ABSTRACT
The Agave genus is composed of about 200 species, but the cultivation of sisal for fiber production in Brazil is restricted to two species: Agave sisalana and Agave fourcroydes, both have several accessions with wide variability. The collection of Agave of Embrapa has 37 accessions maintained in situ and periodically evaluated agronomical traits. Most of these accessions have phenotypic similarities, although they differ in fiber quality, which are widely used for commercial purposes. The identification of promising accesses contributes to the advance in improvement works, focusing on commercial indication. In order to estimate the genetic divergence of this collection, a cluster analyses was performed based on Inter-Simple Sequence Repeat (ISSR) markers and phenotypic traits. Genomic DNA from these accessions were used in polymerase chain reaction (PCR) with thirty ISSR oligonucleotides. For phenotypic characterization, twelve descriptors were adopted based on morphological and agronomic data. The Unweighted Pair Group Method with Arithmetic Mean (UPGMA) and Tocher multivariate methods were adopted. Thirteen groups were formed by the Tocher Method and six by UPGMA; however UPGMA method was more representative in the group formation. The comparison of the band patterns among accessions derived from the shoots showed that genetic variability is generated during asexual reproduction in these plants. The four lines generated from Tatui were the most divergent accessions. These plants are tallest, with higher mass values of fresh and dry mucilage, fresh and dry fiber mass, fiber length and presence of spines at the edges. The accessions from Instituto Agronômico de Campinas (IAC) showed the lowest genetic distances, indicating a possible narrow genetic base and high kinship degree. The crossings between H-RN, H-Kenya, H-400 fls, and H-11648 with Tatuí 1, 2, 3, and 4 can be a valorous strategy to broadening genetic diversity among commercial and native sisal germplasm.
Key words: Sisal, Agave sisalana, molecular markers, morphoagronomic, diversity, cropping breeding.
Agave is a xerophytic and monocot plant that grows natively in semi-arid, subtropical, and tropical regions from the southern United States to northern South America and throughout the Caribbean (Infante et al., 2006). Based on Angiosperm Phylogeny Group (APG) III, Bremer et al. (2009) cited the new classification for the genus Agave, which became part of the Asparragaceae family. The genus Agave, which originated from arid and semi-arid regions of Mexico, contains about 200 species, with restricted geographic distribution (Gentry, 1982; Garcıa-Mendoza and Galvan, 1995). Agave sisalana Perr. and Agave fourcroydes (henequen) are broadly cultivated due to their fiber qualities (Judd et al., 2007; Martin et al., 2009). The reproductive mechanisms of most agaves involve reproduction by seeds and through rhizomes, which appear in early stages of plant development, forming new individuals (Infante et al., 2006). The plant has hermaphrodite flowers and dehiscent fruits. The flowering takes place only once, during the whole cycle. The fiber is the hardest among the fibrous species and has wide use in the artisanal segment, civil construction and automobile industry (Soto et al., 2013). In addition, the mucilage derived from defibration process can be used in ruminant feeding (Silva et al., 1999; Brandão et al., 2011; Macedo et al., 2015).
Brazil is the world’s largest producer and exporter of thread and manufacturer of sisal (A. sisalana) (CONAB, 2016). It is an important crop in semi-arid region, where total rainfall is often scarce and irregular. Despite the widespread use of sisal, information about the genetic basis of genotypes grown in the Northeast region is limited. It is assumed that the cultivars must have the same genetic basis, although A. sisalana is not a genetically pure germplasm (Lock, 1962). According to Moreira and Vieira (1999), the lack of knowledge on heritability of the fiber-related traits has limited the progress of sisal improvement, although they report that the resistance and fiber percentage have high heritability, and therefore can provide selection gains in breeding program. The Brazilian Agricultural Research Corporation (Embrapa) has an Agave collection, containing A. sisalana and A. fourcroydes accessions, maintained in vivo in a semi-arid environment, in the Cariri region of Paraíba, Brazil. Phenotypic descriptors, based on morphological and agronomic traits are periodically recorded. Knowledge of the genetic diversity of the collection is limited, although such information is essential to estimate the potential of the genotypes for later use in breeding program. The analysis of genetic divergence in germplasm banks provides several useful information on genetic resources, based on a data set.
The multivariate methods are important tools to assist the selection procedures in improvement programs and provide broad contribution to classification and identification of germplasm. Among the most used multivariate techniques, the unweighted paired group method using arithmetic averages (UPGMA) is widely adopted by breeders because it is consistent with regard to the allocation of clusters. When different types and number of traits were used, it also revealed higher cophenetic correlation coefficient (Sneath and Sokal, 1973). Other widely used method is the Tocher's optimization method that uses the criterion of optimization, minimizing the average distance intra-cluster and maximizing the average distance inter-cluster (Rao, 1952). However, the Tocher's method does not involve a construction of a phenogram to perform the clustering. The clustering is normally used together with UPGMA, revealing correspondence to the allocation of elements in the groups (Arriel et al., 2006). Although studies involving genetic diversity in sisal collections are limited, some findings are reported in the literature. Navarro-Quezada et al. (2003) analyzed the genetic differentiation of A. deserti complex, comprising A. deserti, A. cerulata and A. subsimplex, using Nei's genetic distance from random amplified polymorphic DNA (RAPD) markers. According to authors, Nei's genetic distances between the three species were low compared to the values obtained from other Agavaceae, and there was no clear correlation with taxonomic divisions.
In an UPGMA analysis, A. subsimplex and A. cerulata formed exclusive monospecific clusters, whereas the A. deserti populations appeared in more than one cluster together with other species. The results were consistent with a pattern of genetic isolation by distance. With the Tocher method, Moreira and Vieira (1999) used agronomic traits collected in seven accessions to estimate the genetic divergence, based on agronomic data. Despite the reduced number of accessions, the authors were able to discriminate them in five groups, with 11648 and IAC 069 being the most divergent and therefore those were recommended for later use in breeding program. The perspective of acquiring heterotic expression with these genotypes is high, considering the genetic base of the genotype 11648 which is an interspecific hybrid between A. angustifolia × A. amaniensis (Doughty, 1938). The Agave collection of Embrapa has been periodically evaluated to agronomical traits and some biochemical studies have been carried out as to mucilage proprieties of A. sisalana accessions (Oliveira et al., 2016). Based on evaluations carried out by Moreira and Vieira (1999), most accessions have phenotypic similarities, although they differ in some fiber traits. The identification of promising accesses is relevant to improvement of sisal, because it contributes to further indication of commercial genotypes. The aim of this work is to determinate the genetic divergence among the Agave accessions from Embrapa performed by a cluster analysis using UPGMA and Tocher methods, based on ISSR markers and phenotypic traits.
Genetic resources and ISRR-PCR assays
In this study, 37 accessions of the sisal collection were used, maintained in Monteiro, PB, Brazil (07°53'22 "S, 37°07'40" W, 599 m). A summary of the phenotypic data of the accessions is found in Table 1. Fresh leaves of each accession were used for DNA extraction based on CTAB method (Ferreira and Grattapaglia, 1998). The PCR assays were performed in a Mastercycler Gradient (Amplitherm Thermal Cyclers). The reaction mixture (25 μl) contained 10× PCR buffer (Fermentas), 25 mM MgCl2, 100 mM dNTP, 1.2 µMol.L-1 of each ISSR primer (USB), 60 ng of genomic DNA, and 1 U of Taq DNA polymerase (Ludwig Biotec). Ten ISSR oligonucleotides were used to PCR assays. The samples were submitted to follow the program: initial cycle of denaturation at 95°C/5 min, followed by 40 cycles of denaturation at 95°C/1 min, annealing at 50°C/1 min, and extension at 72°C/2 min. A final extension cycle was added to reaction at 72°C/7 min. The amplified products were analyzed by electrophoresis on 2.5% agarose gel. All reactions were performed in triplicate.
Analysis of genetic divergence and clustering techniques
For the multivariate analysis, molecular and phenotypic data were used. In the molecular data, each band generated by ISSR-PCR was coded with 1 and 0 for presence and absence, respectively, resulting in a binary matrix, which was used to quantify the genetic similarity (Sij) among the accession pairs based on the coefficient of Jaccard, following the expression:
Where: a means the sum of coincidences of type 1-1 for each pair of accession, b, the sum of the discordances of type 1-0 for each pair of accession, and c, the sum of discordances of type 0-1 for each pair of accession. The arithmetical complement 1- Sij was also used to transform the similarity matrix into a dissimilarity matrix, which is used in cluster analysis. The estimated dissimilarity matrix for the phenotypic data was obtained based on the Gower distance (Gower, 1971), allowing the treatment of phenotypic data simultaneously. The sum of the two matrices was performed to obtain a representative matrix of the molecular and phenotypic data, which was used to perform the cluster analysis. The dissimilarity values ​​of each matrix were standardized through the expression:
Where, dpj is the standardized dissimilarity between an individual i and individual j; dij is the dissimilarity between i and j, and σd is the standard deviation of dissimilarity.
The correlation between the matrices was also made with the purpose of estimating the level of relation between them, obtaining the significance from the t and Mantel tests, indicating or not, the agreement of the two methods in expressing the existing genetic difference between pairs of accessions (Cruz, 2008). The clustering analyses were carried out through Tocher’s optimization and UPGMA methods. The cophenetic correlation coefficient was estimated in order to eliminate the non-hierarchical effects. Cluster analysis was performed using the software GENES, version 2016.6.0 (Cruz, 2013). The following phenotypical traits were used for analysis: number of leaves, plant height, leaf length, fresh leaf mass, dry mucilage mass, fresh fiber mass, dry fiber mass, fiber length, presence of spines on the leaf edge, tillering and high folding endurance.
Phenotypic traits (Table 1) and ISSR markers were used to estimate the genetic divergence among thirty-seven sisal accessions, maintained in situ, in the semi-arid zone Cariri of Paraíba, Brazil (Figure 1A-F). Adult plants were used for agronomical characterization, with subsequent molecular analysis, using leaf tissues. In order to optimize molecular analysis, thirty oligonucleotides were previously tested for polymorphic selection, and therefore contributory to the evaluation processes. Of the total, ten oligos were selected, with high polymorphism rate, revealed by amplicons obtained from agarose gels, with an average of 13/oligo (Table 2). Although we used a relatively small population, maintained asexually, it was possible to identify differences among accessions through profiles obtained via ISSR-PCR. Several bands were common among the accessions; however, some were unique and therefore contributory to the analysis of genetic divergence of the collection. Figure 1G shows a pattern of amplicons obtained with UBC 812, with 11 bands distributed between 0.1 and 1 Kb.
Most molecular markers are robust to identify variability in sexually or asexually propagated populations. In literature, ISSR, amplified fragment length polymorphism (AFLP) and microsatellites markers are the most reported (Barraza-Morales et al., 2006; Abraham-Juárez et al., 2009; Santos et al., 2015). In Agave, the production is supported by intensive clonal propagation and the suppression of the sexual reproduction, leading to reduction in genetic variability plantations (Gil-Vega et al., 2001; Infante et al., 2006; Abraham-Juárez et al., 2009). Abraham-Juárez et al. (2009) analyzed the genetic variability by AFLP markers, using three reproductive forms of A. tequilana, and found 75.08 and 86.06% of polymorphic loci, respectively, from offsets and bulbils generated from the same matrix plant. As to authors, although a significant level of polymorphism was observed between rhizome offsets, the levels were even higher between bulbils, reaching levels comparable to those found between plantlets produced from seeds (90.1%). Infante et al. (2006) explained the origin of the asexual variability in Agave species based on molecular markers. According to authors, in plant shoots, the whole cells are derived from the apical meristem, including the germline cells. In asexual reproduction, meristems are the source of the mitotically derived offprints. The only source of genetic differences in these materials comesfrom somatic mutations.
Although the Agave has both forms of propagation, the sexual process that is responsible for segregation and high variability is not frequent and only occurs under special conditions, such as when the tassel is decapitated before the emission of floriferous branches leading the plants to produce viable seeds and fruits. However, it is a time-consuming process and when it occurs, the progenies often show marginal leaf spines, an undesirable trait for clones destined for commercial exploitation (Abraham-Juárez et al., 2009). The clustering analyses by Tocher and UPGMA are found in Table 3 and Figure 2, respectively. Through Tocher method, 13 groups were formed, of which 6 had only one accession. The group 1 contained the majority of accessions (40.5%), with medium height and high number of leaves. Among them, stands out the hybrids RN, Kenya, 400 fls, 11648 and A. sisalana, all established in Brazilian semi-arid region. According to Silva et al. (2008), the hybrid 11648 and A. sisalana are widely grown in Northeast region, due to acceptable traits to fiber market. The group 2 contained accessions with high height and long fibers, represented by Cabinho and A. fourcroydes. The group 3 clustered the accessions from the Instituto Agronômico de Campinas (IAC 840096, IAC 0069, IAC 0097, IAC 0101) and an ornamental accession (ornamental Sisalana). These materials have medium and short fibers and have high folding endurance.
The group 4 contained two IAC accessions (IAC 84003, IAC 84001/4), all showing low number of leaves and no spines at the edges. The group 5 grouped three lines from Tatuí, all of them are tall plants, with greater leaf weight and extra-long fibers. These attributes are interesting for further use in breeding work. The groups 6 and 7 clustered two accessions each, with tall plants, medium fibers, spines at the edges and tillering, but those of group 6 (Valente, Teixeira) had high folding endurance, which does not occur in group 7 (Espinho, Mutant Paraíba). In clustering analysis via UPGMA, the number of grouping was more condensed, distributed in six clusters. The degree of association between dissimilarity matrices obtained from phenotypic and molecular data was significant, based on t and Mantel test (p≤1%), indicating that both sets of data were adequate to represent the genetic divergence in the collection of sisal. The distribution of accessions through UPGMA was more contributive to discriminate divergent groups than Tocher's and provides more possibilities for choice of parents in breeding work aiming at production and fiber quality. UPGMA method tends to generate higher values ​​of the cophenetic correlation coefficient, allowing to infer that the groups of accessions indicated in the graphic have an adequate fit between the dissimilarity matrix and dendogram obtained (Cruz and Carneiro, 2003).
As shown in Figure 2, the hybrids H-400 fls, H-Kenya, H-RN, and H-11648 were clustered in a group separated from the others. All of them are bred genotypes, with special traits to the natural fiber market. Overall, the fiber of these hybrids shows strength and yield near to 750 g/force and 2.7 t.ha-1, respectively (Amorim Neto and Beltrão, 1999). The most IAC accessions were clustered in group 2 that contained 20 accessions, and most showed short fiber. The groups 3 and 4 contained three accessions each, constituted by mutants collated in Paraíba and Bahia states, and also local types. The group 5 contained A. fourcroydes, Cabinho and Tatuí, all characterized by presence of spines at the leaf edges. The last, group 6, clustered the four lines derived from Tatuí, characterized by high weigh of leaf (fresh mass) and dry mucilage, high weigh of fiber (dry and fresh), large fiber length and presence of spines at the leaf edges. They are similar lines, spreading in different places in Sao Paulo State, located at Sudeste region, Brazil.
Based on results obtained from clustering analysis, we can consider that the variability found in the Embrapa-Agave collection offers possibilities to implement a breeding program in order to meet the demands of the natural fiber market. The richness of this information lies in the fact that, although Agave is originated from Mexico, Brazil has several ecotypes that have as their main skill the broad adaptation to the semi-arid and tropical environments. There is no information on the existence of variability in fiber quality in different environments. However, in the literature, the phenotypical differences in plant canopy are marked when grown in less arid environments (Silva et al., 2008). Thus, depending on the purpose of the breeding program, it is possible to identify in these results divergent genotypes that may contribute to the generation of promising hybrids.
Agronomical and molecular variability was found in Agave collection, maintained by Embrapa, in Monteiro, PB, Brazil. This represent a valorous strategy to broadening genetic diversity among commercial and native sisal germplasm.
The authors declare no conflict of interest between the partners with the release the results.
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