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Revista Brasileira de Fruticultura

versão impressa ISSN 0100-2945versão On-line ISSN 1806-9967

Rev. Bras. Frutic. vol.39 no.5 Jaboticabal dez. 2017  Epub 18-Dez-2017

http://dx.doi.org/10.1590/0100-29452017166 

Genética e Melhoramento

POPULATION GENETIC ANALYSIS OF BRAZILIAN PEACH BREEDING GERMPLASM

ESTRUTURA GENÉTICA DO GERMOPLASMA DE MELHORAMENTO DE PESSEGUEIRO NO BRASIL

LIANE BAHR THUROW2 

MARIA DO CARMO BASSOLS RASEIRA3 

SANDRO BONOW3 

LUIS WILLIAN PACHECO ARGE4 

CAROLINE MARQUES CASTRO3 

2 Eng. Agr., MSc., Doutoranda do Programa de Pós-Graduação em Agronomia/Fitomelhoramento, Universidade Federal de Pelotas,Bolsista CAPES. Pelotas-RS. E-mail: lianepel@yahoo.com.br

3 Eng. Agr., Dr., Pesquisador da Embrapa Clima Temperado, Pelotas-RS. E-mails: maria.bassols@embrapa.br; sandro.bonow@embrapa.br;caroline.castro@embrapa.br

4 Tecg. Fruticult., Dr., Pós-doutorando do Laboratório Nacional de Computação Científica, Petrópolis-RJ. E-mail: l.willianpacheco@gmail.com

ABSTRACT

Peach has great economic and social importance in Brazil. Diverse sources of germplasm were used to introduce desirable traits in the Brazilian peach breeding pool, composed mainly by local selections and accessions selected from populations developed by the national breeding programs, adapted to subtropical climate, with low chill requirement, as well as accessions introduced from several countries. In this research, we used SSR markers, selected by their high level of polymorphism, to access genetic diversity and population structure of a set composed by 204 peach selected genotypes, based on contrasting phenotypes for valuable traits in peach breeding. A total of 80 alleles were obtained, giving an average of eight alleles per locus. In general, the average value of observed heterozygosity (0.46) was lower than the expected heterozygosity (0.63). STRUCTURE analysis assigned 162 accessions splitted into two subpopulations based mainly on their flesh type: melting (96) and non-melting (66) flesh cultivars. The remaining accessions (42) could not be assigned under the 80% membership coefficient criteria. Genetic variability was greater in melting subpopulation compared to non-melting. Additionally, 55% of the alleles present in the breeding varieties were also present in the founder varieties, indicating that founding clones are well represented in current peach cultivars and advanced selections developed. Overall, this study gives a first insight of the peach genetic variability available and evidence for population differentiation (structure) in this peach panel to be exploited and provides the basis for genome-wide association studies.

Index terms Prunus persica; genetic diversity; population structure; low chill germplasm; genetic resources

RESUMO

O pessegueiro tem grande importância econômica e social no Brasil. Diversas fontes de germoplasma foram utilizadas para a introdução de caracteres desejados no pool gênico de pessegueiro do Brasil, constituído principalmente de seleções naturalizadas e acessos selecionados a partir de populações desenvolvidas pelos programas de melhoramento, adaptadas às condições de clima subtropical, de baixa exigência em frio, bem como acessos introduzidos de diversos países. Neste estudo, foram utilizados marcadores SSR, selecionados por seu elevado nível de polimorfismo com objetivo de acessar a variabilidade genética e a estrutura populacional de um painel composto por 204 genótipos de pessegueiro, selecionados com base em fenótipos contrastantes para importantes caracteres no melhoramento do pessegueiro. Um total de 80 alelos foram identificados, com média de oito alelos por loco. Em geral, o valor médio da heterozigosidade observada (0,46) foi menor do que a heterozigosidade esperada (0,63). Análises do STRUCTURE atribuíram 162 acessos em duas subpopulações, majoritariamente com base em caracteres relativos ao fruto: cultivares fundentes (96) e não-fundentes (66). Os acessos restantes (42) foram considerados não-estruturados, utilizando um coeficiente de adesão de 80%. A variabilidade genética foi maior na subpopulação fundente em comparação com a não-fundente. Além disso, 55% dos alelos presentes nas cultivares e seleções do programa de melhoramento também estão presentes nos clones de fundação, indicando que estes clones estão bem representados nas cultivares de pessegueiro e seleções avançadas desenvolvidas. Este estudo apresenta uma primeira percepção da variabilidade genética disponível e evidências para a diferenciação da população (estrutura) neste painel de pessegueiro, que pode ser explorada e servir como base para estudos de mapeamento associativo.

Termos para indexação Prunus persica; variabilidade genética; estrutura de população; germoplasma de baixo frio; recursos genéticos

INTRODUCTION

Peach [Prunus persica (L.) Batsch] is considered an important agricultural crop worldwide, becoming the third most produced temperate tree fruit species, after apple and pear (BYRNE et al., 2012; FONTI FORCADA et al., 2013). The world production of peach and nectarine was approximately 22.8 million tons in 2014 whereas in Brazil was approximately 211 thousand tons (FAOSTAT, 2017).

Peach is native of China where it was domesticated 4,000–5,000 years ago, and later spread around the world (FAUST; TIMON, 1995).For centuries, seed propagation was the main source of plants and consequently landrace genotypes resulted from this movement. This germplasm became the base of productive hybrids adapted to a wide range of different climate regions throughout the world (BYRNE, 2005a; BIELENBERG et al., 2009).

In the Americas, peach was introduced by the Spanish and Portuguese during the sixteenth century, where it was quickly adopted by the native people and spread to a wide variety of environments (BYRNE et al., 2012). According to historical records, peach was introduced in Brazil in 1532 by Martim Afonso de Souza, through seedlings brought from Madeira Island and planted in São Vicente, where it is currently the state of São Paulo (RASEIRA; NAKASU, 2002).

In the 1950s, two breeding programs - one in São Paulo and the other in Rio Grande do Sul state – started in Brazil, aiming the development of fresh market and processing peach cultivars adapted to mild winter regions. Local cultivars were then used as founding clones and had great importance for the breeding programs. All this germplasm probably arrived in Brazil several hundred years ago. Since this initial material was propagated by seed, only the most productive and adapted genotypes were maintained. In this way, locally adapted peaches were developed (BYRNE, 2003). Among them it should be cited the non-melting varieties 'Aldrighi', 'Ambrosio Perret', 'Abóbora', and 'Intermediário', used mainly in the breeding program for processing cultivars and 'Delicioso', 'Precoce Rosado', 'Admirável', and '15 de Novembro', local cultivars used intensively in southern Brazil breeding program for development of fresh market peach cultivars (BYRNE; BACON, 1999). The low chill germplasm composed initially by local varieties and seedlings selections of them was enriched by pollen and seeds (both resulted from crosses or open pollination) from the North American breeding programs and composed the basic genetic material for the Southern Brazil breeding program (RASEIRA et al., 2008a).

The peach germplasm available in Brazil is maintained in the Active Germplasm Bank of stone fruit by Embrapa Temperate Climate, in Pelotas (coordinates 31º40’47”S 52º26’24”W). The origin of this germplasm is diverse, including breeding programs of Embrapa and of the Agronomic Institute of Campinas, introductions from other Brazilian states and old cultivars as well as introductions from countries such as Bolivia, Spain, USA, Italy, Canary Islands, Mexico and Japan (RASEIRA et al., 2008b).

Previous studies, mainly of morphological and phenological characterization, evidenced a high variability among the peach accessions, available for use in breeding, mainly adapted to subtropical climate, with low chilling requirement and accessions currently used as a source of resistance to brown rot, leaf rust and bacterial leaf spot (RASEIRA et al., 2008b).

Over the last two decades, simple sequence repeat markers (SSRs) have been successfully carried out in peach to assess genetic diversity (DIRLEWANGER et al., 2002; ARANZANA et al., 2003; XIE et al., 2010; BOUHADIDA et al., 2011)and more recently, for population stratification (ARANZANA et al., 2010; CAO et al., 2012; FONT I FORCADA et al., 2013; LI et al., 2013; CHAVEZ et al., 2014), providing tools for marker-assisted selection (MAS). However, to date the available peach germplasm in Brazil is still underexplored at the molecular level.

The population structure and genetic relatedness between genotypes has a strong impact on association mapping studies, becoming the main causes of spurious associations. In cases of genotype-phenotype covariance, many markers across the genome will appear to be associated with the trait of interest, when in fact these markers simply capture the genetic relatedness among individuals (MYLES et al., 2009). For this reason, to reduce the confounding effects of complex genetic relatedness among genotypes in breeding populations, it is extremely important to correct for population structure in association mapping tests.

This research is the first attempt to assess genetic diversity, comprising a wider sampling of cultivars and advanced selections, with more informative genetic markers for current peach breeding in Brazil. In the present research, we report the genetic diversity and assess population structure of 204 peach genotypes belonging to peach germplasm bank of Brazil. These results will be useful to improve further breeding strategies and choose more reliable approaches for possible genetic association mapping.

MATERIAL AND METHODS

Plant material.A total of 204 peach cultivars and advanced selections [Prunus persica (L.) Batsch] from the peach breeding program of the Brazilian Agricultural Research Corporation - Embrapa, was evaluated in this study (Table 1). This set is hereafter referred as the peach association panel of Embrapa. Among the total germplasm that composes the Active Germplasm Bank and work collection of the breeding program, these selected 204 genotypes represent the genetic variability available for bacterial spot, brown rot and tolerance to abiotic stresses such as heat tolerance at the flowering stage and chilling requirement. This panel also includes eight of the founding clones of the Brazilian peach breeding program (started in 1953), in order to explore how much of their genome is represented in the current breeding. Among the founders are local varieties such as ‘Aldrighi’, ‘Abóbora’, ‘Edmundo Perret’, ‘Delicioso’, ‘Cristal-Taquari’, ‘Interlúdio’ and North American varieties such as ‘Panamint’ and ‘Sunhigh’.

DNA extraction and microsatellite genotyping.For DNA extraction, fresh young leaves were collected and stored at -80°C. Genomic DNA was isolated from 0.15 to 0.20g frozen leaf tissue, using procedure described by Ferreira e Grattapaglia (1998). The samples were quantitated with the Qubit® 2.0 fluorometer (Life Technologies, Grand Island, NY, USA) and concentrations adjusted to 10 ng/μl for PCR amplification.

The 204 peach genotypes were analyzed using 10 SSR markers: BPPCT020, BPPCT002, BPPCT007, BPPCT015, BPPCT014, BPPCT017 (DIRLEWANGER et al., 2002), pchgms (), USOSINSKI et al., 2000DP98-407 (CIPRIANI et al., 1999), CPPCT022, and CPPCT006 (ARANZANA et al., 2002), previously developed for peach and selected from the Prunus ‘Texas’ almond × ‘Earlygold’ peach (T × E) reference map (DIRLEWANGER et al., 2004). The loci were randomly distributed in the eight linkage groups of the peach genome and chosen on the basis of their high polymorphism and reproducibility.

The forward primer of each pair was tagged with a M13 primer sequence (5’-CACGACGTTGTAAAACGAC-3’) during synthesis. Polymerase chain reactions (PCR) were performed in a total volume of 10 μl containing 20 ng of genomic DNA, 5 μL of 2X GoTaq Green Master Mix (Promega Corporation, USA), and three different primers: 1.0 μM of each reverse and forward primer and 1.0 μM of M13 primer with a fluorescent label ((-29)/IRDye 800-labeled Primer) (LI-COR Biosciences). Amplifications were conducted in a GeneAmp® PCR System 9700 (Applied Biosystems) set as following: pre-denatured at 94°C for 1 min; followed by 30 cycles of 45 s at 94°C, 45 s at primer’s specific annealing temperature, and 2 min at 72°C, finally extended at 72°C for 4 min.

PCR amplified reactions were then diluted with sterile Milli-Q water, in a proportion 1:20, to allow a clear visualization of the amplified fragments.

After, 2 μL of diluted PCR reaction were added to 2 μL of Blue Stop Solution (LI-COR Biosciences) and denatured at 94°C for 3 min, in thermocycler described above. Denatured PCR products (0.8 μL) were then loaded on 6.5% polyacrylamide gel (LI-COR Biosciences) and sized by vertical electrophoresis through a 4300 DNA Analyzer LICOR, programmed to run for a period of 90 min at 1500 V and 40 mA.

Fragment sizes were estimated with the 50-350 bp IRDye 800 Sizing Standard (LI-COR Biosciences) and scoring performed with Saga Generation 2 software (LI-COR Biosciences).

Data analysis.The genetic variability parameters calculated for the whole peach association panel were: number of alleles per locus (NA), effective number of alleles per locus (NE), observed heterozygosity (Ho), expected heterozygosity (He), and Wright’s fixation index (F) calculated with POPGENE software version 1.32 (YEH et al., 1997).Polymorphism information content (PIC), allele frequency and number of different genotypes amplified by each marker were calculated using Powermarker 3.25 software (LIU; MUSE, 2005).

For genetic comparison between founders and breeding germplasm, all cultivars released by Embrapa breeding program, since its inception, in 1953, as well as the founders, composed a group.

This group, of 81 peach and nectarine cultivars, was sub-divided according to its breeding period. Founder varieties were identified as “Pre-1960s” and cultivars released by the breeding program were classified into five groups according to their releasing decade (1960s to 2000s). To assess changes in allelic counts for each SSR locus analyzed, the total number of alleles for a given breeding period was calculated by adding the numbers of alleles present in the cultivars in that period. To detect the alleles that were lost in a given period, we calculated the number of alleles absent in cultivars of the period and present in the founders (Pre-1960s). Similarly, gained alleles were calculated by the number of alleles present in cultivars of the period and that were absent in the founders (Pre-1960s).

Population structure on the whole peach association panel was addressed using two different approaches. The first method based on a Bayesian clustering approach was performed on the 204 peach genotypes using STRUCTURE version 2.3.4 software (PRITCHARD et al., 2000). Twenty independent runs per K value were performed by setting the number of subpopulations (K) ranging from 1 to 10. Each run started with 10,000 burn-in period followed by 100,000 Monte Carlo Markov Chain (MCMC) interactions, adopting an admixture model and correlated allele frequencies.

STRUCTURE HARVESTER software (EARL; VONHOLDT, 2012) was used for visualizing STRUCTURE output and implementing the Evanno method to analyze for population stratification results (EVANNO et al., 2005).

The membership coefficients for the most optimal number of subpopulations were permuted to match the 20 replicates for that K value as closely as possible, using CLUMPP version 1.1.2 (JAKOBSSON; ROSENBERG, 2007). Genotypes were assigned to a subpopulation when their membership coefficient (Q) was higher than 0.8.

Results were plotted with DISTRUCT version 1.1 (ROSENBERG, 2004).

The second method to assess population structure was a factorial analysis, more specifically a Principal Coordinate analysis (PCoA) of all inferred subpopulations, based on similarity matrix, calculated using Jaccard coefficient and further construction of the 3D scatter plot using scatterplot3d package (LIGGES; MÄCHLER, 2003) performed with the R software (http://www.R-project.org).

RESULTS AND DISCUSSION

Genetic diversity analysis In the current study, a set of 204 peach accessions were genotyped with 10 selected SSR markers distributed across the eight linkage groups of the peach genome. All loci evaluated in this study were polymorphic amplifying a total of 80 alleles, and produced a maximum of two bands per genotype, in accordance with the diploid level of this species.

An average of 8.0 alleles per locus was observed, which is higher than the values reported in previous studies on genetic diversity in peach [4.2 alleles per locus (DIRLEWANGER et al., 2002);7.3 (ARANZANA et al., 2003); 6.36 ();6ARANZANA et al., 2010.5 (ANDERSON, 2010); 6.73(BOUHADIDA et al., 2011); 6.4 (CAO et al., 2012); 5.1 (FONT I FORCADA et al., 2013)].

Chavez et al. (2014) reported that a total of 237 alleles were amplified for 168 peach and nectarine cultivars and selections of the University of Florida (UF) peach germplasm using 36 SSR markers. Comparing three SSR markers (BPPCT014, BPPCT017 and CPPCT022) in common with their work, we were able to distinguish an average of 24 genotypes per marker while only 16 genotypes per marker were observed in UF peach germplasm. Amplified band sizes were similar to those reported by Bouhadida et al. (2011) in 94 peach cultivars including Spanish native peach and foreign commercial cultivars, however, the genetic variability parameters were lower than those observed in this study.

Our study demonstrates that 42 alleles (52.5%) showed low frequencies (less than 5%) including nine unique alleles detected in seven genotypes. The cultivars ‘Amarillo’ and ‘Arlequim’ presented two unique alleles each. Four unique alleles were detected in ‘Tarumã’, ‘Springcrest’, ‘Natal’ and ‘Vila Nova’, which are introductions from other countries or old cultivars, probably not used very much for breeding purposes due to some undesirable traits. ‘Tarumã’ is a late ripening variety, whereas ‘Springcrest’, an introduction used at the beginning of the breeding program, requires higher chilling hours than what accumulates in Southern Brazil. The cultivar ‘Natal’ has not been used in the program and ‘Vila Nova’ is interesting but susceptible to brown rot and bacterial spot. A unique allele was also found in a recent advanced selection of flat peaches, Cascata 1511, originated from a cross with pollen from China.

Low allelic frequencies resulted in a low effective number of alleles (Ae) with an average of 3.05 representing 38.13% of the total identified alleles. The observed heterozygosity (Ho) ranged from 0.28 (CPPCT006) to 0.62 (BPPCT015), with an average of 0.46. These were lower than the expected heterozygosity values (He), which ranged between 0.50 (BPPCT002 and BPPCT007) and 0.80 (BPPCT017), with an average of 0.63. Consequently, Wright’s fixation indices (F) showed positive values, with a mean of 0.28 for all loci (Table 2).

Table 2 also shows that BPPCT017 had the highest polymorphism information content (PIC) of 0.77 and BPPCT002 had the lowest PIC of 0.45.

The number of different genotypes identified by each marker ranged between 7 (CPPCT006) and 36 (BPPCT015), with an average of 19.8.

These results are also consistent with Aranzana et al. (2010), using 224 peach and nectarine cultivars from around the world, when comparing genetic variability parameters using eight SSR markers common to both studies. The estimated genetic diversity of Brazilian peach breeding germplasm was higher than reported by Aranzana’s study, measures of diversity for A (8.0 vs. 7.6), Ae (2.99 vs. 2.56), Ho (0.46 vs. 0.42), He (0.63 vs. 0.55), respectively, and an average of 19.88 genotypes identified per marker in our study vs.14.25 in theirs. High values of all the measures of diversity indicated allelic richness in the analyzed germplasm, which can provide additional tools for breeding. However, results from Oriental germplasm as reported in Li et al. (2013) study, using 653 peach accessions around the world amplified with 48 SSRs, showed that the estimated genetic diversity of the Brazilian peach germplasm was lower when the two studies were compared based on eight SSR markers in common: A (8.0 vs. 14.5), Ho (0.46 vs.0.51) and genotypes amplified by marker (19.87 vs.31.75), respectively. Probably, these high values can be explained by the large sample set covering peach cultivars from Oriental and Occidental geographic regions, including wild, landraces, breeding peach accessions and peach related species. Similarly, the value of F (0.20 vs. 0.28) was higher in Brazilian peach germplasm. The effective number of alleles per locus Ae (2.99 vs. 3.02) and the expected heterozygosity He (0.63 vs. 0.64) was consistent across both studies. As reported by those same authors, the Oriental accessions contributed most to the variability of the sample, especially landraces from the northwest of China, region where peach was originated. An average of 10 alleles per locus was amplified from 146 Chinese landraces. This is higher than the average of 6.4 alleles per locus reported by Cao et al. (2012), in 104 landraces distributed across six geographical regions of China analyzed with 53 SSRs and thus found an average genetic diversity of 0.567 with is lower than 0.63 found in this study.

In addition, this higher genetic variability observed in our study could be explained by the origin of germplasm sources. As these sources of low chill had generally poor commercial characteristics, they were crossed with high chill cultivars with higher commercial quality. Thus, previous studies indicate that, in general, the low chill germplasm is more diverse than the high chill USA germplasm, since it combines the high chill USA germplasm with local cultivars from Brazil and the exotic low chill southern China germplasm (BYRNE; BACON, 1999).

Among the total of 204 peach cultivars and advanced selections analyzed, the used set of 10 SSR markers enable to distinguish unambiguously 198 P. persica genotypes, with the exception of three groups with two cultivars each, which present identical genotypes. From these three groups, the first one includes the peach selections Bolinha 9 and Bolinha 17, both found with identical fingerprints and described as open pollinated seedlings of ‘Bolinha’.

In the second group, the selection Cascata 1423 had identical genotype as the cultivar Fascínio, probably because the selection is an open pollinated seedling from ‘Fascínio’. Finally, in the third group, the cultivars Ametista and Esmeralda were described with the same molecular profiles, probably due to the first being originated from an open pollination of ‘Esmeralda’. The same occurred with Aranzana et al. (2010) that used a set of 50 SSRs and were able to identify only 209 different genotypes among the 224 cultivars studied. These results demonstrate that a reduced number of informative SSR loci are efficient to genotype a peach germplasm collection and additional markers sometimes do not generate an effective increase in relevant information.

Genetic comparison between founders and breeding peach germplasm Eight founder varieties of the Embrapa peach breeding program were studied in order to explore how much of their genome is represented in the peach breeding cultivars. We observed that 55% of the alleles present in the breeding germplasm were also present in the founder varieties, and only 20% of these alleles shared are considered rare in the breeding varieties (it refers to alleles present at a frequency < 0.05). We observed that the genetic diversity of the Embrapa germplasm panel including founder varieties did not change significantly across the decades of breeding with total number of alleles by breeding period ranged from 40 to 53 (Table 3).

There was a slight decrease in the number of alleles for cultivars released during the 1970s and 2000s than those released before the 1960s, comprising the founders of the breeding program, as well as the alleles absent when comparing to the founders, that were higher than the observed gained alleles. On the other hand, allelic counts increased during the 1960s, 1980s and 1990s comparing to the initial breeding period before the 1960s (Table 3).

Population Structure Population stratification for all 204 peach genotypes representatives of the Brazilian peach breeding germplasm were analyzed by successively increasing the number of subpopulations (K) from 1 to 10, with K=2 followed by K=3 having the highest delta K (ΔK) values using the Evanno method (EVANNO et al., 2005).

CLUMPP alignment on the outcomes of 20 independent replicates for K=2, showed a similarity of 99%, indicating that the assignment of accessions to the subpopulation was well correlated among runs. Population stratification suggested a clear subdivision by correspondence with flesh type: melting (yellow bars) and non-melting flesh cultivars/ selections (red bars) (Figure 1, K=2).

Genotypes were assigned to a population if its membership coefficient was ≥ 80%. Therefore, for all peach germplasm analyzed, 96 accessions were clustered into melting subpopulation, including peach cultivars for fresh consumption, nectarines with melting flesh, flat peach selections and three of the founding clones used in the breeding program (‘Delicioso’, ‘Interlúdio’ and ‘Sunhigh’), while 66 accessions grouped with non-melting subpopulation, mainly including peach cultivars for canning, nectarines with non-melting flesh and three founder varieties (‘Abóbora’, ‘Aldrighi’, and ‘Cristal-Taquari’).

Considering K=2, the remaining 42 accessions could not be assigned under the 80% membership coefficient criteria and were considered to be unstructured suggesting allele sharing. Mainly accessions clustered within this admixed group are cultivars with dual purpose (fresh market and canning) as ‘Eldorado’, ‘Leonense’, ‘Maciel’ and ‘Riograndense’, also comprises genotypes that have one or more of this dual purpose cultivars as parents or that present crosses between melting x non-melting cultivars in the genetic background.

Ten of the 33 nectarines analyzed in this study and two founder varieties, ‘Edmundo Perret’ (canning peach) and ‘Panamint’ (the only founding clone of nectarine genotyped in this study), were also included in this admixture group. A clear agreement between population stratification at K=2 and genetic diversity can be visualized with the PCoA analysis that also shows the relative close relationships between clusters obtained by STRUCTURE (Figure 2).

With increasing K to three subpopulations, the non-melting flesh group remained almost invariable (in red, Figure 1 K=3), whereas the cluster of melting accessions split into two subpopulations, one principally melting flesh peaches (in yellow) and the other, a more diverse cluster composed mainly by nectarines (20 accessions of the total 33 nectarines analyzed), peach germplasm introductions from USA, Japan, Mexico and Bolivia, and advanced selections that have introductions as one of the parents. Moving to K=3, the percentage of admixed accessions also increased, suggesting a more complex structure than with K=2. No additional major subpopulations were observed with other K values.

Similar results associating genetic structure with fruit characteristics were also reported before by other authors (ARANZANA et al., 2010; LI et al., 2013; CHAVEZ et al., 2014) analyzing peach germplasm.

Aranzana et al. (2010) identified three main groups: melting flesh peaches, melting flesh nectarines, and non-melting cultivars. Corroborating with the results of this study, these same authors with K=2 detected a subdivision between melting and non-melting cultivars. Moving to K= 3, the cluster of melting cultivars split into peaches and nectarines. In our study it was not detected clear genetic evidence for nectarine vs. peach, but moving to K=3, melting cultivars also split into peaches and nectarines, with the more diverse subpopulation being the one that groups most of the nectarines, while for those authors the non-melting subpopulation was considered the most diverse.

The Brazilian peach breeding program does not maintain separate breeding efforts for peaches and nectarines, this could explain why the founding clone nectarine variety ‘Panamint’ grouped together with the melting peach founders ‘Interlúdio’ and ‘Sunhigh’, when considered the subdivision of the germplasm into three subpopulations (K=3).

However, only the founder variety ‘Delicioso’, the main cultivar used to develop melting flesh peach for fresh consumption, grouped with the melting subpopulation. The same was observed with the nonmelting founder varieties ‘Abóbora’ and ‘Aldrighi’ that cluster together in non-melting subpopulation, showing that different genetic resources were used in order to develop peach for fresh consumption and canning purposes in early breeding times. This may also be due to the longest period of breeding for melting and non-melting peach cultivars, with most of agronomic and fruit quality traits economically important already fixed, thus crosses are realized mainly between genotypes belonging to the same subgroup.

Li et al. (2013) assigned accessions to three subpopulations: Oriental, Occidental, and Landraces.

The Occidental breeding subpopulation was subdivided into nectarine and peach subpopulations and Oriental subpopulation into ‘Yu Lu’ and ‘Hakuho’, were ‘Yu Lu’ derived cultivars include mostly accessions from Zhejiang Province and ‘Hakuho’ grouped Japanese cultivars or pedigreeassociated with ‘Hakuho’ high-chill Japanese cultivar. Three main groups were also identified by Chavez et al. (2014) in UF peach germplasm: outgroups and landrace germplasm, melting cultivars, and non-melting cultivars.

The principal coordinate analysis (PCoA) was used as a second approach to detect population structure. Once again the same subpopulations were identified as before with the Bayesian analysis.

PCoA plot using all 204 peach genotypes is showed in Figure 2. In the PCoA, it is obvious that the primary axis separates the accessions based on the peach flesh type (melting and non-melting). It is observed that genetic variability is greater in the melting subpopulation. This difference between melting and non-melting subpopulations is due their genetic background. At the beginning of the program, breeding of melting peach cultivars used as basis several introductions from different countries and breeding programs, whereas the non-melting peach breeding efforts used mainly local varieties. Thus, the gene pool to select genotypes for processing purpose was probably smaller than the gene pool available to select peaches for fresh consumption. Also, it may contribute the fact that, worldwide, the number of peach breeding programs for processing purpose are only a few compared to peach breeding programs aiming melting peaches for fresh consumption.

Thus, the source of germplasm exchange is limited.

In the development of non-melting peaches for both the processing and fresh markets located in low chill zones, the longest running program is the nonmelting Embrapa peach program, in Brazil. Later, breeding efforts in Mexico were initiated. It should be noted that fresh market cultivars with non-melting flesh have been used for centuries in Latin America and Spain (BYRNE, 2005b). This approach has more recently been promoted in U.S by the Florida’s fruit breeding program focused on non-melting texture peaches for fresh market.

In our study majority of the cultivars introduced from North America breeding programs were structured in the melting subpopulation suggesting a common gene pool. These results are in agreement, considering the active exchange of germplasm between the breeding programs of these two countries and the use of North American cultivars in Embrapa peach breeding program.

We also observed that the genetic diversity does not appear to have decreased analyzing allele frequencies grouping cultivars by decades of release.

Among the germplasm present in this collection are a wide range of local peach cultivars, North American cultivars and germplasm introgressed from other countries indicating continuous enrichment of the breeding pool. The distribution of the founders in the three dimensions graph (Figure 2), suggests that founder varieties are still a direct source of current peach variability for breeding and release of new cultivars.

As the population structure is considered strong within melting and non-melting flesh genotypes, correction for population stratification will be necessary in order to preclude false positives in association studies. This research is a first step and a reference to choose genotypes for association mapping studies and enable marker assisted selection in peach breeding.

FIGURE 1 Population stratification of 204 peach cultivars based on Bayesian clustering approach. For K=2, accessions in yellow indicates melting peach subpopulation, accessions in red indicates non-melting peach subpopulation. For K=3, each color refers to a different subpopulation inferred by STRUCTURE analysis. 

FIGURE 2 Genetic relationships between the 204 P. persica genotypes visualized using a principal coordinate analysis (PCoA). The individuals are coloured with respect to their subpopulations inferred by STRUCTURE analysis (membership coefficient ≥ 80%): black indicates melting peach, red indicates non-melting peach, green indicates unstructured individuals (admixture), blue represents the founder varieties. The light blue and pink color represent the overlaid with fruit morphology traits, nectarines dispersion and flat peaches respectively. The first, second and third principal coordinates account for 17.62%, 10.24% and 7.52% of the total variation, respectively. 

TABLE 1 Cultivars and advanced selections of peach breeding germplasm, breeding period of cultivars released by the Embrapa breeding program and fruit characteristics.  

ID Cultivar Decade a Fruit trait b ID Cultivar Decade a Fruit trait b
1 Abóbora Pre-1960s PYNRC 44 Coral 2 1980s PWMRC
2 ágata 1970s PYNRC 45 Cristal-taquari Pre-1960s PWNRC
3 Aldrighi Pre-1960s PYNRC 46 Delicioso Pre-1960s PWMRF
4 Alpes 1980s PYNRC 47 Della Nona 1990s PWMRF
5 Amarillo - N??RS 48 Diamante 1970s PYNRC
6 Anita 1990s NWMRC 49 Dulce 1990s NWMRS
7 Apote - P?NR? 50 Edmundo Perret Pre-1960s PWMRC
8 Arlequim - PWMRF 51 Eldorado 1980s PYNRC
9 Atenas 2000s PYNRC 52 Eragil - PYMRF
10 Aurora 1 - PYNRC 53 Esmeralda 1980s PYNRC
11 Aurora 2 - PYMRC 54 Ewtrin - NWMRC
12 Yellow_Babcock* - PYNRC 55 Farrapos 1960s PYNRC
13 Barbosa 1990s PWMRF 56 Flordabella - PYMRF
14 Bolinha 1980s PYNRC 57 Flordaglo - PWMRC
15 BR1 1970s PWMRC 58 FlordaGrande - PYMRS
16 BR3 1970s PWMRS 59 Flordaking - PYMRC
17 Cai 1960s PWMRC 60 Flordaprince - PYMRC
18 Capdeboscq 1960s PYNRC 61 Galaxy - PWMFC
19 Cardeal 1960s PYMRC 62 Gaúcho 1980s PWMRS
20 Regalo 2000s PWMRC 63 Gaúcho de Porto Alegre 1980s PWMRS
21 Cascata 805 - PWMRS 64 Gaudério 1960s PYMRC
22 Cascata 828 - PWMF? 65 Granada 1990s PYNRC
23 Cascata 838 - PYMFS 66 Interlúdio Pre-1960s PYMRS
24 Fascínio 2000s PWNRS 67 Jade 1980s PYNRC
25 Cascata 1303 - PYNRC 68 Josefina - NWMRF
26 Cascata 1373 - PYNFC 69 Jubileu 1990s PYNRC
27 Cerrito 1960s PYNRC 70 Kampai 2000s PWMRS
28 Chimarrita 1980s PWMRS 71 Leonense 1990s PYNRC
29 Chiripá 1970s PWMRF 72 Libra 2000s PYNRC
30 Conserva 334 - PYNRC 73 Linda 1980s NYMRC
31 Conserva 594 - PYNRC 74 Lord 1970s PYNRC
32 Conserva 672 - PYNRC 75 Maciel 1990s PYNRC
33 Conserva 930 - PYNRC 76 Madrugador 1960s PYNRC
34 Conserva 947 - PYNRC 77 Magno 1970s PYNRC
35 Conserva 1566 - PYNR? 78 Mara 1990s NYMRS
36 Conserva 1578 - PYNRC 79 Marli 1980s PWMRS
37 Conserva 1596 - PYNRC 80 Minuano - PYMRS
38 Conserva 1600 - PYNRC 81 Mollares Hierro - PWMRF
39 Conserva 1612 - PYNRC 82 Morro Redondo 1960s PYNRC
40 Conserva 1666 - PYNRC 83 Natal - PWMRC
41 Conserva 1798 - PYNRC 84 Necta 468 - NWMRC
42 Convênio 1960s PYNRC 85 Necta 480 - NYNRC
43 Coral 1960s PWMRS 86 Necta 496 - NWMRC
ID Cultivar Decade a Fruit trait b ID Cultivar Decade a Fruit trait b
87 Necta 511 - NYNRC 132 Bonão 2000s PYNRC
88 Necta 512 - NYNRC 133 c-2006-198-48 - ?????
89 Necta 528 - NWNRS 134 c-2006-201-2 - ?????
90 Necta 532 - NY?RF 135 c-2006-201-9 - ?????
91 Okinawa - PWMRF 136 Carapuça 1960s PYMRS
92 Olímpia 2000s PYNRC 137 Cascata 349 - PYMRS
93 Ônix 1980s PYNRC 138 Cascata 700 - PYMRC
94 Pampeano 1990s PWMRS 139 Cascata 727 - PWNRC
95 Panamint Pre-1960s NYMRF 140 Cascata 1005 - PYNR?
96 Pepita 2000s PYNRC 141 Cascata 1015 - PYNR?
97 Pérola de Itaquera - PWNRC 142 Cascata 1055 - PWNR?
98 Pilcha 1980s PYMRC 143 Cascata 1067 - PWNR?
99 Piratini 1960s PYNRC 144 Cascata 1423 - PWMRS
100 Planalto 1990s PWNRC 145 Cascata 1429 - PWMRS
101 Precocinho 1980s PYNRC 146 Cascata 1493 - PWNRC
102 Premier 1960s PWMRS 147 Cascata 1511 - PWMFC
103 Princesa 1960s PYMRS 148 Cascata 1513 - PWMRS
104 Real - PYNRC 149 Cascata 1577 - PWMRS
105 Riograndense 1990s PYNRC 150 Cascata 1669 - PWMRS
106 Rubimel 2000s PYMRS 151 Chato 10 - PWMFF
107 Safira 1980s PYNRS 152 Chato 11 - PYMFC
108 San Pedro - PYMRS 153 Chato 13 - PWMFF
109 Santa áurea 2000s PYNRC 154 Chula 1990s PWMRF
110 Sentinela 1980s PWMRC 155 Conserva 657 - PYNRC
111 Sinuelo 1970s PYMRS 156 Conserva 1127 - PYNRC
112 Sulina 1980s PWMRC 157 Conserva 1218 - PYNRC
113 Sunblaze - NYMRS 158 Conserva 1278 - PYNRC
114 Sunhigh Pre-1960s PYMRS 159 Conserva 1526 - PYNRC
115 Sunmist - NWMRC 160 Conserva 1556 - PYNRC
116 Taquari 80 - PWMRS 161 Conserva 1806 - PYNRC
117 Tarumã 1960s PYNRC 162 Conserva 1812 - PYNRC
118 Topázio 1970s PYNRC 163 Douradão - PYMRF
119 Tropic Beauty - PYMRC 164 Dourado 2 - PYMRF
120 Tropic Blush - PYNRC 165 Early Diamond - NYMRC
121 Tropic Snow - PWMRS 166 Flor da star - PYMRC
122 Turquesa 1970s PYNRC 167 Granito 1990s PYNRC
123 Vanguarda 1980s PYNRC 168 Ingo - PWMRF
124 Alvorada 1960s PYMRC 169 July Elberta - PYMR?
125 Âmbar 2000s PYNRC 170 La Feliciana - PYMRF
126 Ametista 1990s PYNRC 171 Lotus - NYMRF
127 AztecGold - PYNRC 172 Maravilha - PWMRC
128 Babygold 7 - PYNRC 173 Marfim 1990s PWNRC
129 Babygold 9 - P?NR? 174 Necta 422 - NYNRC
130 Baronesa 1960s PYMRS 175 Necta 466 - NYNRC
131 Blancona - PWMRF 176 Necta 508 - NYMRS
ID Cultivar Decade a Fruit trait b ID Cultivar Decade a Fruit trait b
177 Necta 529 - NYMRS 191 Springcrest - PYMR?
178 Necta 531 - NWMRS 192 Suncoast - NYMRF
179 Necta 543 - NWMRF 193 Sunlite - NYMRS
180 Necta 3973 - NWMRC 194 Sunred - NYMRC
181 Morena - NYMR? 195 Super morena - NYMRS
182 Nectared 5 - NYMRC 196 Talismã - PWMRC
183 Norman - PYMRF 197 Taq 98 - PWMRC
184 Piazito - PWMRS 198 Tsukuba - ?????
185 Rayon - PYMRC 199 Tx 1A 95 - PYMRC
186 Rei Del Monte - PYMR? 200 Tx 1A 100 - PYMR?
187 Sel. Bolinha 9 - PYNRC 201 Tx 1A 125 - PYMRC
188 Sel. Bolinha 17 - PYNRC 202 Tx 1A 150 - PYMRC
189 Sel. Bolinha 25 - PYNRC 203 Tx 2A 232 LWN - NWMRC
190 Sel. Bolinha 26 - PYNRC 204 Vila Nova 1960s PYMRF

a Decade = Refers to the breeding period that the cultivar was released (only for cultivars released by the Embrapa Breeding Program).

bFruit traits are as follows = First letter: P peach, N nectarine. Second letter: W white, Y yellow. Third letter: N non-melting flesh, M melting flesh. Fourth letter: R round shape, F flat shape. Fifth letter: C clingstone, S semi-cligstone, F freestone

?= No information available

TABLE 2 Summary statistics of variability calculated for the 10 SSR markers in 204 peach genotypes. 

SSR Marker A Ae Ho He F PIC Nºgenotypes
pchgms3 7 2.10 0.34 0.52 0.35 0.50 19
BPPCT020 7 2.75 0.45 0.64 0.29 0.58 13
BPPCT002 7 1.98 0.35 0.50 0.30 0.45 14
BPPCT007 7 2.01 0.38 0.50 0.24 0.46 12
BPPCT015 14 3.58 0.62 0.72 0.14 0.70 36
BPPCT014 5 2.39 0.44 0.58 0.25 0.54 11
BPPCT017 9 4.95 0.59 0.80 0.27 0.77 27
UDP98-407 9 4.58 0.57 0.78 0.27 0.75 25
CPPCT022 11 3.91 0.59 0.74 0.21 0.72 34
CPPCT006 4 2.25 0.28 0.56 0.50 0.46 7
Average 8 3.05 0.46 0.63 0.28 0.59 19.8
Total 80 30.5 - - - - 198

A = number of observed alleles;

Ae = effective number of alleles;

Ho = observed heterozygosity;

He = expected heterozygosity;

F = Wright’s fixation index;

PIC = polymorphism information content.

TABLE 3 Changes in allelic counts by breeding period using SSR markers to evaluate P. persica cultivars released by the Embrapa Peach Breeding Program. 

SSR marker Total nº; of alleles a Nº; of rare alleles b Allelic counts by breeding period
Pre-1960s (8) c 1960s (18) 1970s (10) 1980s (18) 1990s (16) 2000s (11)
pchgms3 6 2 3 4 4 6 5 4
BPPCT020 6 2 4 6 3 4 5 4
BPPCT002 6 3 3 6 3 5 4 3
BPPCT007 4 1 3 3 2 4 4 3
BPPCT015 9 4 7 7 6 7 8 5
BPPCT014 4 0 4 4 4 4 3 4
BPPCT017 8 3 6 6 6 7 6 7
UDP98-407 8 3 6 7 5 6 6 5
CPPCT022 9 5 5 6 5 6 6 6
CPPCT006 4 2 3 2 2 4 3 2
Observed total 64 25 44 51 40 53 50 43
Observed lost d 5 7 3 5 8
Observed gain e 12 3 12 11 7

a Total number of alleles in 81 peach cultivars released by the Embrapa Breeding Program including eight founder varieties.

b Refers to alleles present at a frequency <0.05.

cNumbers in parenthesis indicate the total number of cultivars analyzed in a given period.

dNumber of alleles absent in cultivars of a given period that were present in the founders (Pre-1960s).

eNumber of alleles present in cultivars of a given period that were absent in the founders (Pre-1960s).

CONCLUSIONS

The genotypic information described here demonstrates a high level of genetic diversity available in Embrapa germplasm collection to be exploited for breeding. The both ways to investigate population stratification indicate strong population structure between melting and non-melting flesh genotypes. These results are essential in understanding the genomic relationships among the different peach genotypes and a very important tool for adjusting association tests. Future studies will include SNP discovery and genotyping to search for markers associated with valuable traits in peach.

ACKNOWLEDGMENTS

This work was supported by funds from the Project “Melhoramento genético de pessegueiro, nectarineira e ameixeira para adaptação às condições brasileiras” N° 562400/2010-9 granted by Capes/ MTC/ CNPq – REPENSA.

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Received: July 27, 2016; Accepted: December 13, 2016

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