Genetic diversity, yield, and fruit quality of persimmon in the tropics

Abstract The objective of this work was to determine the genetic diversity, yield, and fruit quality of persimmon genotypes grown in the tropics, in order to select promising genetic materials. DNA extraction was performed on young leaves of 19 persimmon genotypes. For pomological characterization, 15 genotypes were selected. From each genotype, 50 fruit at the physiological maturity stage were harvested in the morning, in order to determine the following parameters: physicochemical characteristics; and the productive variables number of fruit per plant, average fruit fresh mass, average yield, and estimated average yield in two seasons. Twenty SSR markers were tested, out of which 12 were selected to evaluate genetic similarity, which allowed of the identification of distinct groups. The mean genetic diversity value found was 0.41, which is an indicative of low diversity among the analyzed persimmon genotypes. The 'Guiombo', 'Iapar 125', 'Kakimel', 'Mikado RJ', 'Rama Forte Tardio', and 'Taubaté' genotypes show a high yield. The genotypes classified as pollination-constant astringent ('Pomelo', 'Regina', 'Rubi', and 'Taubaté') and those classified as pollination-variant astringent ('Rama Forte', 'Guiombo', and 'Cereja') are potential materials for selection and genetic breeding programs due to their excellent fruit physicochemical characteristics. The investigation through molecular markers is an efficient approach to study the genetic diversity of persimmon genotypes grown in the tropics.


Introduction
Persimmon (Diospyros kaki L.f.) fruit tree is traditionally grown in temperate or subtropical regions (Martínez-Calvo et al., 2013).Persimmon was introduced in Brazil in the late 19 th century.However, its cultivation only expanded around 1920 with the arrival of Japanese immigrants, who provided new cultivars and production techniques (Blood et al., 2020).In Brazil, programs for the conservation of persimmon genetic materials are located at the IAC (Instituto Agronômico, in the municipality of Campinas, in the state of São Paulo, Brazil), which was the institution responsible for disseminating this fruit in Brazilian subtropical regions (Peche et al., 2016a(Peche et al., , 2016b)).However, the last genotype launched by IAC is the persimmon 'Fuyuhana ' -in 1983.One of the main problems related to the diversity management of persimmon genetic resources is the assignment of genotype identity, due to the existence of synonyms and homonyms among local varieties, misleading Japanese translations, and incorrect labeling in the past.
The main characteristics used as references for the classification of persimmon genotypes are the fruit astringency losses related to cross-pollination that resulted in four genotypes groups (Yonemori et al., 2008b), as follows: pollination constant nonastringent (PCNA), pollination variant nonastringent (PVNA), pollination constant astringent (PCA), and pollination variant astringent (PVA).
The germplasm banks are the first step for implementing a program of selection.One of the most important requirements of germplasm banks is the ability to identify the accessions, for which techniques of molecular genetics have become a set of tools for the identification, characterization, and genetic study of species.Molecular markers are important to detect the variability, since they can be used to indicate polymorphisms at the DNA level, and to establish links between the presence or absence of genes that control a particular characteristic (Badenes et al., 2016).
SSR markers provide an ideal tool for this purpose, since they have desirable molecular marker properties, mainly because they are highly polymorphic, reproducible, abundant, and codominant (Soriano et al., 2006).The development of techniques that use codominant markers as simple sequence repeats (SSRs) for persimmon has provided reliable markers for persimmon genetic and diversity studies (Naval et al., 2010;Gil-Muñoz et al., 2018).The characterization of pomological traits is not sufficient for the estimation of available genetic variability, but it can be used in an additive way for the identification by molecular markers.
The objective of this work was to determine the genetic diversity, yield, and fruit quality of persimmon genotypes grown in the tropics, in order to select promising genetic materials.

Materials and Methods
The plant materials came from the germplasm bank located in the municipality of São Bento do Sapucaí, in the state of São Paulo, Brazil (22°41'S, 45°43'W, at 886 m altitude), with mesothermal climate -Cwa, according to the Köppen-Geiger's classification, with dry winters and concentrated rains from October to March, with greater intensity between the months of December and February (Alvares et al., 2013).
A total of 2 μL of loading buffer (0.02% bromophenol blue, 40% glycerol) was added to 20 μL of each PCR volume after amplification; 17 μL were loaded into 2% agarose gel (dissolved in 1X TBE -89 mmol L -1 Tris, 89 m mol L -1 boric acid, 2.5 m mol L -1 EDTA, pH 8.3) and subjected to horizontal electrophoresis at 200 V, 200 mA, and 100 W for approximately 95 min.The gels were stained in a solution containing ethidium bromide (0.02 μL water) for 60 min for visualization under ultraviolet light.The 1 kb molecular weight marker (Promega Corporation, Madison, WI, USA) was used as the standard molecular-weight size marker.
The results were visualized on Locophy L-pix HE photographic documentation equipment (Loccus Biotecnologia, São Paulo, SP, Brazil).The electrophoretic profile of each SSR primer was transformed into a binary matrix.The presence of a fragment was represented by 1, and the absence of fragment was represented by 0. Binary data were used to perform all subsequent analyses.
To evaluate the information of the microsatellites used, the polymorphism information content (PIC) was calculated from allele frequencies for all accessions.The matrix of genetic similarities in pairs was made with the software GeneAlEx v. 6.41.The resulting matrix was used to generate a principal coordinate analysis (PCoA).A phylogenetic tree was constructed by applying the distance matrix computed with the software Phylogenetic Computer Tools v. 1.3.To perform a neighbor-joining (NJ) analysis the software Phylip v. 3.695 was used.Tree stability was tested with 1000 bootstrap data arrays.
The following analyses were performed on the pulp of the 15 persimmon genotypes: titratable acidity (TA, in percentage of malic acid) was determined by titration with 0.1 N NaOH solution and 1% phenolphthalein as an indicator, with values expressed as the percentage of malic acid, and soluble solids (SS, °Brix) were measured using an Atago digital refractometer (Atago CO., Shiba-koen, Minato-ku, Tokyo, Japan).
The evaluated productive variables of the 15 persimmon genotypes were as follows: number of fruit per plant; fruit fresh mass (g); yield (kg per plant); and estimated average yield (Mg ha -1 ) in two seasons.Fruit Pesq.agropec.bras., Brasília, v.58, e03242, 2023 DOI: 10.1590/S1678-3921.pab2023.v58.03242 were harvested weekly, counted, and weighed using a semi-analytical balance.Total fruit mass was used to determine plant yield.Subsequently, the estimated productivity was attained by multiplying the plant yield by the number of plants per hectare (410 plants ha -1 ).
Data were subjected to the analysis of variance by the F-test, and the experimental design was completely randomized with 15 treatments (genotypes) and six replicates.The means were compared y the Scott-Knott's test, at 5% probability.The analyses were performed using the computer program for analysis of variance Sisvar, version 5.6.(Ferreira, 2014).

Results and Discussion
In the analysis of 17 persimmon genotypes, 141 alleles were identified by 12 SSR polymorphic markers.The allele sizes ranged from 134 to 365 bp.
The PIC results for each marker confirmed their utility for identifying differences among the samples analyzed in the present study (Table 1).Moreover, they ranged from 0.6289 (ssrdk04) to 0.926 (ssrdk03), averaging 0.86755.All 12 markers except for ssrdk04 were highly polymorphic, having a PIC value equal to or higher than 0.78.The PIC values of a locus were associated with the number of detected alleles; for instance, the highest PIC value corresponded to ssrdk03 (18 alleles), and the lowest one corresponded  2013) evaluated the genetic diversity of 48 genotypes for seven species of the Diospyros genus, using 11 SRAP primers, and obtained 303 totally polymorphic fragments that allowed of the identification of all the species and the genotypes into species.Similarly, Naval et al. ( 2010) evaluated 71 persimmon genotypes, using SSR primers, and obtained 206 fragments with 100% polymorphism.A lower percentage (75%) was obtained by Yang et al. (2015), using 14 SSR primers in different species of Diospyros.
The genetic distance values determined by Nei (1972) varied from 0.32 (for 'Trakoukaki' compared with 'Erma Rideo') to 0.67 (for 'Trakoukaki' compared with 'Chocolate').In the present study, the higher distances corresponded to the outgroups D. lotus and D. virginiana with the D. kaki genotypes, and the distance ranged between 0.78 and 0.97 (Table 2).
Nei's index (Nei, 1972) is a genetic diversity identifier ranging from 0 to 1, with 0 indicating no genetic diversity, and 1, showing the maximum genetic diversity (Giustina et al., 2014).The average genetic diversity value found in the present study was 0.41, which suggests low diversity among the analyzed genotypes.The greatest variation found between the Nei's genetic distance values occurred between the D. lotus and D. virginiana outgroups and the D. kaki genotypes.
The PIC value ranges from 0 to 0.25 for markers considered less informative, and it ranges from 0.25 to 0.5 for moderately informative markers, and above 0.5, for markers that show high information content.The PIC value allows of primer classification and indicates the primer efficiency in the detection of polymorphisms (Costa et al., 2015).Thus, in the present study, the primers were very informative (PIC=0.866).Markers that show lower PIC values can also be considered markers of interest, since they can identify genotypes separately.This is an important issue in non-Asian countries, where the introduction of persimmon varieties led to a high number of poorly identified genotypes (Naval et al., 2010).The SSRs used in the present study allowed of the identification of several persimmon genotypes, including 'Paraguai', Based on microsatellite data, the genetic distances among persimmon accessions were used to generate a neighbor-joining cladogram (Figure 1).The cladogram showed two major groups.Group I comprises mainly PCA genotypes ('Pomelo', 'Regina', 'Rubi', 'Trakoukaki' and 'Taubaté').Group II, with 63.5% bootstraps, included mostly PVA type genotypes ('Kakimel', 'Chocolate', 'Mikado', and 'Rojo Brillante'), except for the genotypes 'Kyoto' and 'Vaniglia', both of which are PVNA type genotypes; within this group, the genotypes 'Rojo Brillante' and 'Mikado' grouped together with 56.9% probability.Moreover, the 'Fuyu' PCNA genotype was grouped with a probability of 64.3%, and the genotype 'Paraguai' was isolated from the others.
Microsatellite data were subjected to PCA to obtain an alternative view of the relationships among the accessions (Figure 2).As expected, the results of this analysis agreed with the neighbor-joining cladogram.PC1 accounted for 16.25% of the variability, while PC2 accounted for 13.8% of the variability.The plot of the genotypes in the space of PC1 and PC2 resulted in groups similar to those in the cladogram.
Cluster and principal component analysis grouped the genotypes by astringency type, showing a high level of genetic relatedness.Similar results were obtained by Yonemori et al. (2008a) and Gil-Muñoz et al. (2018).In both studies, the PCNA genotypes grouped together.
However, in another study, Yonemori et al. (2008b) analyzed a large number of persimmon accessions, including Japanese, Korean, and Chinese accessions, using AFLP markers; their findings showed a unique clade of PCNA Japanese genotypes, which suggests an independent evolution, although the authors did not report the bootstrap value that supported the clade groupings.
Naval et al. ( 2010) studied 71 persimmon cultivars, and Liang et al. (2015) analyzed 133 ones.They used SSR markers and obtained similar results, for which PCNA genotypes grouped together.This can be explained by the fact that the PCNA type is a recessive mutation that arose in Japan.The short history of the mutation and its low spread resulted in low genetic variability among genotypes of the same type (Yonemori et al., 2008b).
For the other groups, the results were similar to those found by Naval et al. (2010), Liang et al. (2015) and Gil-Muñoz et al. (2018), in which groups of PCA and PVA types were not separated as clearly as the PCNA type.The selection of characters of interest related to the pomological and chemical properties of fruit, phenology, and yield that are associated with genetic diversity data is important for the selection of promising genotypes.Curi et al. (2017) analyzed the characteristics and environmental effects on persimmon genotypes, in a subtropical region, and determined different parameters for fruit size.We found similar results to those reported by Martínez-Calvo et al. (2013), who reported fruit weights between 150 and 160 g and average diameters ranging from 65 to 70 mm in different persimmon genotypes.
Based on the genetic markers, 'Rama Forte', 'Pomelo', 'Regina', 'Rubi', 'Guiombo', 'Cereja', and 'Taubaté' genotypes, classified as PCA and PVA, formed the group I (Figures 1 and 2).These genotypes were characterized by firmer fruit and uniform and reddish maturation; however their fruit weighed less, hence, these genotypes were less productive than those from group II.Nevertheless, these genotypes show fruit with high contents of soluble solids, total sugar, and phenolics (Table 3).
The groups formed by the cladogram and the principal component analysis suggest a correlation between the genetic characterization and the quality attributes of persimmon fruit, indicating a relationship between the fruit characteristics and the genetic origin of the genotypes.This evidence underscores the importance of characterization at both genetic and pomological levels, for the germplasm management and the selection of promising genotypes for later use in genetic improvement programs.

Conclusions
1.The average genetic diversity value found was 0.41, which suggests low diversity among the analyzed persimmon (Diospyrus kaki) genotypes.
5. The investigation of molecular markers is efficient approach to study of the genetic diversity of Brazilian persimmon genotypes grown in the tropics.

Table 1 .
Summary of microsatellite allele data revealed by 12 microsatellite loci in 17 genotypes of Diospyros kaki.