Genetic diversity of nuts traits and fatty acids of pecan (Carya illinoinensis) germplasm resources


 Pecan (Carya illinoinensis) is the most economically valuable nut tree growing in many countries of the world. 10 nut quantitative traits and 15 fatty acid components of 112 pecan accessions were determined to analyze the morphometric and fatty acids genetic diversity in this study. The measured nuts traits of single nut mass, nut transverse, longitudinal and lateral diameter, nut aspect ratio, single nuts kernel mass, kernel yield and shell thinness were found highly variable. 15 fatty acids were detected among 36 tested fat acids in the nut kernel of pecan, and 14 fatty acids were found high variation except for the C12:0. Plenty of these traits are significant economic importance and could be used as breeding targets to improve the pecan variety. The positive correlations were observed between each pair of single nut mass, nut transverse diameter, nut longitudinal diameter and nut lateral diameter. Single nuts kernel mass is significantly positively correlated with single nut mass, nut transverse diameter, nut longitudinal diameter and nut lateral diameter. The 2D PCA plot successfully grouped the samples according to their phenotypic resemblance and morphological characteristics. 112 accessions were grouped into 4 and 3 major clusters according to the nut quantitative traits and fatty acids components and contents, respectively. Based on these results, we suggest that multidisciplinary research team should be set for genetic breeding of pecan to promote the conservation of local genetic diversity and improve the nuts production and commercialization in China.


Introduction
Pecan (Carya illinoinensis (Wangenh.) K. Koch), which is native from north America and below to Juglandaceae family, is one of the most economically valuable nut trees in the world (Grauke et al., 2016;Poletto et al.,2015). Breeding promoted the development of improved varieties of pecan. Pecan growers have conquered grafting technology and established the rst commercial orchard through grafting 'Centennial' pecans in 1846, improving exciting pecan culture (Taylor, 1905). Plenty of pecan varieties of the world were bred in USA (Bentley et al., 2019). The geographic range of species distribution re ects broad adaptation. Regional constraints factors including short growing seasons, soil acidity-alkalinity, disease pressure associated with increased moisture, and so on, should be considered in the cultivation of pecan. To date, the pecan trees were grown in many climate zones, including subtropical, tropical and temperate regions.
Pecan trees are large, longer lived, outcrossing, highly heterozygous, and slow-to-bear. The trees are monoecious and dichogamy. There are two types, protandrous and protogynous. The population manifests heterodichogamy, to maximize cross pollination and outbreeding between individuals. Previous report showed that dichogamy is controlled by a single locus with simple dominance (Thompson 1985). This pattern of oral biology increases heterozygosity in natural stands (Bentley et al., 2019), thus, pecan germplasm resources have complicated genetic background and have many traits with genetic diversity.
The pecan average breeding cycle need 35 years from initial cross to release due to the long juvenility (Bentley et al., 2019).
The pecan nut oil is rich in unsaturated fatty acids (UFA) and considered as a healthy oil (Villarreal-Lozoya et al., 2007; Ros and Mataix 2006). The content of monounsaturated fatty acids (MUFA) was higher than that of polyunsaturated fatty acids (PUFA) in pecan. Many studies have shown that eating of MUFA could lesser low-density lipoprotein (LDL) cholesterol, guards against coronary heart disease (CHD), controls the blood pressure, and might have valuable effects on in ammation (Sujatha et al., 2001;Alonso et al., 2006). PUFA have antithrombotic and antiatherosclerotic properties (Simopoulos 1991 Pecan has been introduced to China for more than 110 years, and this species has been cultivated in several provinces, including Jiangsu, Zhejiang, Anhui, and so on. Some cultivars were introduced from the United States by breeders in 20th century. And small number of varieties were selected from seedlings. 42 cultivars and 70 advanced breeding selections of pecan with very large range of nuts shapes and sizes were collection and conservation in Nanjing Lvhe District pecan ne variety base of Jiangsu Provincial. Morphological measurements are relatively valuable tools used in systematic analysis and cultivars identi cation (Rubini Pisano et al., 2018; Igbari et al., 2019). 10 nut quantitative traits and 36 fatty acid components of 112 pecan accessions were determined to analyze the morphometric and fatty acids genetic diversity in this study.

Materials And Methods
Materials 42 cultivars and 70 advanced breeding selections of pecan were evaluated in this study (Table 1). Experiments were carried out in 2020 on 9 to 11 -year-old pecan trees trained with standard horticultural practices at the experiment farm of at Nanjing (Nanjing Lvhe District pecan ne variety base of Jiangsu Provincial, Nanjing Green Universe Pecan Science & Technology Co. Ltd) with plant spacing 6 m 8m. The site of the experimental orchard is located at 32°27′N latitude and 118°34′E longitude. Average temperatures were 21 to 29 and 19 to 27 in summer or autumn, respectively. Total annual precipitations were 351mm and 403 mm in summer or autumn, respectively. Soil characteristics were including: Soil Texture=clay loam, pH value=7. Drip irrigation system was performed to guarantee abundant water for pecan growth and development throughout the growing season. The nuts were harvested upto pericarp dehiscence and dried at low temperature (30 oC) in oven for 24 hours for investigating their characters. Evaluation for each species was performed separately based on a randomized complete blocks design (RCBD) with three replications and 30 nuts in each replicate. Nut quantitative traits evaluation The following ten nut quantitative traits were studied in this study: single nut mass (SNM), nut transverse diameter (NTD), nut longitudinal diameter (NLOD), nut lateral diameter (NLAD), nut shape index (NSI), ratio of transverse longitudinal (RTO), single nuts kernel mass (SNKM), kernel yield (KY), shell thinness (ST) and oil yield (OY). Nut shape index= nut longitudinal diameter/ nut transverse diameter, kernel yield (%) = nuts kernel mass/ nut mass x 100%.
Pecan oil was extracted from nuts kernel with Soxhlet extraction method using petroleum ether (Toro-Vazquez et al., 1999;Jabar et al., 2015). 10 g of minced nuts kernel per collections was put separately in a Soxhlet apparatus with 220 ml of petroleum ether. Oil was extracted during 12 hours at 75∘C. Oil was obtained by solvent evaporation in a Rotavapor at 70∘C and then measured. The oil was stored at 4∘C until analyses. Oil yield (%) = solvent free oil/kernel mass x 100%. The oil was stored at 4∘C until analyses.

Fatty acids componenst and content determination
Pecan fatty acids were determination by gas chromatography mass spectrometry (GCMS) using internal standard method (El Riachy et al., 2019). Extracted oil samples (1 mg), which were after saponi cation and methyl esteri cation, was analyzed by GC-MS using an Agilent 7890A, 5975C, using an HP-5MS column (30m×0.25mm, 0.25µm; Agilent). Helium was used as a carrier gas with a ow rate of 1.0 mL/min. The column heat gradient was 60 o C to 200 o C at 8 o C /min, followed by 3 o C /min gradient to 280 o C, which was held for 5 min. A 1 µl injection was used for each sample analyzed by GC-MS. 36 fatty acids were measured in this study (Supplementary Table S1).

Statistical analysis
Data processing, using the below statistical analyses, was performed using the SPSS Statistics version 17. The data were subjected to basic statistics and an ANOVA (analysis of variance) was executed to provide the signi cant differences of characters between accessions. Duncan's multiple range tests were used to compare means for each trait. A correlation analysis was performed to study the relationship between traits. Principal component analysis (PCA) was done to study patterns of variation in a set of interrelated traits through the identi cation of subsets of these traits. Ward's cluster analysis using the squared euclidean distance was performed to classify the genotypes (Crossa and Franco, 2004). Genetic similarity computing and the construction of respective 2D plots were also performed.

Statistics and correlations for the 10 nut quantitative traits variables
The 10 nut quantitative traits of 112 C. illinoinensis germplasm were determined and the descriptive statistics of means, mean standard error, variance, range, maxima, minima, standard deviations and coe cients of variation (CV) are analyzed in this study ( Table 2, Supplementary Table S2). The results showed that some nut traits had high CVs, representing extensive morphological variability. These included single nuts kernel mass (32.75%), single nut mass (28.80%), shell thinness (25.03%). The remaining traits showed comparatively low CV values (<20%). Oil yield and ratio of transverse longitudinal had the lowest CVs of 5.38% and 2.91%, respectively. The ANOVA showed that accessions effects in pecan were signi cant on all nut quantitative traits. Statistically signi cant correlations among all the 10 nut traits were analyzed ( Table 3). The positive correlations were observed between each pair of single nut mass, nut transverse diameter, nut longitudinal diameter and nut lateral diameter. Nut shape index is signi cantly positively correlated with nut longitudinal diameter, but negatively correlated with nut transverse diameter and nut lateral diameter. Single nuts kernel mass is signi cantly positively correlated with single nut mass, nut transverse diameter, nut longitudinal diameter and nut lateral diameter. There is a very signi cant negative correlation between shell thinness and kernel yield (-0.793). Oil yield is signi cantly positively correlated with single nut mass, single nuts kernel mass and kernel yield.  Because the rst two PCs explained 64.5% of the total variation among the cultivars (Table 4), the approximation of the real multivariate diversity of the cultivars on the 2-PC axis is quite acceptable for the most important discriminating (contributing) traits. The scatter plot of the rst two principal components (Fig. 1) shows geometrical distances among the cultivars in the plot that re ect a genetic similarity for the 10 measured nut traits and the relations between the four groups delivered by the cluster analysis. According to phenotypic resemblance and morphological characteristics, the samples were plot grouped. For example, accessions SD08, SD09, SD38, SD50 and Kanza had the smallest nut transverse diameter and lateral diameter, minimum single nut mass and nuts kernel mass. SD58, SD59 and SD60 had the smaller single nut mass, nuts kernel mass and lateral diameter. SD04, SD13, SD14 and Zhongshan 25 had the thickest shell thinness (> 1.0mm) and minimum kernel yield. SD07, Choctaw, Jackson, Kiowa, Success and Waco had the biggest nut lateral diameter and nut transverse diameter, bigger nut fruit weight, higher single nuts kernel mass and medium shell thinness. Mahan, Wichta, Melrose, Nacono, Pownee, Western shelly, Desirable, Mohawk and EI mart had largest nut longitudinal diameter and single nuts kernel mass, biggest nut weight, thinner shell thinness. These results demonstrate that single nut mass, nut transverse diameter, nut longitudinal diameter, nut lateral diameter, single nuts kernel mass, kernel yield and shell thinness are highly positively correlated and as a result, these morphological traits led to the highest loading factors in this PCA analysis. The distribution of the cultivars in Fig. 1 showed that their rich genetic diversity (pro les) for the studied traits.
SD49 had the highest total of fatty acids (713.911 mg/g) and total of unsaturated fatty acids (668.853 mg/g), followed by Pawnee (620.707 mg/g and 568.167 mg/g) and SD64 (587.681 mg/g and 547.813 mg/g). Ratio of unsaturated fatty acids in all Carya illinoinensis germplasm were more than 91%, and the highest was SD45 (99.64%). The maximum of mono-(406.776 mg/g) and polyunsaturated fatty acids (262.077 mg/g) were in SD49, followed by Pawnee (384.170 mg/g) and Oconee (326.585 mg/g) for monounsaturated fatty acids, and by SD42 (244.645 mg/g) and SD64 (239.717 mg/g) for polyunsaturated fatty acids. The maximum of monounsaturated fatty acid ratio was Forkert (80.88%), followed by Sauber (78.24%) and SD04 (77.74%). The ANOVA showed that accessions effects in pecan were signi cant on all detected 15 fatty acids components. The descriptive statistics of means, mean standard error, variance, range, maxima, minima, standard deviations and CV of 15 fatty acids are also analyzed ( Table 5). The results showed that some fatty acids had high CVs, representing extensive fatty acid component variability. These included eicosanoic acid (37.67%), heptadecanoic acid (37.47%), hexadecenoic acid (33.64%), decanoic acid (31.8%) and octadecatrienoic acid (31.65%). The remaining fatty acids showed comparatively low CV values (<30%). Ratio of unsaturated fatty acids had the lowest CV of 0.81%.
A 2D PCA plot was made according to the rst two components (Fig. 2). The samples were plot grouped according to fatty acids content.

Cluster analysis
Two dendrograms were obtained from the Ward's method using the euclidean distance based on the 112 accessions nut quantitative traits (Fig. 3A) and fatty acids content (Fig. 3B). Based on the nut quantitative traits, the 112 accessions were grouped in four distinct groups (Fig. 3A). C1 contained 31 accessions which had larger relatively singer nut mass and higher singer nuts kernel mass. C2 contained 12 accessions which had maximum singer nut mass and singer nut kernel mass, the longer nut transverse and lateral diameter. C3 contained 28 accessions which had minor singer nut mass and singer nut kernel mass, shorter nut transverse and longitudinal diameter. C4 contained 41 accessions which had miner singer nut mass and singer nut kernel mass, shorter nut longitudinal diameter, miner nut shape index, and thicker shell thinness.
Based on the 15 fatty acids contents, the 112 accessions were grouped in three major clusters (Fig. 3B). Cluster D1 included 32 accessions which had higher contents of C16, C18:1, C20:1, total of fatty acids, total of UFA and total of MUFA. Cluster D1 included two accessions which had highest contents of C16, C18:1, C20:1, total of fatty acids, total of UFA and total of MUFA. However, cluster D3 included 78 accessions, which had lower contents of C16, C18:1, C20:1, total of fatty acids, total of UFA and total of MUFA.

Discussion
Nut quantitative traits and fatty acids components and contents of 112 pecan accessions were analyzed to determine patterns of diversity, characterize cultivars, and identify plants with commercial or nutritional potentials. Comparing to the walnuts (Juglans regia L.) ( (Prasad 2003). The fatty acid pro le of the ve nuts (brazil, pecan, pine, pistachio and cashew nuts) were determined by capillary-column GC, and the results showed that 11 fatty acid compositions were detected in pecan nuts and ratio of unsaturated/ saturated fatty acids of pecan (13.54) higher than those in brazil (2.79), pine (6.81), pistachio (9.75) and cashew (3.92) nuts (Ryan et al., 2006). The ratio of unsaturated fatty acids (93.55%) of pecan in our study was similar to that obtained (93.66%) in the percent study (Ryan et al., 2006). 15 fatty acids (Table 5, Supplementary Table S3) were detected among 36 tested fat acids in the nuts kernel of 112 pecan germplasm (Supplementary Table S1), and signi cant variations exist in many fatty acid components except for the C12:0. And the UFA, MUFA, PUFA contents variation coe cients were 14.67, 15.55 and 25.02 respectively, indicating these fatty acids components were genetic variation among different pecan.
Pecan is an exotic species in China, the acreage areas have been continued increase in Jiangsu, Anhui and other provinces of our country. Pecan morphometric and fatty acids genetic diversity were a very large range in this study. New pecan individuals were originated from directed or spontaneous crossing among different cultivars during the past mor than one century cultivating in China. Such new individuals were usually not registered, however, have unique valuable one or more characters, for instance, SD49 had the highest total of fatty acids (713.911 mg/g), total of UFA (668.853 mg/g), MUFA (406.776 mg/g) and PUFA (262.077 mg/g). SD02 (0.45 mm) had the thinnest shell thinness and highest kernel yield (62.25%). SD05 had the highest oil yield (73.82%).
These individuals with excellent characterizations should be conservated and used for pecan development and breeding in future. Multidisciplinary research team should be set for genetic breeding of pecan to promote the conservation of local genetic diversity and the traits neglected, improve the nuts production and commercialization in China.
In conclusion, this study consolidated that pecan present genetic diversity in nut quantitative traits and fatty acids components and contents. Pecan nut kernel contains 15 fatty acids components. The ratio of unsaturated fatty acids was up to 93.55%, and the ration of MUFA was higher than that of PUFA. And the UFA, MUFA, PUFA contents were genetic variation among different pecan. Favorable fatty acids and morphometric characteristics should be considered in pecan breed.

Figure 1
Two dimensional PCA plot based on the rst two components for 10 nut quantitative traits of 112 pecan germplasm.

Figure 2
Two dimensional PCA plot based on the rst two components for 15 fatty acids contents in 112 pecan germplasm.