Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 74(4): 51-58, 2021 Firenze University Press www.fupress.com/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.36253/caryologia-1129 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: Melika Tabasi, Masoud Sheidai, Fahimeh Koohdar, Darab Hassani (2021) A meta-analysis of genetic divergence versus phenotypic plasticity in walnut cultivars (Juglans regia L.). Caryologia 74(4): 51-58. doi: 10.36253/ caryologia-1129 Received: November 06, 2020 Accepted: November 25, 2021 Published: March 08, 2022 Copyright: © 2021 Melika Tabasi, Masoud Sheidai, Fahimeh Koohdar, Darab Has- sani. This is an open access, peer- reviewed article published by Firenze University Press (http://www.fupress. com/caryologia) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All rel- evant data are within the paper and its Supporting Information files. Competing Interests: The Author(s) declare(s) no conflict of interest. A meta-analysis of genetic divergence versus phenotypic plasticity in walnut cultivars (Juglans regia L.) Melika Tabasi1, Masoud Sheidai1,*, Fahimeh Koohdar1, Darab Hassani2 1 Department of Plant Sciences and Biotechnology, Faculty of Life Sciences and Biotech- nology, Shahid Beheshti University, Tehran, Iran 2 Temperate fruits research Center, Horticultural Science Research Institute, Agricultural Research, Education and Extension Organizations, Karaj, Iran *Corresponding author. E-mail: msheidai@sbu.ac.ir Abstract. Persian walnut (Juglans regia L.), is very auspicious plant species of Iran from both economical and food points of views. Both wildly grown as well as cultivated forms of this plant species are scattered in different geographical regions of the country and are a valuable source for edible nut as well as job employment. Scattered published data on genetic diversity of this important plant species are mainly based on different molecular data analyses; therefore a meta-analysis of the same cultivars based on sev- eral different molecular markers including DNA-sequences and multi-locus markers was conducted to provide a detailed insight on genetic structure of walnuts. The results indicated a moderated genetic variability of about 40 percent in the studied cultivars; however these cultivars are genetically differentiated as revealed by Fst and AMOVA. HGT analyses revealed that the cultivars phylogeny differs to some degree by different markers and therefore a heat map was constructed to reveal the cultivars relationships based on combined molecular data. A higher Pst value was obtained compared to that of Fst genetic differentiation, therefore, it seems that local adaptation and selection have played role in the walnut cultivars’ morphological divergence. LFMM analysis identified some adaptive multi-locus alleles in the studied walnut cultivars. Keyword: adaptive alleles, HGT, LFMM, Persian walnut, Pst. INTRODUCTION Persian walnut (Juglans regia L.), is one of the main economically cultivat- ed crop plants in Iran. We have a rich walnut germplasm with extensive culti- var genetic variability (Tabasi et al. 2020). Both wildly grown as well as culti- vated forms of this valuable plant species are scattered in different geographical regions of the country. Now and then we encounter new and limited reports on genetic features some of these populations by using different molecular markers (See for example, Maghsoodi et al. 2018; Tabasi et al. 2020). We therefore carried out the present meta-analysis study based on a set of Persian walnut cultivars which were used in previous investigation to pro- 52 Melika Tabasi et al. vide data for: 1- How much genetic diversity is present in these cultivars based on combined data, 2-Determine the phylogenetic relationship of these cultivars based on combined molecular data, 3-Provide data on phenotypic plasticity of these cultivars. A meta-analysis is a statistical analysis that com- bines the results of multiple scientific studies address- ing the same question. Each individual study reporting measurements that are expected to have some degree of error, and therefore, The aim is to derive a pooled estimate closest to the unknown common truth based on how this error is perceived (Nakaoka et al. 2009). Meta-analysis is mainly a method for systematically combining pertinent qualitative and quantitative data from several selected studies to develop a single con- clusion that has greater statistical power. This conclu- sion is statistically stronger than the analysis of any single study, due to increased numbers of subjects, greater diversit y a mong subjects, or accumu lated effects and results (Greenland et al. 2008; Walker et al. 2008). Meta-analysis would be used for the follow- ing purposes: To establish statistical significance with studies that have conflicting results, to develop a more correct estimate of effect magnitude, to provide a more complex analysis of harms, safety data, and benefits (Nakaoka et al. 2009). MATERIAL AND METHODS Plant materials Morphological, ISSR, IRAP, REMAP, SCoT, trnH- psbA and ITS data of 6 populations used (Table 1) are collected according to following studies: Maghsoodi et al. 2018; Aghaei et al. 2019 in press; Tabasi et al. 2020. Data analyses Genetic analyses Detrented correspondance analysis (DCA) was per- formed to study the suitability of molecular markers for population genetic study as performed in PAST version 2.17 (Podani 2000). Genetic distance and Fst values were obtained from published data and also determined by us for DNA sequences. We used DNA sequence compari- son non-parametric Kruskal–Wallis test as performed in PAST, and also by DNAsp program (Rozas et al. 2019). Horizontal gene transfer (HGT) was used to reveal the phylogenetic trees discordance as performed in TREX online (Boc et al. 2012). Similarly, the Mantel test was used to indicate association between genetic distance and geographical distance in the studied culti- vars. PAST program was used for this purpose. A heat map was constructed based on combined genetic data by R-Package. 4.2. We used LFMM program (Frichot. et al. 2013) to identify multi-locus alleles with potential adaptive value. This program includes an integrated framework based on population genetics, ecological modeling and statisti- cal techniques using latent factor mixed model. Quantitative morphological characters For Pst (Phenotypic plasticity) analysis, we used four quantitative morphological characters in Persian wal- nut cultivars. These characters are: 1-Nut length (mm), 2-Nut diameter (mm), 3-Nut length/diameter ratio, and, 4-Thickness of shell (mm). Pst values were determined by R-Package 4.2. Table 1. Juglans regia populations. No Type Name of population Province Locality Longitude Latitude Altitude 1 Wild Nahavand Hamadan Nahavand 23°48’ 23°48’ 1627 2 Cultivated Soozani Markazi Tafresh 59°49’ 42°34’ 1838 3 Cultivated Basloghi Markazi Tafresh 59°49’ 42°34’ 1837 4 Wild Astara Gilan Astara 52°48’ 20°38’ -26 5 Cultivated Kaghazi Markazi Tafresh 59°49’ 42°34’ 1838 6 Wild Khoy West Azarbaijan Khoy 57°44’ 33°38’ 1135 53A meta-analysis of genetic divergence versus phenotypic plasticity in walnut cultivars (Juglans regia L.) RESULTS Genetic divergence DCA (Dentrented correspondance analysis) plot (Fig. 1) of combined molecular markers (ISSR, IRAP, REMAP, SCot, cp-DNA, and ITS data), produced a well scattered diagram, indicating that these molecular markers in combination can differentiate walnut popula- tions well-enough. In Maghsoodi et al. (2018) study on walnut popula- tions, the mean Nei’ genetic distance for ISSR data was 0.3, while the mean genetic distance based on chloro- plast-DNA as well as nuclear ITS (Maghsoodi et al. 2018) sequence analysis were 0.1. The Fst analysis for all genetic markers produced a significant difference among walnut cultivars (P<0.05). For example, the Kruskal-Wallis test for equal medi- ans in nuclear ITS DNA sequences as well as chloro- plast DNA, produced a significant difference. (P = 0.03, and P<0.001, respectively). Similarly, dnaSP population’ genetic differentiation test produced significant Fst value for most of the pair-wise population compared (See for example Table 2). The Mantel test produced significant correlation (r = 0.14, P = 0.04) between genetic distance and geographi- cal distance of the studied populations. These results indicate that an extensive genetic changes have occurred during walnut cultivar divergence. Cultivar phylogeny Cultivar grouping obtained by different molecular markers differed in some degree from each other. The HGT (Horizontal gene transfer) analysis between chloro- plast DNA and combined multi locus data (ISSR, REM- AP, and SCoT), revealed in total two HGT events due to phylogenetic dis-agreement (Fig. 2). Similarly, HGT analysis revealed that three HGT events occurred between cp-DNA and nuclear ITS-DNA sequences (Fig. 3). The Mantel test performed among chloroplast and nuclear ITS-DNA sequences as well as combined multi- locus molecular markers, after 10000 permutation, did not produce significant association between phylogenetic trees obtained from these (Partial correlation r: 0.50, P = 0.14). Moreover, Robinson and Foulds dis- tance between phylogenetic trees produced RF =6. These results are in agreement with HGT results, and indicate that phylogenetic relationship of walnut cultivars based on different molecular markers differ to some extent, and care should be taken for drawing concrete conclu- sions in these types of investigations. Figue 1. DCA plot of walnut cultivars based on combined sequence and multi-locus data. 54 Melika Tabasi et al. A phylogenetic heat map constructed based on com- bined molecular data (Fig. 4), revealed that the cultivar 1 is genetically stands far from the others due to genetic difference. Genetic diversity and local adaptation The LFMM did not produce a significant associa- tion between IRAP and REMAP loci with geographical distribution of the studied walnut cultivars. However, a significant association was obtained between three ISSR loci as well as two SCoT loci with geographical features (Longitude, latitude and altitude) in walnut populations (Table 3, Fig. 5). Phenotypic plasticity versus genetic divergence We obtained a significant correlation (P<0.01) between morphological characters studied (Table 4). Moreover, ANOVA revealed that the studied cultivars differ significantly in these morphological features (P = 0.01). The Pst values obtained for the studied quantita- tive morphological characters were much higher than genetic Fst value (Mean = 0.25). For example, rep- resentative Pst values for the nut height and width are provided in Tables 4 and 5. The higher Pst values compared to that of Fst indicate that the studied mor- phological characters diverged among walnut cultivars under influence of either local environmental condi- tions, or due to cultivating and selection practice avail- able in the region (Table 6). Table 2. Pair-wise Fst value for ITS DNA sequences among walnut cultivars (the number of cultivars are according to Table1). 1 2 3 4 5 6 1 — 2 0.47 — 3 0.05 0.29 — 4 0.05 0.29 1 — 5 0.24 0.30 0.43 0.43 — 6 0.04 0.08 0.09 0.09-0 0.04 — Figure 2. HGT between cp DNA and combined multi locus mark- ers in walnut cultivars. Figure 3. HGT tree between chloroplast and ITS-DNA sequences in walnut cultivars. Figure 4. Heat map of walnut cultivars based on combined molecu- lar data. 55A meta-analysis of genetic divergence versus phenotypic plasticity in walnut cultivars (Juglans regia L.) DISCUSSION Genetic diversity and cultivars phylogeny We obtained almost close value for genetic diver- sity of Persian walnut cultivars by both DNA-sequences analysis as well as multi-locus molecular markers data. A significant genetic difference observed in both kinds of data indicates genetic differentiation of the studied cultivars which can be utilized in future breeding and hybridization projects. Detailed data on genetic structure and phylogenetic relationship of economically crop plants like the Persian walnut is of immediate importance for genetic conser- vation and breeding programs. Moreover, knowledge about the effect of environmental/geographical features on genetic diversity as well as agronomic features of crop plants can improve above mentioned tasks. Meta-analysis is an approach which either increase the number of observation in a particular study, or com- bine scattered data from several sources on a similar subject. Both cases can improve insight about a particu- lar problem or task (Heidari et al. 2018). The present study revealed that a combined data analy- sis based on different molecular markers may improve our insight on both genetic structure as well as phylogenetic relationship of important crop plants like Persian walnut. We also noticed that walnut cultivar agronomic features may be affected by both artificial as well as natural selec- tion. A proper QTL (Quantitative Trait Locus) study can also reveal association of important agronomic characters. The present study identified some of multi-locus molecular markers are either adaptive or are the genomic sites in the vicinity of adaptive genes. This is an impor- tant finding for QTL investigation of Persian walnut. Meta-analysis concerned with plant genetic studies have been performed with regard to genetic correlations between plant resistances to multiple natural enemies (Leimu et al. 2006). These studies are considered impor- tant to determine the mode of selection that natural ene- mies impose on a host plant, the structure of herbivore and pathogen communities, all of which determine the success of plant breeding for resistance to multiple dis- eases and pests (Leimu et al. 2006). Similarly, a meta- analysis was performed to understand the genetic con- trol of flavor in tomato cultivars (Zhao et al. 2019). These authors used data of genome-wide association studies (GWAS) using 775 tomato accessions and 2,316,117 SNPs from three GWAS panels and reported several signifi- cant associations for the contents of sugars, acids, amino acids, and flavor-related volatiles. They also concluded that fruit citrate and malate contents were affected by selection during domestication and improvement. Table 3. LFMM results for ISSR data in walnut populations. ISSR loci Zscore -log10(p-value) p-value SNP_1 0.215472 0.0812363 0.829399 SNP_2 1.07686 0.550458 0.281541 SNP_3 0.257655 0.0987196 0.796673 SNP_4 0.640301 0.282349 0.521977 SNP_5 0.090856 0.032636 0.927607 SNP_6 1.69642 1.04669 0.0898066 SNP_7 0.309536 0.120954 0.756914 SNP_8 3.24327 2.92751 0.001181 SNP_9 0.369296 0.147577 0.711907 SNP_10 0.014174 0.00493936 0.988691 SNP_11 0.00968666 0.00336958 0.992271 SNP_12 0.651046 0.288179 0.515017 SNP_13 0.162106 0.0598711 0.871222 SNP_14 0.502927 0.211114 0.615015 SNP_15 0.538672 0.229065 0.590113 SNP_16 3.17389 2.82271 0.0015041 SNP_17 0.551459 0.235586 0.581319 SNP_18 2.34492 1.72054 0.019031 SNP_19 0.63401 0.278953 0.526074 SNP_20 0.477576 0.198629 0.632952 SNP_21 2.72343 2.18971 0.00646084 SNP_22 0.498368 0.208854 0.618224 SNP_23 0.383171 0.153915 0.701593 SNP_24 0.590575 0.255859 0.554805 SNP_25 0.798828 0.372235 0.42439 SNP_26 0.449796 0.185181 0.652858 SNP_27 0.083641 0.0299592 0.933342 SNP_28 1.82734 1.16974 0.0676484 SNP_29 0.838597 0.396103 0.401695 SNP_30 0.0258046 0.00903406 0.979413 SNP_31 1.89246 1.23337 0.0584291 SNP_32 1.44793 0.830805 0.147637 SNP_33 1.45811 0.839203 0.144809 SNP_34 1.38228 0.777582 0.166885 SNP_35 0.0207987 0.00726703 0.983406 SNP_36 0.451128 0.185821 0.651898 SNP_37 1.03731 0.523472 0.299591 SNP_38 1.55968 0.925052 0.118836 SNP_39 0.576949 0.248741 0.563974 SNP_40 1.16006 0.609022 0.246024 SNP_41 0.0331206 0.0116291 0.973578 SNP_42 1.06123 0.539726 0.288585 SNP_43 1.23962 0.667327 0.215116 SNP_44 0.928131 0.451808 0.35334 SNP_45 0.906725 0.43824 0.364552 SNP_46 0.908105 0.43911 0.363823 SNP_47 0.643651 0.284162 0.519802 56 Melika Tabasi et al. In a meta-analysis with respect to plant crop culti- vars genetic diversity (Van de Wouw et al. 2010), it was concluded that in the long run no substantial reduction in the regional diversity of crop varieties which were released by plant breeders has taken place, and a gradual narrowing of the genetic base of the varieties was not observed. Genetic versus phenotypic differentiation We obtained a higher value for Pst versus Fst, in quantitative morphological characters of fruit among representative walnut cultivars studied. The Pst is taken as index for morphological local adaptation under the influence of natural selection imposed by environment (Brommer 2011). When Pst = FST, it is believed that morphological divergence is due to genetic drift; but while Pst > Fst, it indicates the role of directional selec- tion among the studied populations. However, a lower Figure 5. LFMM Manhattan plots showing three ISSR loci. (A) and two SCoT loci (B), with -log10(p) >2, which indicate association with geographical features in walnut populations. Table 4. Pearson’ coefficient of correlation among morphological characters studied. (Characters A-D are: The nut height, nut width, ration of nut height/nut width, and the nut diameter. All values are in mm). A B C D A — 2.5062E-05 0.026767 0.31372 B 0.77027 — 1.0055E-10 0.021356 C -0.53937 -0.92392 — 0.17431 D -0.38474 -0.55061 0.42827 — Bellow diagonal = r value, above diagonal = P value. Table 5. Pst values for the nut height among studied walnut culti- vars. (The mean Fst value = 0.25). 1 2 3 4 5 6 7 8 9 1 -- 2 0.05 — 3 0.88 0.70 — 4 0.73 0.40 0.70 — 5 0.88 0.70 0.04 0.71 — 6 0.84 0.65 0.07 0.56 0.16 — Table 6. Pst values for the nut width (mm) among studied walnut cultivars. (The mean Fst value = 0.25). 1 2 3 4 5 6 7 8 9 1 -- 2 0.18 — 3 0.77 0.77 — 4 0.76 0.74 0.20 — 5 0.89 0.87 0.46 0.73 — 6 0.90 0.86 0.17 0.73 0.38 — 57A meta-analysis of genetic divergence versus phenotypic plasticity in walnut cultivars (Juglans regia L.) value of Pst in contrast to Fst, indicates that the same phenotypes are favored in different populations due to stabilizing selection. We may therefore, conclude that, due to some local environmental conditions/geographi- cal coordinates, or local practices of cultivation or selec- tion, some adaptive changes have occurred in walnut cultivars. Similar studies have shown that morphological and agronomical traits divergence have been occurred in many taxa (See for example, Leinonen et al. 2013; Sto- janova et al. 2018; Caré et al. 2018). Stojanova et al. (2018), found an adaptive differentia- tion in phenotypic traits across the climatic gradient in different populations of Festuca rubber. Similarly, Caré et al. (2018), reported morphological differentiation in crown architecture in geographical populations of Ger- man Norway spruce. They reported a high Pst value (0.952–0.989) between the neighboring autochthonous and allochthonous stands of similar age in contrast to a very low neutral genetic differentiation (Fst = 0.002– 0.007; G”st = 0.002–0.030) probably due to the effect of directional selection on adaptive gene loci involved in phenotypic differentiation. In conclusion, the meta-analysis based on published data on different molecular markers concerned with the same Persian walnut cultivars, revealed that a combined analysis of molecular data matrix (including chloroplast and nuclear DNA, as well as multi-locus markers, like ISSR, IRAP, REMAP, and SCoT markers), produce more accurate and significantly improved result for genetic diversity analysis as well as finding the cultivars’ phylo- genetic relationship. 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