Physicochemical Characterization and Fatty Acid Profiling of Different Philippine Pili Nut (Canarium ovatum,Engl.) Varieties

Canarium ovatum, commonly known as Pili nut, is an endemic crop in the Philippines. This study focused on the assessment of the physical, nutritional, and fatty acid profile of the pulp and kernel of Pili nut varieties. The study included 7 varieties that are cultivated in a single soil condition at the Albay Research and Development Center, Buang, Tabaco City, Philippines. Standard method Association of Official Analytical Chemist for nutritional analysis and DNA barcoding were used in the study. Molecular authentication through DNA barcoding shows that studied Pili nut varieties belong to genus C. ovatum. Split component of the fruit reveals that pulp is the major component (12.6–25.3%) of the fruit, followed by shell (5.97–12.3%), kernel (1.75–3.49%), and testa (0.17–0.34%). Lyophilized Pili nut kernel contains 67.2–74.1% fat, 11.5–13.2% protein, 3.43–9.97% dietary fiber, and 2.93–3.37% ash, and provides 704–749 cal 100 g^?1 energy. Pili nut pulp contains 43.4–53.1% dietary fiber, 20.2–31.7% fat, 8.32–10.9% ash, and 4.53–6.32% protein, and provides 361–432 cal 100 g^?1 energy. Pili nut pulp is an excellent source of dietary fiber. Prominent fatty acid is oleic acid (C18:1) for all varieties, which is higher in the pulp than in the kernel. Other major fatty acids include palmitic (C16:0), linoleic (C18:2), stearic (C18:0), and linolenic (C18:3) acids. Most of the physical and chemical characteristics measured depend significantly on the variety.     

Fatty acid composition of seed oil triglycerides inCoincya (Brassicaceae)

Oil and triglyceride contents and fatty acid composition were determined for seeds in nine taxa belonging to the genusCoincya (Brassicaceae) on the Iberian Peninsula (Spain and Portugal). The oil content ranges from 11.1 to 24.6%, triglycerides from 68.7 to 88.5%. The major fatty acids were erucic (24.6–30.5%), linolenic (17.7–27.7%), linoleic (13.9–24.6%) and oleic acid (12.3–21.8%).     

Fatty acid and triacylglycerol compositions of seed oils of five Amaranthus accessions and their comparison to other oils

This paper reports the fatty acid and triacylglycerol (TAG) compositions of five Amaranthus accessions (RRC1011, R149, A.K343, A.K432, and A. K433) representing two species and a cross between one of these and a third species. Seed oils of these were analyzed by gas chromatography and reversed-phase high-performance liquid chromatography, and their compositional properties compared with buck-wheat (Fagopyrum esculentum), corn (Zea mays), rice bran (Oryza sativa), soybean (Glycine max L. Merr.), sesame (Sesamum indicum), quinoa (Chenopodium quinoa), and cottonseed (Gossypium hirsutum) oils. All Amaranthus accessions were relatively high in palmitic (21.4–23.8%) and low in oleic (22.8–31.5%) and linolenic (0.65–0.93%) acids when compared to most of the grain and seed oils. The fatty acid composition of Amaranthus accessions K343, K433, and K432 (group I) were different from R149 and RRC1011 (group II) in mono and polyunsaturated fatty acids, but the saturate/unsaturate (S/U) ratios were very similar. All Amaranthus accessions were similar in TAG type, but showed slight differences in percentage. High similarities in UUU, UUS, and USS composition were observed among Amaranthus K343, K433 and K432, and between R149 and RRC1011. The fatty acid compositions of Amaranthus oil (group I) and cottonseed oil were similar, but their TAG compositions were different. The grain and oilseed oils were different from each other and from the Amaranthus accessions oils in terms of fatty acid composition, S/U, and TAG ratios. The UUU, UUS, and USS percentages were very diverse in grain and seed oils. The percentages of squalene in the TAG sample from the Amaranthus accessions were 8.05% in K343, 11.10% in K433, 11.19% in K432, 9.96% in R149, and 9.16% in RRC1011. Squalene was also tentatively identified in quinoa and ricebran oils at levels of 3.39 and 3.10%, respectively.     

Kernel fatty acid and triacylglycerol composition for three almond cultivars during maturation

The kernel oil content, kernel FA and TAG composition, kernel moisture content, and kernel weight as well as fruit weight of three almond cultivars (Achaak, Mazetto, and Perlees) were monitored during the maturation of kernels. Lipid fractions of all almond samples were extracted using a mixture of chloroform and methanol. FAMF and TAG contained in these fractions were analyzed by GC and HPLC, respectively. The ratio of kernel to fruit weight appears to be a good indicator of almond kernel development. The total lipid content of developing almond kernels exhibited a sigmoidal pattern with time, similar to seeds and kernels of other higher plants; the cultivar Achaak showed a higher rate of lipid accumulation. The proportion of eleic acid (0) dominated at the later stage of maturation for all three almond cultivars. Although there was no significant difference in the FA composition for the three cultivars studied, marked differences were observed in their TAG profiles. Ten TAG species identified were LLL, LLO, LnOO, LOO, LOP, PLP, OOO, POO, POP, and SOO, where L represents linoleic acid; Ln, linolenic acid; P, palmitic acid; and S, stearic acid. The difference in the TAG profile can be useful for distinguishing various cultivars. The oil of Mazetto cultivar kernes exhibited a TAG composition comparable to that of olive oil.     

Fatty acid composition, extraction, fractionation, and stabilization of bullfrog (Rana catesbeiana) oil

The oil extracted from the fat-storage organ (fat body) of the bullfrog (Rana catesbeiana) was characterized for its fatty acid composition. The main fatty acids were palmitic (18.1%), stearic (4.1%), myristic (2.7%), oleic (31.7%), and linoleic (12.9%) acids. Long-chain polyunsaturated fatty acids were also present in significant amounts, i.e., eicosapentaenoic (1.5%) and docosahexaenoic (4.7%), and were probably derived from the fish meal content of the diet. A partially fractionated oil was extracted from the homogenized and frozen fat body with an oleic acid content of 43.2%. The natural alkaloid boldine, added at 0.5 mg/g oil level, improved the oxidative stability by a factor ranging from 1.7 to 2.4, as assessed by the Oil Stability Index method between 90 and 110°C. The stabilization effect of boldine was higher than that of naringenin, morin, and quercitin and for the synthetic antioxidant butylated hydroxytoluene at the same concentration level.     

Fatty acid composition and its relationship with physicochemical properties of pecan (Carya illinoensis) oil

The composition and physicochemical properties of pecan (Carya illinoensis) kernels and oils from different native trees of the central region of Mexico were investigated. The main compositional characteristic of the kernel was the high lipid content (70–79% w/w on dry basis) with elevated concentration of oleic acid (55–75% w/w). The results confirmed the relationship in the biosynthesis of linoleic and linolenic acids from oleic acid existing in oilseeds. Our results indicate that in pecans such relationship is a function of pecan tree age. The proportion of oleic, linoleic, and linolenic fatty acids determined the oxidative stability, viscosity, and melting/crystallization behavior of pecan oil. In general, these properties in pecan oils were similar or superior to extra-virgin olive oil and unrefined sesame oil. Although all native pecan oils studied showed a significant concentration of oleic acid, a particular group of native Mexican pecan trees produces an oil with a fatty acid composition with the nutritional appeal that consumers demand nowadays (i.e., very high oleic acid, 60–75%), with excellent natural oxidative stability (i.e., induction time for oxidation between 8.5 and 10.8 h), and substantially higher concentrations of α-, γ-, and δ-tocopherol than in pecan varieties previously reported in the literature.     

Positional analysis and determination of triacylglycerol structure ofArgania spinosa seed oil

The distribution of fatty acids between the sn-1, sn-2 and sn-3 positions of triacylglycerols fromArgania spinosa seed oil of Morocco has been determined. Saturated fatty acids showed a preference for external positions. The sn-1 position contained slightly more palmitic acid than the sn-3 position, whereas stearic acid was preferentially esterified at the sn-3 position. Linoleic acid occurred predominantly in the sn-2 position with lesser amount evenly distributed between the sn-1 and the sn-3 positions, as generally found in vegetable oils. Oleic acid was distributed with a slight preference shown for the internal position, whereas the distribution between the external positions revealed a slight preference for the sn-1 position. The distribution of the triacylglycerols determined from high-performance liquid chromatography (HPLC) is at variance with that calculated from the 1-random 2-random 3-random distribution theory. This is particularly true for trioleoyl and trilinoleoylglycerols. In contrast, the agreement between theory and experiment is good for triacylglycerols containing two oleoyl and one linoleoyl chains, one oleoyl, one linoleoyl and one palmitoyl chains or one oleoyl, one palmitoyl, and one stearoyl chains.     

Canarium schweinfurthii Engl.: Chemical composition of the fruit pulp

Canarium schweinfurthii Engl. (Burseraceae) is a wild tree found mostly in Africa, which produces fruit similar to olives and which is barely used. On a dry matter basis, the fruit pulp from Côte d’Ivoire (the Ivory Coast) was found to contain 5.6% protein, 30–50% fat, 8.2% starch, 11.8% cellulose and 8.3% ash (the highest mineral elements being potassium, 1.2% and calcium, 0.4%). The melting and solidification points of the extracted fat (44.5°C and 35.2°C, respectively) are higher than those of all the commercial and otherCanarium-species oils. This oil shows low iodine, peroxide and carotene values (36, 17 meq-g and 2 mg, respectively). The fatty acid composition of the oil revealed a high content of oleic (89.4%) or stearic (67.7–84.0%) acids in the liquid, semi-solid and solid forms of the oil. Consequently, the content of these two acids is much higher inCanarium schweinfurthii oil than in any other vegetable oil. The three forms (liquid, semi-liquid and solid) of the oil depend on the maturity of the fruit and these stages will be investigated further in future work.     

Fatty acid composition of some pine seed oils

The fatty acid composition of seeds from seven species of the genusPinus (P. pinaster, P. griffithii, P. pinea, P. koraiensis, P. sylvestris, P. mughus, andP. nigra) was established. Pine seeds are rich in oil (31–68% by weight) and contain several unusual polymethylene-interrupted unsaturated fatty acids with acis-5 ethylenic bond. These are thecis-5,cis-9 18:2,cis-5,cis-9,cis-12 18:3,cis-5,cis-11 20:2, andcis-5,cis-11,cis-14 20:3 acids, with a trace ofcis-5,cis-9,cis-12,cis-15 18:4 acid. Their percentage relative to total fatty acids varies from a low of 3.1% (P. pinea) to a high of 30.3% (P. sylvestris), depending on the species. The majorcis-5 double bond-containing acid is generally thecis-5,cis-9,cis-12 18:3 acid (pinolenic acid). In all species, linoleic acid represents approximately one-half the total fatty acids, whereas the content of oleic acid varies in the range 14–36% inversely to the sum of fatty acids containing acis-5 ethylenic bond. The easily available seeds fromP. koraiensis appear to be a good source of pinolenic acid: their oil content isca. 65%, and pinolenic represents about 15% of total fatty acids. These values appear to be rather constant.Pinus pinaster, which is grown on several thousand acres in the southwest of France, is an interesting source ofcis-5,cis-11,cis-14 20:3 acid (7% in the oil, which isca. 35% of the dehulled seed weight), an acid sharing in common three double bonds with arachidonic acid. Apparently,P. sylvestris seed oil contains the highest level ofcis-5 double bond-containing acids among pine seed oils that have ever been analyzed.     

13C nuclear magnetic resonance spectroscopic analysis of the triacylglycerol composition ofBiota orientalis and carrot seed oil

¹³C nuclear magnetic resonance (NMR) spectroscopic analysis of the whole oil (triacylglycerols) ofBiota orientalis seeds confirms the presence of oleate [18:1(9Z)], linoleate [18:2(9Z, 12Z)], linolenate [18:3((9Z, 12Z, 15Z)], 20:3 (5Z, 11Z, 14Z), 20:4(5Z, 11Z, 14Z, 17Z), and saturated fatty acids in the acyl groups by comparing the observed carbon shifts with previously established shift data for model triacylglycerols. This technique shows that the saturated, 20:3 and 20:4 fatty acids are distributed mainly in the α-acyl positions, whereas oleate, linoleate, and linolenate are randomly acylated to the α- and β-positions of the glycerol “backbone”. Stereospecific hydrolysis of theBiota oil with pancreatic lipase, followed by chromatographic analysis of fatty esters, reveals the presence of trace amounts of 16:0(0.7%), 18:0(0.5%), 20:3 (0.4%), and 20:4 (1.3%) in the β-position of the glycerol “backbone”, which are undetectable by¹³C NMR technique on the whole oil. Semiquantitative assessment of the¹³C NMR signal intensities gives the relative percentages of the fatty acid distribution as: saturated 16:0, 18:0 (12.0% α-acyl), oleate (7.7% α-acyl 8.7% β-acyl), total linoleate and linolenate (31.7% α-acyl; 24.2% βacyl), total 20:3 and 20:4 (15.7% α-acyl). The¹³C NMR spectroscopic analysis of carrot seed oil identifies the presence of saturated (18:0), 18:1(6Z), 18:1(9Z), and 18:2(9Z, 12Z). The saturated fatty acid is found in the α-acyl positions. Semi-quantitative assessment of the signal intensities gives the relative percentages of the fatty acids as: 18:0 (4.5% α-acyl), 18:1(6Z) (49.6% α-acyl; 19.7% β-acyl), oleate (6.5% α-acyl; 8.6% β-acyl) and linoleate (5.2% α-acyl; 6.9% β-acyl).     

Effect of fatty acid positional distribution and triacylglycerol composition on lipid by-products formation during heat treatment: II. Trans isomers

This study examined the effect of the fatty acid positional distribution and of the triacylglycerol (TG) composition on heat-induced trans isomerization of linoleic and linolenic acids. For this, we synthesized diacid TG molecules that were acylated only with linoleic acid (L) or with linolenic acid (Ln) along with palmitic acid (P). The fatty acid of interest was positioned either in the central position (PLP and PLnP, respectively) or in one of the two outer positions (PPL and PPLn, respectively). Monoacid TG, i.e., trilinolein and trilinolenin, were also synthesized and mixed with tripalmitin in a 1:2 ratio. This model TG was also compared to another TG model, which consisted of a canola oil and its randomized counterpart whose fatty acid positional distribution and TG composition were determined by means of high-performance liquid chromatography. After heating, the content of trans isomers was determined by gas-liquid chromatography with a polar capillary column. In model TG, polyunsaturated fatty acids in monoacid TG (LLL and LnLnLn) exhibited the highest degree of isomerization, compared to diacid TG, and this effect was greatest at 220°C. At this temperature, an effect of the TG structure was observed only with linolenic acid. In that situation, 18:3n-3 acylated in the central position of the TG molecule (PLnP) displayed the highest sensitivity to trans geometrical isomerization. Although to a lesser extent, the same trends as for the pure TG model were observed with the canola oil model with regard to the influence of the fatty acid positional distribution and TG molecular species.     

Effect of fatty acid positional distribution and triacylglycerol composition on lipid by-products formation during heat treatment: II. Trans isomers

This study examined the effect of the fatty acid positional distribution and of the triacylglycerol (TG) composition on heat-induced trans isomerization of linoleic and linolenic acids. For this, we synthesized diacid TG molecules that were acylated only with linoleic acid (L) or with linolenic acid (Ln) along with palmitic acid (P). The fatty acid of interest was positioned either in the central position (PLP and PLnP, respectively) or in one of the two outer positions (PPL and PPLn, respectively). Monoacid TG, i.e., trilinolein and trilinolenin, were also synthesized and mixed with tripalmitin in a 1:2 ratio. This model TG was also compared to another TG model, which consisted of a canola oil and its randomized counterpart whose fatty acid positional distribution and TG composition were determined by means of high-performance liquid chromatography. After heating, the content of trans isomers was determined by gas-liquid chromatography with a polar capillary column. In model TG, polyunsaturated fatty acids in monoacid TG (LLL and LnLnLn) exhibited the highest degree of isomerization, compared to diacid TG, and this effect was greatest at 220°C. At this temperature, an effect of the TG structure was observed only with linolenic acid. In that situation, 18:3n-3 acylated in the central position of the TG molecule (PLnP) displayed the highest sensitivity to trans geometrical isomerization. Although to a lesser extent, the same trends as for the pure TG model were observed with the canola oil model with regard to the influence of the fatty acid positional distribution and TG molecular species.     

Chemical composition and physicochemical and hydrogenation characteristics of high-palmitic acid solin (low-linolenic acid flaxseed) oil

The physicochemical characteristics and FA compositions were determined for refined-bleached-deodorized (RBD) high-palmitic acid solin (HPS) oil, RBD solin oil, and degummed linseed oil. The predominant FA in HPS oil were palmitic (16.6%), palmitoleic (1.4%), stearic (2.5%), oleic (11.3%), linoleic (63.7%), and linolenic (3.4%). HPS oil was substantially higher in palmitic acid than either solin oil or linseed oil, and similar to solin oil in linolenic acid content. HPS, solin, and linseed oils exhibited similar sterol and tocopherol profiles. The physicochemical characteristics of the three oils (iodine value, saponification value, m.p., density, specific gravity, viscosity, PV, FFA content, color) reflected their FA profiles and degree of refinement. During hydrogenation of HPS oil, the proportion of saturated FA (palmitic and stearic) increased, and that of unsaturated FA (oleic, linoleic, and linolenic) decreased as the iodine value declined. This resulted in an inverse linear relationship between m.p. and iodine value. Hydrogenation also generated trans FA. The proportion of trans FA was inversely related to iodine value in partially hydrogenated samples. Fully hydrogenated HPS oil (i.e., HPS stearine, iodine value <5) was devoid of trans FA.     

Effect of rosehip extraction process on oil and defatted meal physicochemical properties

The physicochemical properties of oil from Rosa affinis rubiginosa seeds were analyzed after extraction by (i) organic solvent, (ii) cold pressing, and (iii) cold pressing assisted by enzymatic pretreatment using a mixture of the Novo-Nordisk A/S products Cellubrix (cellulase and hemicellulase activities) and Olivex (pectinase, cellulase, and hemicellulase activities). There were no significant differences in oil quality parameters, such as iodine value, refractive index, saponification value, unsaponifiable matter, and FA profile, when applying any of the three extraction processes. Although significant variations were observed in FFA content (acid value) and PV of the oil obtained by both of the cold-pressing oil extraction processes, these results were lower than the maximum value established from the Codex Alimentarius Commission. All-trans-retinoic acid content improved by 700% in rosehip oil obtained through cold pressing, with and without enzymatic pretreatment, in comparison with organic solvent extraction. This result is quite important for cosmetic oil because all-trans-retinoic acid is the main bioactive component responsible for the regenerative properties of this oil.     

Fatty acid selectivity of lipases: γ-linolenic acid from borage oil

The γ-linolenic acid (Z,Z,Z-6,9,12-octadecatrienoic acid, GLA) present in borage oil free fatty acids was concentrated in esterification reactions that were catalyzed by several preparations of the acyl-specific lipase ofGeotrichum candidum. In this manner, a 95% recovery of the GLA originally present in borage oil (25% GLA) was obtained as a highly enriched fatty acid fraction with a GLA content of >70%. Other fatty acids concentrated in this fraction were the monounsaturated fatty acids with chainlengths of C-20 and longer that were present in the oil. An immobilized preparation ofG. candidum on silica gel also was used for the enrichment of GLA in borage oil. In this instance, a 75% recovery of GLA was obtained, and the supported lipase was reusable (three cycles) with minimal loss in activity. Presented in part at the 84th Annual Meeting of the American Oil Chemists’ Society, Anaheim, California, May 1993.     

Investigation of the technological properties ofNigella sativa (black cumin) seed oil

Three commercially cultivatedNigella sativa seed varieties of Turkish origin were analyzed, and the characteristics and constituents of the seed oils were reported. Presence of lipase enzyme in seed results in enzymatic hydrolysis at ordinary temperature; the free acid content of oil may increase up to 40% or higher. Black cumin seed oil might serve as a source of semi-drying oil and fatty acids of technical grade, and the removal of free fatty acids from oil and the recovery of fatty acids were investigated.     

New preparation of pure petroselinic acid from fennel oil (Foeniculum vulgare)

Pure petroselinic acid (cis-6-octadecenoic acid) has been isolated from fennel oil by acid soap crystallization at 4°C in methanol, followed by two urea segregations at room temperature and crystallization at −30°C in acetone. The purity control of petroselinic acid was effected by combined gas chromatography, and¹³C nuclear magnetic resonance. This petroselinic acid preparation was compared to other previous crystallization or enzymatic methods, showing that this method is both short (four steps) and easy to apply.     

General properties and the fatty acid composition of the oil from the mophane caterpillar, Imbrasia belina

A preliminary investigation of the bulk properties of the oil from the edible mophane caterpillar (phane), Imbrasia belina, showed a significant difference in the iodine values of the oils from mature and young phane. Detailed analysis of the fatty acid composition of the two oil samples was thus carried out by capillary gas chromatography (GC) and complemented with ¹H and ¹³C nuclear magnetic resonance (NMR) studies to investigate the degree of unstauration in the two oil samples. While these studies showed that the oil samples from the mature and young mophane caterpillar were much the same in fatty acid composition, the data revealed a significant divergence from a literature report on phane oil. This earlier report puts the ratio of total saturated to total unsaturated fatty acids at approximately 1:1 (48.2:48.8, in percentages) and estimates the fatty acid composition for the major fatty acids as 16:0 (31.9%), 18:0 (15.2%), 18:1 (20.4%), 18:2 (9.9%), and 18:3 (19%). The data collected from the present work, however, showed the fatty acid composition for total saturated and total unsaturated fatty acids to be 40.5 and 57.0%, respectively. This work estimated the fatty acid composition for the major fatty acids as 16:0 (27.2%), 18:0 (12.3%), 18:1 (16.1%), 18.2 (10.7%), and 18:3 (29.0%). Thus, linolenic acid was the most abundant fatty acid in the phane oil. The GC results of the present analysis were largely corroborated by studies of the composition of fatty acid classes in the phane oil estimated from integrals of ¹H and ¹³C NMR signals. Oils from other edible Lepidoptera larvae are also known to be much richer in unsaturated than saturated fatty acids.     

Development of calibration equations to predict oil content and fatty acid composition in brassicaceae germplasm by near-infrared reflectance spectroscopy

The use of nondestructive analytical methods is critical for the evaluation of very small seed samples such as those from germplasm collections. The objective of this study was to evaluate the potential of near-infrared reflectance spectroscopy (NIRS) for the simultaneous analysis of seed oil content and concentration of major fatty acids in intact-seed samples of the family Brassicaceae. A total of 495 samples from 56 genera and 128 species were analyzed by NIRS. The fatty acid composition of the seed oil was determined in all the samples by gas-liquid chromatography (GLC). The total seed oil content was determined by solvent extraction in 129 samples from 22 genera. Calibration equations for oil content (n=97) and individual fatty acids (n=410) were developed and tested through external validation with the samples not included in the calibration sets. The calibration equations for oil content (r ²=0.97 in validation) and concentrations of C_18:1 (r ²=0.93), C_18:3 (r ²=0.95), and C_22:1 (r ²=0.94) showed very good performance and provided reliable estimations of these traits in the samples of the validation set. The calibration equations for C_16:0, C_18:0, and C_18:2 content were less reliable, with r ² from 0.67 to 0.73. There was practically no response of NIRS to differences in C_20:1 (r ²=0.31). These results demonstrated that the oil content and concentrations of C_18:1, C_18:3, and C_22:1 can be estimated reliably within the family Brassicaceae by using NIRS calibration equations integrating broad taxonomic variability.     

Varietal and crop year effects on lipid composition of walnut (Juglans regia) genotypes

The FA, unsaponifiable, and volatile constituents of oil from three walnut varieties from two consecutive crop years were studied. The walnut oils (WO) were rich in PUFA and low in saturated FA. The tocopherol fraction consisted mainly of γ-tocopherol. High contents of β-sitosterol were found, together with campesterol and Δ⁵-avenasterol in similar amounts. Methylsterols present in WO were identified as cycloartenol, cyclolaudenol, cycloeucalenol, and 24-methylenecycloartanol. The hydrocarbon fraction was characterized by the predominance of C₁₄–C₂₀ n-alkanes. The major volatiles were aldehydes produced through the linoleic acid oxidative pathway. FA, methylsterols, and some hydrocarbons presented statistically significant differences among varieties. Most of this variation was due to the genotype. The Franquette variety was noteworthy by its higher oil and oleic acid contents. In contrast, tocopherols and volatile compounds showed minor differences among varieties; they were strongly influenced by the crop year. Chemical data were subjected to principal component analysis. The parameters that gave the greatest discrimination between the walnut varieties were oleic and linolenic acids, tetradecane, eicosane, tetracosane, cycloartenol, and 24-methylenecycloartanol. These components presented the major varietal influences and could be useful to determine the identity of walnut genotypes.