other Prosopis species (P.alba, P. caldenia, P. ferox, P. hassleri, P. nigra, P. ruscifolia, and P. torquata) and the range of alleles found from zero to five, was very similar to that of our algarroba population that ranged from zero to four alleles per locus.Similarly, the loci developed by Alves et al. (2014) for P. ruscifolia and P. rubriflora detected between one and five alleles per locus, and only one locus (Prb9) of P. rubriflora amplified to algaroba with just one allele, probably due of the genetic distance between them.In a study carried out by Bessega et al. (2013), microsatellite loci developed for P. alba and P. chilensis also obtained a higher concentration between two and five alleles per locus.These loci were not used in this work for algaroba.
Although the loci developed by Torales et al. (2013) for P. alba have amplified algaroba DNA of the species tested, these loci did not show data of allelic diversity.Thus, the reduced number of alleles detected in the present study is explained by the low allelic diversity of the species of the genus Prosopis.
Despite the low allelic diversity, low inbreeding levels were found even in the phase of the introduction, history of the species, with possible genetic bottleneck events and founder effects.This fact can be explained on the basis of two hypotheses.The first one is related to the fact that the "F" evaluates the result of the crossings of individuals from one generation to another; thus, this indicates that there was no intersection between relatives in the previous generation.The second hypothesis that can have a joint action with the first is the reproductive system of the species that favors heterozygosis, because it is allogamous and self-incompatible.
As for the associated pairs in the analysis of linkage disequilibrium, we suggest the use of X, Y, and Z only because they contain a higher ICP or because they have a greater number of alleles.
The data of the grouping of the individuals evidences the dispersion of the accessions sampled by the genetic distance.Of the three groups, one has individuals that were more closely connected, indicating the possibility of having origins of the same region in cases of cultivated individuals or of individuals those have been naturally dispersed by the region.It is possible that these individuals may be the fruit of crossing individuals that are physically close and the other groups otherwise.Despite the genetic distance shown in the analysis, the 18 followed the same pattern of the other accessions collected.
The results of this study add value to the literature, showing the importance of having molecular tools from species with a close phylogenetic relationship, because the algaroba (P.juliflora-P.pallida) has no SSR developed and the present work contributed 22 cross-amplified SSR loci.
The results of the diversity of the different populations of Prosopis may be related to the low number of alleles found in the Prosopis species, for which molecular data are known.Another observation is that, among these species, only the population used in the present study presented an estimate of H O higher than that of H E .Because the area sampled in the present study is a growing area, introductions of different accessions may have favored the observed heterozygosity, even with low allelic diversity.
In addition to our comparison with the literature, we added new genotyping data of a Brazilian population of algaroba.However, there are populations of algaroba in different states of northeastern Brazil.Therefore, the following question remains open for future investigation: are the different Brazilian populations formed by diploid individuals?Do these populations have the general tendency of low polymorphism in SSR loci?There were multiple introductions of algaroba in Brazil?The microsatellite loci that showed cross-amplification and PIC in algaroba in the present work are useful to delineate new studies aimed to answer these questions.
Although the different studies carried out at distinct areas such as natural populations, anthropic areas and recovered areas, there was no correlation pattern with H O values.The natural population of P. chilensis showed the highest mean value of H O (0.607) of the presented studies, however it was lower than the mean H E value (0.647).This information reinforcing that the low genetic diversity is not strictly related to the environmental conditions of the study area, but with a natural characteristic of the genus.

Table 1 .
SSR primers derived from Prosopis species cross-amplified to algaroba complex with allele amplitude and optimized hybridization temperature