**3.1 Primers selection**

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 700 pb.

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.

Genetic Diversity Analysis of *Heliconia psittacorum* Cultivars

Subgroup B.

green area distally.

between *H. psittacorum* x *H. marginata*.

cv. Lady Di.

and Interspecific Hybrids Using Nuclear and Chloroplast DNA Regions 17

Fig. 3. Cluster analysis on 11 genotypes of the UFRPE Heliconia Germplasm Collection, used in this study, through ITS. G I= Group I; G II= Group II; SG A= Subgroup A; SG B=

The first group GI, consisting of *Heliconia* sp., that according to farmers, is identified as *H. psittacorum* cv. Sassy and was more divergent from the other genotypes. The hypothesis that it is a new cultivar is supported by the fact that it was the only one that came from the state of Alagoas. It presents floral features intermediate between the triploid cultivars (Costa *et al*., 2008) of the subgroup SG A, bracts of pink and lilac, which presents individual characteristics such as, ovarian (OV) green distally and yellow green proximally, pedicel (PD) yellow green and sepals (SE) orange with indistinct blackish

In the second group GII, subgroup SG A, formed by triploid cultivars of *H. psittacorum*: cvs. Suriname Sassy and Sassy, that present bracts with pink and lilac, was also included genotypes from the state of São Paulo, *H. psittacorum* cv. St Vincent Red and *H. psittacorum*

The subgroup SG B was formed by the hybrid *H. psittacorum* x *H. spathocircinata* cvs. Golden Torch, Golden Torch Adrian and Red Opal, with bracts yellow and red. In this subgroup on an external position, it was observed *H.* x *nickeriensis*, that is supposed to be an hybrid

The hybrids showed low levels of similarity, around 12% of these comparisons reached levels above 50% (Table 3), probably because they are the result from supposed crosses

between genetically distant parents or even the influences of epigenetic factors.

Fig. 1. Nuclear region amplifications that includes the spacers EF11-EF22. Cultivars and interspecific hybrids: A- *H. psittacorum* cv. Sassy; B- *H. psittacorum* cv. Red Gold; C- *H. psittacorum* x *H. spathocircinata* cv. Golden Torch Adrian; D- *H. psittacorum* cv. Suriname Sassy; E- *H. psittacorum* x *H. spathocircinata* cv. Red Opal; F- *H.* x nickeriensis; G- *H. psittacorum* x *H. spathocircinata* cv. Golden Torch; H- *Heliconia* sp.; I- *H. psittacorum* cv. Lady Di; J- *H. psittacorum* cv. Strawberries e Cream; K- *H. psittacorum* cv. St. Vincent Red. (M = 1 kb DNA ladder).

Fig. 2. Chloroplast regions with *primers trnL-trnF* amplifications. Cultivars and interspecific hybrids: A- *H. psittacorum* cv. Sassy; B- *H. psittacorum* cv. Red Gold; C- *H. psittacorum* x *H. spathocircinata* cv. Golden Torch Adrian; D- *H. psittacorum* cv. Suriname Sassy; E- *H. psittacorum* x *H. spathocircinata* cv. Red Opal; F- *H.* x nickeriensis; G- *H. psittacorum* x *H. spathocircinata* cv. Golden Torch; H- *Heliconia* sp.; I- *H. psittacorum* cv. Lady Di; J- *H. psittacorum* cv. Strawberries e Cream; K- *H. psittacorum* cv. St. Vincent Red. (M = 1 kb DNA ladder).

#### **3.2 Internal transcribed spacers**

From the data generated by ITS markers and the analysis of the dendrogram (Fig. 3), it was observed the formation of two main groups (GI and GII) well sustained. The GI group, is constituted by *Heliconia* sp., while, the other, more representative, GII, is subdivided into two other subgroups, SG A and SG B.

Fig. 1. Nuclear region amplifications that includes the spacers EF11-EF22. Cultivars and interspecific hybrids: A- *H. psittacorum* cv. Sassy; B- *H. psittacorum* cv. Red Gold; C- *H. psittacorum* x *H. spathocircinata* cv. Golden Torch Adrian; D- *H. psittacorum* cv. Suriname Sassy; E- *H. psittacorum* x *H. spathocircinata* cv. Red Opal; F- *H.* x nickeriensis; G- *H.* 

*psittacorum* x *H. spathocircinata* cv. Golden Torch; H- *Heliconia* sp.; I- *H. psittacorum* cv. Lady Di; J- *H. psittacorum* cv. Strawberries e Cream; K- *H. psittacorum* cv. St. Vincent Red. (M = 1

Fig. 2. Chloroplast regions with *primers trnL-trnF* amplifications. Cultivars and interspecific hybrids: A- *H. psittacorum* cv. Sassy; B- *H. psittacorum* cv. Red Gold; C- *H. psittacorum* x *H. spathocircinata* cv. Golden Torch Adrian; D- *H. psittacorum* cv. Suriname Sassy; E- *H. psittacorum* x *H. spathocircinata* cv. Red Opal; F- *H.* x nickeriensis; G- *H. psittacorum* x *H. spathocircinata* cv. Golden Torch; H- *Heliconia* sp.; I- *H. psittacorum* cv. Lady Di; J- *H.* 

*psittacorum* cv. Strawberries e Cream; K- *H. psittacorum* cv. St. Vincent Red. (M = 1 kb DNA

From the data generated by ITS markers and the analysis of the dendrogram (Fig. 3), it was observed the formation of two main groups (GI and GII) well sustained. The GI group, is constituted by *Heliconia* sp., while, the other, more representative, GII, is subdivided into

kb DNA ladder).

ladder).

**3.2 Internal transcribed spacers** 

two other subgroups, SG A and SG B.

Fig. 3. Cluster analysis on 11 genotypes of the UFRPE Heliconia Germplasm Collection, used in this study, through ITS. G I= Group I; G II= Group II; SG A= Subgroup A; SG B= Subgroup B.

The first group GI, consisting of *Heliconia* sp., that according to farmers, is identified as *H. psittacorum* cv. Sassy and was more divergent from the other genotypes. The hypothesis that it is a new cultivar is supported by the fact that it was the only one that came from the state of Alagoas. It presents floral features intermediate between the triploid cultivars (Costa *et al*., 2008) of the subgroup SG A, bracts of pink and lilac, which presents individual characteristics such as, ovarian (OV) green distally and yellow green proximally, pedicel (PD) yellow green and sepals (SE) orange with indistinct blackish green area distally.

In the second group GII, subgroup SG A, formed by triploid cultivars of *H. psittacorum*: cvs. Suriname Sassy and Sassy, that present bracts with pink and lilac, was also included genotypes from the state of São Paulo, *H. psittacorum* cv. St Vincent Red and *H. psittacorum* cv. Lady Di.

The subgroup SG B was formed by the hybrid *H. psittacorum* x *H. spathocircinata* cvs. Golden Torch, Golden Torch Adrian and Red Opal, with bracts yellow and red. In this subgroup on an external position, it was observed *H.* x *nickeriensis*, that is supposed to be an hybrid between *H. psittacorum* x *H. marginata*.

The hybrids showed low levels of similarity, around 12% of these comparisons reached levels above 50% (Table 3), probably because they are the result from supposed crosses between genetically distant parents or even the influences of epigenetic factors.


Table 3. Genetic similarity between 11 cultivars of *H. psittacorum* and interspecific hybrids of the UFRPE Heliconia Germplasm Collection used in this study

Genetic Diversity Analysis of *Heliconia psittacorum* Cultivars

**4. Discussion** 

molecular markers in heliconia.

**4.2 Internal transcribed spacers** 

these four genotypes in primer combination ITS3-ITS4.

**4.1 Primers selection** 

clade.

1991).

and Interspecific Hybrids Using Nuclear and Chloroplast DNA Regions 19

It was not possible to obtain DNA with acceptable quality from *Heliconia* using the conventional methodology, as mentioned by Kumar *et al*. (1998), in an earlier study with

Band patterns variation may be related to high occurrence rate of base substitution and the great possibility of indels accumulation (events of inserts and/or deletions of nucleotides), moreover, these sequences are difficult to identify (Albert *et al*., 2002). The study with a great number of genotypes aims to explain the inheritance of the chloroplast, which may vary according to the subgenus and be useful for genetic diversity studies of the group. These *primers* (*trnL-F*) have been successfully used in genetic diversity analysis of

In the absence of more precise evidence, it was decided to keep the genotype, here called *Heliconia* sp., as a specie not yet identified. It is assumed as a new cultivar of *H. psittacorum* cv. Sassy that occurred due to different geographic conditions. In fact, this finding requires further studies. Other molecular markers can be used to solve this issue, as did Kumar *et al*. (1998), that using RAPD, found that two triploid cultivars, Iris and Petra were the same genotype. Sheela *et al*. (2006) by using RAPD, found that cvs. St Vincent Red and Lady Di, were also grouped in the same subgroup. Thus, assuming that these genotypes formed a subgroup brother of triploid cultivars *H. psittacorum* cv. Sassy and cv. Suriname Sassy, presenting 2n = 36 (Costa *et al*., 2008), leads to the assumption that cvs. St Vincent Red and Lady Di are supposed to be triploid, corroborating with the similar banding pattern among

The group that gathered the hybrids *H. psittacorum* x *H. spathocircinata* cvs. Golden Torch, Golden Torch Adrian and Red Opal was expected, once the nrDNA has biparental inheritance, and it is a nuclear molecular marker. *H.* x *nickeriensis* belongs to the *Heliconia* subgenus and *Pendulae* section (Kress *et al*., 1993), this subdivision is based on the consistency of vegetative structure, and staminodes and style shape, especially in the pending heliconia. *H. marginata*, alleged parent, has pending inflorescence, and yet, differ from other hybrids that are crosses between *H. psittacorum* x *H. spathocircinata* and belongs to the *Stenochlamys* subgenus and *Stenochlamys* section (Kress *et al*., 1993). Using RAPD markers to study genetic variability and relationship between 124 genotypes of the genus *Heliconia*, Marouelli *et al*. (2010), managed to gather interspecific hybrids of *H. psittacorum* in the same

The hybrids showed small similarity that can be explained by the coevolution hypothesis, which considers the great genetic diversity of the genotype in the center of origin, once in northeast Brazil is frequently encountered native populations of *H. psittacorum*. Moreover, there is a wide variety of *H. psittacorum* hybrids described in literature, especially *H. spathorcircinata*, confirming the potential of this specie to form hybrids (Berry and Kress,

Orchidaceae (Kocyan *et al*., 2004) and Bromeliaceae groups (Sousa *et al*., 2007).
