**2. Materials and methods**

#### **2.1 Plant material and genomic DNA extraction**

The *Heliconia* Germplasm Collection (UFRPE-HCG) is located in Camaragibe-PE at 8°1'19'' South, 34º59'33'' West and 100 m above the sea level, in a 0.3 ha experimental area. The

The natural variation among heliconia individuals or populations has led to taxonomic identification doubts among farmers and researchers. Thus, genebanks have played an important role in genetic diversity conservation, providing raw material for crop breeding, including landraces and their wild relatives. DNA markers, which allow the access to variability at DNA level, emerge as an efficient alternative for plant species characterization

The choice on which molecular marker technique shall be used depends on its reproducibility and simplicity. Kumar *et al*. (1998) distinguished three cultivars of the hybrid *H. psittacorum* x *H. spathocircinata* cvs. Golden Torch, Red Torch and Alan Carle which showed only slight differences in RAPD markers profile from *H.* x *nickeriensis* Maas and de Rooij (*H. psittacorum* x *H. marginata*), they also observed similarities in RAPD profiles and morphology. The authors concluded that two triploid *H. psittacorum*: cv. Iris and Petra, are

Genetic diversity studies grew up in interest during the last years (Jatoi *et al*., 2008; Kladmook *et al*., 2010). As a result, nucleotide sequences of ribosomal genes (rDNA) and chloroplast genes (cpDNA) have been exploited to investigate several individuals of the Zingiberales order (Kress, 1990, 1995; Kress *et al*., 2001) once they are not capable of lateral transfers and are not subject to the same functional limitations, they allow greater confidence in the results (Camara, 2008). The unit of ribosomal eukaryotic organisms consists of three genic and three non-genic regions. On one hand, the genic regions (18S, 5,8S and 26S) are conserved and evolve slowly. On the other hand, non-genic regions, known as ITS - Internal Transcribed Spacer (ITS-1 and ITS-2), evolve rapidly, showing high polymorphism and, therefore, allowing its use at higher hierarchical levels. The variability found in these regions could be the result of mutations in these areas, since they suffer less selection pressure and may be well used to

This molecular marker is important from the genetic variability assessment point of view, because the rDNA mutltigenic family once subjected to a rapid evolution in concert event, allows greater precision in the reconstruction process of the relationship between species based on sequencing, since this phenomenon increases the intragenomic uniformity (Baldwin *et al*., 1995). These authors also affirm that due to the biparental inheritance of the nuclear genome it is possible to study the origin of hybrids and their parents. Moreover, chloroplast genes (cpDNA), such as the leucyn and fenilalanyn of RNA transporter (*trnLtrnF*), the treonyn and leucyn of RNA transporter (*trnT-trnL*) and the protein small 4 (*rps4*), have been used successfully to solve genetic diversity doubts in taxonomic lower levels. Johansen (2005), for example, studying the genetic diversity in Zingiberales order, using

The aim of this study was to evaluate genetic diversity involving *Heliconia psittacorum* cultivars and interspecific hybrids of the Federal Rural University of Pernambuco *Heliconia*

The *Heliconia* Germplasm Collection (UFRPE-HCG) is located in Camaragibe-PE at 8°1'19'' South, 34º59'33'' West and 100 m above the sea level, in a 0.3 ha experimental area. The

cpDNA, has positioned all Heliconiaceae and Musaceae within a same clade.

Germplasm Collection (UFRPE-HCG), using nuclear and chloroplast DNA regions.

by quantifying diversity and determining its genetic structure (Bruns *et al*., 1991).

supposed to be the same genotype.

**2. Materials and methods** 

**2.1 Plant material and genomic DNA extraction** 

study genetic diversity in plants (Bruns *et al*., 1991).

average annual temperature is 25.1ºC and monthly rainfall of 176 mm, with maximum of 377 mm and minimum of 37 mm (ITEP, 2008). This study evaluated 11 *Heliconia psittacorum* cultivars and interspecific hybrids (Table 1) obtained by exchange with research institutions and farmers from the states of Pernambuco (PE), Alagoas (AL) and Sao Paulo (SP) in Brazil. The analyzed genotypes presented short size, musoid habit and erect inflorescence disposed at a single plan (Berry and Kress, 1991).


*<sup>a</sup>*Identification based on Berry and Kress (1991) and Castro *et al*. (2007); *b*Based on Kress *et al*. (1993); *c*BC: bract color; OV: ovary; PD: pedicel; SE: sepals. *d*Ploidy (Costa *et al*., 2008).

Table 1. Genotypes, location, classification and description for 11 *Heliconia psittacorum* cultivars and interspecific hybrids of the UFRPE Heliconia Germplasm Collection used in this study

Genetic Diversity Analysis of *Heliconia psittacorum* Cultivars

2004)*; trnL-trnF* (Sang *et al.*, 1997); *trnS-trnF* and *trnS-trnL.* 

**2.2 PCR amplification** 

milli-Q water to complete the reaction.

Vilber Lourmat photographer.

**2.3 Statistical analysis** 

program Gene (Cruz, 2006).

**3.1 Primers selection** 

to the Laboratory for immediate DNA extraction.

**3. Results** 

700 pb.

and Interspecific Hybrids Using Nuclear and Chloroplast DNA Regions 15

*al*. (1990); and EF11-EF22; and for chloroplast genes analysis: *rps3'-rps5'* (Sanchez-Baracaldo,

The DNA amplification using PCR was performed to a final volume of 25 L containing 1 L template DNA, 0.3 L Taq-DNA polymerase (Invitrogen), 2.51 L Tris-HCl (pH 8, 0), and 0.75 L MgCl2, 2 L of each dNTPs, 1 L primer, 1 L oligonucleotide 1 and 2; and 15.45 L

Amplifications were performed in a thermocycler MJ Reseach, Inc., PTC100 under the following conditions: step 1 - following a denaturation step of 95°C for 3 minutes; step 2 – 94°C for 1 minute; step 3 – 58°C for 1 minute for annealing temperature; step 4 – 72°C for 1 min (repeat steps 2/3/4 for 29 cycles) followed by a final extension at 72°C for 10 minutes and 10°C for 24h. The PCR product visualization was performed in 1.5% agarose gel stained with SYBER Gold (Invitrogen), visualized under ultraviolet light and recorded on a digital

Through the interpretation of gels, molecular data were tabulated as presence (1) or absence (0) of DNA fragments by primers for each genotype. Genetic similarities among genotypes were determined based on the Jaccard (1908) coefficients. A dendrogram was then constructed using the unweighted pair-group method of the arithmetic average (UPGMA) based on the similarity matrix. The cluster analyses were conducted using the computer

The best condition for heliconia DNA extraction was using leaves in the earlyest stage of development, harvested, packed in a polystyrene box containing liquid nitrogen and taken

Primer combination ITS4-ITS3 resulted in most of the polymorphic band region, while for the primer combination ITS4-ITS5 it was observed the least polymorphism. The primers used amplified from 1 to 6 band regions, with clear polymorphism between the genotypes. The amplifications of the nuclear region that includes the spacers ITS1-ITS2 and EF11-EF22 (Fig. 1) generated fragments of approximately 396 to 506 pb, which agrees with Baldwin *et al*. (1995), by claiming that ITS markers have numerous small sized copies, reaching up to

Chloroplast regions amplifications that used the primers tRNA of leucine and phenylalanine (*trnL-trnF*) generated fragments of approximately 1636 pb (Fig. 2). For the spacers regions *rps3'*-*rps5'* as well as for the regions *trnS-trnL* and *trnS-trnF*, it was observed monomorphic

and polymorphic band patterns for the evaluated cultivars and hybrids.

Molecular markers analyses occurred in the Plant Biotechnology Laboratory - UFRPE. The optimization of the DNA extraction protocol was performed using fresh young leaves samples of heliconia, harvested in the earliest stage of development and treated under three conditions: harvested, packed in a polystyrene box containing liquid nitrogen and taken to the Laboratory for immediate DNA extraction; harvested and frozen at -20°C for 1 day before extraction; harvest and preserved in silica gel for 5 days before extraction.

In the DNA extraction, Doyle and Doyle (1990) protocol were used with modifications, which was prepared at a 2x CTAB (hexadecyltrimethylammonium bromide) buffer solution. It was added 700 microliter extraction buffer to 200 mg of macerated leaves in test tubes and taken to bath at 65°C. The tubes, after cooled at room temperature, were centrifuged and the supernatant transferred to new tubes. Supernatant was added to 700 microliter (L) CIA (Chloroform-Isoamyl Alcohol) and then centrifuged was performed.

The supernatant was added to 700 microliter (L) CIA (Chloroform-Isoamyl Alcohol) and then centrifuged. After this process, supernatant was added to 500 L of cold isopropanol and stored for 24 hours in a freezer at -20ºC. Subsequently, it was washed twice with 70% ethanol and with 95% ethanol. The precipitate was dried at room temperature for 20 minutes and then resuspended with 300 L TE containing RNAse, incubated at 37°C for 30 minutes, then 5M NaCl and 300 L of cooled isopropanol were added, in which the DNA was precipitated. Solution was incubated at 4°C throughout the night and the pellet resuspended in 300 L TE. The DNA was quantified in 0.8% agarose gel.


Table 2. Sequence of primers used in amplification reactions in genotypes of the UFRPE Heliconia Germplasm Collection used in this study

A set of 12 primers (Table 2) and 10 combinations of this primers were selected and tested for the ITS analysis: ITS1-ITS4; ITS5-ITS4; ITS1-ITS2; ITS5-ITS2; ITS3-ITS4 based on White *et*  *al*. (1990); and EF11-EF22; and for chloroplast genes analysis: *rps3'-rps5'* (Sanchez-Baracaldo, 2004)*; trnL-trnF* (Sang *et al.*, 1997); *trnS-trnF* and *trnS-trnL.* 
