Genetic variability in pecan genotypes in Brazil

ABSTRACT Pecan crops has been expanding in recent years, mainly in southern Brazil. Genotypes that compose Brazilian orchards come from the USA and from selections of plants made by Brazilian producers. This study aimed to characterize with microsatellite markers pecan cultivars registered for cultivation in Brazil, including some selections made in the country. It is important to know the genetic variability of the pecan tree, as it facilitates the identification of possible phytotechnical deficiencies due to the genetic similarity between the plants, in addition to helping in the conservation of the species, among other. Thirty-four, out of forty collected accessions, were genotyped with 11 selected SSR (Simple Sequence Repeats) loci. Twenty-four polymorphic alleles were identified. The genetic similarity matrix, based on the Jaccard coefficient, ranged from 0.125 to 1.0; general mean of similarity was 0.46. The cluster analysis, which was carried out by the Unweighted Pair Group Method with Arithmetic Mean (UPGMA), classified pecan accessions into four main groups. Results showed that there is high genetic variability in germplasm evaluated, although some accessions may be duplicates.

These differences between pecan tree plants can be seen in the characteristics, such as fruit shape and size, quality, plant architecture and reproduction.In Brazil, 43 cultivars have been registered at the Ministry of Agriculture, Livestock and Food Supply (Mapa, 2020).In addition, there is genetic material of unknown origin which resulted from selections carried out by farmers and nurserypersons, taking into account morphological, productive and genetic aspects (Hamann et al., 2018;Poletto et al., 2020).
Rafaela Schmidt de Souza et al.
Pecan is a perennial plant, deciduous and monoecious fruit tree (having pistillate and staminate flowers on the same plant).In addition, the pecan has a natural mechanism called dichogamy, where the period of pollen release and stigma receptivity are different, and this period may be complete or partial.However, there is a possibility that a small percentage of self-pollination will occur, normally pollination is anemophilous (Thompson & Romberg, 1985;Sparks, 1992;Wells, 2017b).
To know the magnitude of the genetic variability available in the country is an important step to ensure the development of pecan crop in the future.An alternative to help to identify and characterize genetic diversity of a species is the use of molecular markers, which enable differences among plants to be revealed by DNA analysis.
Regarding the different types of markers based on the PCR, microsatellite markers or Simple Sequence Repeats (SSR), are the most successful ones in studies of genetic variation, mapping, genotype identification and others (Caixeta et al., 2009;Wang et al., 2010;Librelon et al., 2013).
This study aimed to characterize with microsatellite markers pecan cultivars registered for cultivation in Brazil, including some selections made in national territory, to know magnitude of the genetic variability cultivated in the country.

MATERIAL AND METHODS
Forty pecan genotypes were evaluated.Twenty-one are cultivars registered at the Ministry of Agriculture, Livestock and Food Supply (Mapa, 2020), while 19 are selections found in orchards in southern Brazil (Table 1).
Considering all cultivars under analysis, farmers believe that about 38% of them are the most grown in the region.
Plant material was collected from young leaves (disease-free folioles were thoroughly extended) and taken to the Laboratory of Molecular Biology and stored in an ultrafreezer at -80 ºC, up to DNA extraction.
Leaf genomic DNA was extracted with the protocol of DNA isolation based on cetyltrimethylammonium bromide (CTAB), in agreement with the methodology proposed by Ferreira & Grattapaglia (1996).
Amplifications were carried out by the Gene Amp® PCR System 9700 thermocycler (Applied Biosystems).
Amplification cycles were a denaturation step at 94 °C for 3 min, followed by 35 cycles at 94 °C for 30 seconds; in the annealing phase, temperatures ranged to be adequate for every primer for 30 seconds, at 72 °C for 1 min and ended with an extension cycle at 72 °C for 3 min and a cycle at 4 °C up to sample removal from the thermocycler (Grauke et al., 2003).
Amplification products were separated by 2% agarose gel electrophoresis with the use of the molecular marker 1 Kb plus DNA Ladder (Invitrogen™).Amplified fragments were visualized by the Gel Logic 2200 Imaging System (Kodak).

Amplification results of every locus under analysis
were registered qualitatively, i. e., presence (1) or absence (0).Although SSR markers are codominant markers, due to the amplification of some locis in more than one region, the markers were treated as dominant.
The genetic distance matrix was generated using the complement of the Jaccard similarity coefficient.Based on the resulting genetic distance matrix, the cluster analysis was performed by the Unweighted Pair Group Method with Arithmetic Mean (UPGMA), with the help of the NTSYS-pc Numerical Taxonomy and Multivariate Analysis System, version 2.02 (Rohlf, 1998).A total of 24 polymorphic bands were identified in the 11 selected primer pairs.Due to their low DNA quality, six genotypes -'Caddo', 'Chickasaw', 'Mohawk', Success 02, 'Importada' and Selection Mi01 -also had to be removed from the analysis.Therefore, 34 out of 40 collected accessions were evaluated.

Genetic variability in pecan genotypes in Brazil
Mean similarity of genotypes under study was 46%.
It shows that the percentage of genetic difference among accessions is higher than the similarity among them.
The similarity matrix (Figure 1) shows that some pecan genotypes exhibited more than 80% genetic divergence, i.
Based on the cluster analysis, with a cophenetic correlation coefficient was 73.10% with the genetic similarity matrix, four large groups were identified (Figure 2).The cut off point corresponSded to the mean genetic similarity of about 46%.
Group I comprised exclusively by 'Success'.This cultivar was genetically very different from the other genotypes under study.It was also found to be distant from genotypes with which it has some parental in common, such as cultivars 'Barton', 'Gratex', 'Desirable', 'Choctaw' and 'Jackson'.The hypothesis that can explain the distance from 'Success' is that the markers used may be in distinct, random regions, and perhaps not linked to the region where the common ancestor is located.
Group II comprised only two cultivars: one of unknown origin ('Sumner') and 'Shoshoni', which resulted from a cross between 'Evers' and 'Odom'.Both cultivars Summer and Shoshoni exhibited 67%.It was higher than the mean, which was 0.46, revealing the proximity of both materials.
Group III was composed of cultivars Stuart, Barton (2, 3 and 4), Elliot and Cheyenne.The Selection Crioula is a genotype that was selected by farmers and probably is a duplicate of 'Cowley', a further study, with the aid morphological descriptors can help to elucidate this.
Both cultivars, Apache and Shawnee, were classified into the same group.Both cultivars have an ancestral in common, the cultivar Schley.'Shawnee', results from a cross between 'Schley' and 'Barton', is a cultivar that exhibits moderate yield, precocity and low tolerance to scabies.The cultivar Apache, which results from a cross between 'Burkett' and 'Schley', is more tolerant to this disease (Walker et al., 2016).Sharma & Sharma (2001) studied genetic divergence of Juglans regia L. plants and grouped them in 16 groups according to their characteristics.According to the authors, the difference among plants is often chosen as a requirement to conduct a good selection process.Ghanbari et al. (2019) also evaluated genetic difference among 31 European nut trees with the use of ISSR markers.In this case, they were classified into three large groups and 26 alleles; mean per locus was 7. Their results were different from the ones found by this study, but they showed differences among other species of fruit trees.
The most common cultivars in Brazilian orchards are 'Barton', 'Melhorada', 'Imperial', 'Importada', 'Jackson' and 'Shawnee' (Crosa et al., 2020).Among them, there are some genetically very similar.For instance, 'Melhorada' is closer to 'Jackson', 'Imperial' and 'Shawnee', with 1.000, 0.667 and 0.538, respectively (Figure 1).Nevertheless, 'Barton' 03 and 04 exhibited high genetic divergence in this group of cultivars, since its values of genetic similarity were 0.250 ('Melhorada'), 0.294 ('Imperial') and 0.333 ('Jackson').Oliveira et al. (2021) highlight different studies and advances in the technology used in the characterization or genetic identification in the pecan culture, along with the increased need to seek improvements in production aspects among other characteristics that influence nut production.The authors also noted a progress in recent years in relation to studies directed to the area of molecular biology in relation to culture.
Since Pecan crop introduction in Brazil, several selections were made in the nurseries, resulting in an important source of germplasm (Bilharva et al., 2018;Poletto et al., 2020).Our study shows that, although some of the selections analyzed were very similar to some cultivars, others are genetically distinct, showing a unique molecular profile, highlighting the importance of this genetic variability for the development of pecan crop in Brazil.

CONCLUSIONS
Pecan genotypes under evaluation exhibit high genetic variation.
Some potential duplicates were identified in germoplasm evaluated.
The SSR loci analyzed in this study are good markers for genetic identification pecan germplasm cultivated in Brazil.
Based on the loci under evaluation and the number of identified alleles, genotypes could be classified into four main groups.
Conner & Wood (2001) used RAPD markers to study pecan cultivars and found similarity between'Elliot' and   'Barton' (0.46).The comparison of their results and the ones found by this study shows that values were similar, since genetic similarity between Elliot and Barton 3 and 4 was 0.47.Both studies confirm that there is similarity between these cultivars, based on the analysis of the DNA level.Subgroup IV-A comprised most genotypes that exhibited proximity in their origins.For instance, cultivars Desirable, Choctaw and Gratex had a progenitor in common, i. e., Success.Thus, they were included in the same group.Besides, cultivars Choctaw, Wichita and Tejas, whose origin had the participation of the cultivar Mahan, and were grouped close to each other, mainly Wichita and Tejas, which had high degree of genetic similarity.In addition, they were quite close to Mahan since the degree of similarity was 0.833.Grauke et al. (2003) used SSR and studied some pecan accessions, such as cultivars Wichita and Mahan, genetically.Both exhibited genetic similarity (0.63).The results found in the present study does not corroborate those observed by Grauke et al. (2003), since similarity was higher (0.833).High similarity between both cultivars was expected because Mahan is in the origin of Wichita.Jia et al. (2011) also observed the close grouping relationship of the cultivars Mahan and Wichita, which presented a genetic similarity of 0.86.Confirming this close genetic relationship between these two cultivars, as can also be observed in the present study.The cultivar Cowley and the Selection Crioula showed the same molecular profile and formed a group whose genetic similarity was 0.667 in relation to Desirable.

Table 2 :
Sequence, size and annealing temperatures of SSR primers used for genetically characterizing pecan accessions Grauke et al., 2003.mer pairs and annealing temperature calculated in agreement with fragment size.However, some primers underwent some adaptation.Source:Grauke et al., 2003.