Pistacia Species Research Articles

SCoT markers provide insight into the genetic diversity, population structure and phylogenetic relationships among three Pistacia species of Iran

Genetic variability, phylogenetic relationship and population structure were evaluated among 50 accessions of three Iranian Pistacia species using start codon targeted (SCoT) primers and morphological traits. Genetic similarity and structure of the germplasm were evaluated based on the molecular data and their association with phenotypic characters were estimated by multiple regression analysis. The SCoT primers amplified 76 fragments on the germplasm with high level of polymorphism (98.46%). The average of RP, PIC, and MI were 3.30, 0.38, and 2.12, respectively representing the effectiveness of primer set for genetic discrimination among the Pistacia species. A high level of genetic variability was observed among the accessions. The germplasm belonging to the same species were grouped together using different clustering methods. Among the studied species, P. khinjuk Stocks. proved to be the most diverse species and had the highest gene diversity (h = 0.33) and Shannon’s information indices (I = 0.483) followed by P. atlantica Desf. and P. vera L., respectively. According to results, P. vera and P. khinjuk were the most closely related species, while P. atlantica and P. vera had the highest genetic differentiation. Significant association was observed between some of the morphological attributes and a number of SCoT generated fragments. Three species-specific amplicons were detected in P. atlantica and P. vera which can be used for designing sequence characterized amplification regions (SCAR) primers and subsequent marker assistant selection as well as species assignment in controversial cases. This study indicates the effectiveness of SCoT technique for genetic characterization of Pistacia germplasm, which could be used for genetic diversity and evolutionary assessments, detecting and tagging of agronomically important traits as well as quick, reliable and more precise authentication of Pistacia species.

Morphometric analysis and genetic diversity in Pistacia species populations using sequence related amplified polymorphism

The Anacardiaceae family includes 11 or more species, including the genus Pistacia. Pistacia vera L. is the most commercially developed of them all, thanks to its tasty nut seeds. Pistacia khinjuk Stocks, Pistacia atlantica Dsef, Pistacia vera, Pistacia eurycarpa Yalt. (Pistacia atlantica subsp. Kurdica Zoh.) and all have their origins in Iran. The present study aimed to investigate the SRAP (Sequence-related amplified polymorphism) markers in 13 wild pistachio accessions, which comprised three different species: Pistacia khinjuk, Pistacia vera, and Pistacia atlantica. Through polymerase chain reaction amplifications (PCR) of three Pistacia species, a total of 170 (Number of total loci) (NTL) DNA bands were obtained. Ten different selective primers were combined to generate these bands. The number of amplified pieces ranged from nine to twenty-six. The projected impartial gene diversity (UHe) ranged from 0.053 (Pistacia khinjuk) to 0.417 (Pistacia khinjuk) (Pistacia atlantica subsp. Kurdica). The genetic similarity of three species is estimated to be between 0.61 to 0.90. Two significant groupings emerged from the clustering findings: Pistacia khinjuk and Pistacia atlantica subsp. Kurdica exhibited the least similarity in the SRAP markers study. Our findings revealed excellent molecular recognition of all genotypes tested, indicating that a significant amount of genetic variety exists among pistachio accessions. This discovery might be useful in breeding management techniques for genetic preservation and cultivar improvement.

Transcriptome Sequencing and Development of Novel Genic SSR Markers From Pistacia vera L.

In this study, we aimed to develop novel genic simple sequence repeat (eSSR) markers and to study phylogenetic relationship among Pistacia species. Transcriptome sequencing was performed in different tissues of Siirt and Atl cultivars of pistachio (Pistacia vera). A total of 37.5-Gb data were used in the assembly. The number of total contigs and unigenes was calculated as 98,831, and the length of N50 was 1,333 bp after assembly. A total of 14,308 dinucleotide, trinucleotide, tetranucleotide, pentanucleotide, and hexanucleotide SSR motifs (4–17) were detected, and the most abundant SSR repeat types were trinucleotide (29.54%), dinucleotide (24.06%), hexanucleotide (20.67%), pentanucleotide (18.88%), and tetranucleotide (6.85%), respectively. Overall 250 primer pairs were designed randomly and tested in eight Pistacia species for amplification. Of them, 233 were generated polymerase chain reaction products in at least one of the Pistacia species. A total of 55 primer pairs that had amplifications in all tested Pistacia species were used to characterize 11 P. vera cultivars and 78 wild Pistacia genotypes belonging to nine Pistacia species (P. khinjuk, P. eurycarpa, P. atlantica, P. mutica, P. integerrima, P. chinensis, P. terebinthus, P. palaestina, and P. lentiscus). A total of 434 alleles were generated from 55 polymorphic eSSR loci with an average of 7.89 alleles per locus. The mean number of effective allele was 3.40 per locus. Polymorphism information content was 0.61, whereas observed (Ho) and expected heterozygosity (He) values were 0.39 and 0.65, respectively. UPGMA (unweighted pair-group method with arithmetic averages) and STRUCTURE analysis divided 89 Pistacia genotypes into seven populations. The closest species to P. vera was P. khinjuk. P. eurycarpa was closer P. atlantica than P. khinjuk. P. atlantica–P. mutica and P. terebinthus–P. palaestina pairs of species were not clearly separated from each other, and they were suggested as the same species. The present study demonstrated that eSSR markers can be used in the characterization and phylogenetic analysis of Pistacia species and cultivars, as well as genetic linkage mapping and QTL (quantitative trait locus) analysis.

Determination of Phenolic Compounds’ Extraction Conditions from Pistacia palaestina Leaves at Two Different Stages of Maturity

Background: Phenolic compounds are distributed throughout the plant kingdom. Numerous investigations have proved that Pistacia species have secondary metabolites, and these contain different classes of bioactive phenolic compounds. Objective: The objective of the present work was to optimize the extraction conditions of phenolic compounds from Pistacia palaestina leaves cultivated in Syria and find out the effects of the solvent type, extraction time, and temperature on total phenolic content (TPC) and in vitro antioxidant activity% (AA%). Two different growth stages were studied. Methods: Antioxidant activity and total phenolic content values were estimated using 2,2’-diphenyl- 1-picrylhydrazyl and Folin-Ciocalteu methods, respectively. Results: The highest TPC and AA% were found in fresh leaves (14.32g/100g and 92.69%, respectively). The optimum extraction solvent for phenolic recovery and antioxidant capacity was acetone 40%. Changes both in temperature (25, 40, 60 and 80°C) and time (15, 30, 45 and 60 min) had no significant influence on TPC and AA% of Pistacia extracts. Conclusion: Phenolic compounds could be extracted in a short time at a low temperature and could be used as natural antioxidant agents.

Comparison and evaluation of oil content, composition and antioxidant properties of Pistacia atlantica and Pistacia khinjuk grown as wild.

The growing demands of consumers for edible seed oils containing high unsaturated fatty acid and antioxidant content have resulted in considerable efforts to investigate plants as possible sources of oils and nuts. In this research, the amount of fatty acid compositions, total flavonoid, phenol and antioxidant properties of Pistacia atlantica and Pistacia khinjuk were evaluated. The kernel oil content of P. atlantica and P. khinjuk were 24.33 ± 0.333% and 31.00 ± 0.577%, respectively. Palmitic acid, Stearic acid, Oleic acid, Linoleic acid, and Palmitoleic acid were the main components in the oil of the two Pistacia species. The results showed that unsaturated fatty acids accounted for approximately 77.65% and 74.87% of total fatty acids in P. atlantica and P. khinjuk, respectively. The two Pistacia species were rich in phenolic compounds (130.77± 3.11 and 126.91± 4.41 mg quercetin/100 g oil) and had high antioxidant properties (4.545± 0.655 and 15.733± 0.689 mg/g oil) in P. atlantica and P. khinjuk, respectively. Oil content and Oleic acid of the two species of Pistacia are shown/known to be higher than some other edible oils. This research showed that the kernel oil of the two species of Pistacia have the same value in terms of quality, taste and natural antioxidant qualities with other edible oils.

Chemometric discrimination of three Pistacia species via their metabolic profiling and their possible in vitro effects on memory functions

Alzheimer’s disease (AD) is the most widespread neurodegenerative disease; there are around ten million new cases of Alzheimer yearly worldwide especially in middle or low-income countries. Pistacia is a genus of flowering plants including the well-known, economically important P. chinensis Bunge, P. lentiscus L. and P. khinjuk. In this study, the metabolic profiling of Pistacia leaves extracts was achieved via UHPLC-ESI-MS analysis and GC–MS analysis employing chemometric analysis for their discrimination. In addition, the methanolic extracts of different Pistacia species were assessed for their anti-cholinesterase and anti-inflammatory activities by various in vitro assays. 37 and 30 metabolites belonging to different classes were identified by UHPLC-ESI-MS and GC–MS analyses respectively. Chemometric analysis revealed that P. lentiscus and P. khinjuk were more closely related chemically to each other. All studied Pistacia leaves extracts showed apparent anti-cholinesterase and anti-inflammatory activities, which promotes their use in the prevention and management of AD.

Morphological, ecophysiological and photosynthetic diversity of some Pistacia species for use in breeding programs

Evaluation of genetic diversity is the basis of any breeding programs in all plants. The aim of this study was to evaluate and investigate the morphological, ecophysiological and photosynthetic diversity of some Pistacia species which can be used in breeding programs. A total of 42 genotypes belonging to Pistacia atlantica Desf., P. mutica Fischer & C. A. Meyer, Pistacia vera L., Pistacia terebinthus L. and P. khinjuk Stocks species were studied. The results showed that the P. terebinthus species is more efficient than other species in terms of leaf weight, chlorophyll a and b and total chlorophyll, as well as chlorophyll fluorescence and photosynthesis efficiency index. Among the varieties of pistachio belonged to P. vera, ‘Siirt’ and ‘Uzun’ cultivars had the highest levels of chlorophyll a, b, and total, photosynthesis rate and transpiration. In addition, P. mutica species had the highest leaf phenol content and stomatal conduction. For most traits measured, the lowest value was observed in P. atlantica species. Moreover, the results showed that there was a positive and significant correlation between morphological and photosynthetic traits as well as morphological and ecophysiological ones so that, the leaf length had a positive and significant correlation with the quantum yield of photosystem II (Fv/Fm) (r = 0.87), the efficiency index of photosynthesis (pi) (r = 0.76), total chlorophyll (r = 0.92), and carotenoid (r = 0.91). Cluster analysis divided the genotypes into three distinct groups. The first group consisted of P. vera species and its genotypes plus P. khinjuk species. The second group included the species of P. mutica and P. atlantica, and the third one just consisted of the species of P. terebinthus. In general, the results of this study showed that P. terebinthus specie, as well as ‘Akbari’ and ‘Siirt’ cultivars from P. vera can be used as parents in breeding programs to improve the rootstocks and cultivars resistant to environmental conditions.

Effects of dormancy-breaking treatments on seed germination of Pistacia species

This study was carried out to determine the effects of different dormancy-breaking treatments including stratification, sulfuric acid scarification, dehulling and gibberellic acid (GA3), on seed germination of different Pistacia species e.g. Pistacia vera cv. Kırmızı, Pistacia vera cv. Siirt, P. khinjuk-A, P. khinjuk-B, P. atlantica, P. terebinthus and P. mutica using as rootstock for pistachio trees. Seed dormancy-breaking treatments were shelled (control), shelled + GA3, dehulled, dehulled + GA3, Sulfuric acid scarification and sulfuric acid scarification + GA3 applications in the experiment. The seeds of Pistacia species were stratified at 4 °C for 50 days after the dormancy-breaking treatments. Stratified seeds were sown in the seedling viols filled with peat in the greenhouse to determine the germination percentage. The highest germination rate was obtained from sulfuric acid scarification in P. khinjuk-A (96.7%), and followed by dehulled application of P. vera cv. Kırmızı and P. mutica species with 94.6% and 93.2%, respectively. On the other hand, the lowest seed germination was observed for shelled + GA3 application of P. mutica with 6.7%. When the seed germination rates of Pistacia sp. were examined, the highest germination occurred in P. vera cv. Kırmızı, and followed by P. khinjuk-B. The lowest germination rate was observed on P. atlantica.

Whole genomes and transcriptomes reveal adaptation and domestication of pistachio

BackgroundPistachio (Pistacia vera), one of the most important commercial nut crops worldwide, is highly adaptable to abiotic stresses and is tolerant to drought and salt stresses.ResultsHere, we provide a draft de novo genome of pistachio as well as large-scale genome resequencing. Comparative genomic analyses reveal stress adaptation of pistachio is likely attributable to the expanded cytochrome P450 and chitinase gene families. Particularly, a comparative transcriptomic analysis shows that the jasmonic acid (JA) biosynthetic pathway plays an important role in salt tolerance in pistachio. Moreover, we resequence 93 cultivars and 14 wild P. vera genomes and 35 closely related wild Pistacia genomes, to provide insights into population structure, genetic diversity, and domestication. We find that frequent genetic admixture occurred among the different wild Pistacia species. Comparative population genomic analyses reveal that pistachio was domesticated about 8000 years ago and suggest that key genes for domestication related to tree and seed size experienced artificial selection.ConclusionsOur study provides insight into genetic underpinning of local adaptation and domestication of pistachio. The Pistacia genome sequences should facilitate future studies to understand the genetic basis of agronomically and environmentally related traits of desert crops.

The Effect of Oleander (Nerium oleander L.) Extracts on Seed Germination and Seedling Growth of Four Pistacia Species

In this study, the effects of the extracts obtained from flower, stem, leaf, branch and their mixture of Nerium oleander L. on the seed germination and seedling growth of four Pistacia species (Pistacia terebinthus L., P. vera L., P. khinjuk Stocks., P. atlantica Desf. and P. terebinthus L.) were investigated. Five hundred grams of N. oleander was taken from the plant parts and the extracts were mixed with distilled water (1.5 L). The extracts were applied to Pistacia species for 24 and 48 hours. The results showed that the extract had positive effects on their germination and growths of the species mentioned above. The highest germination rate for all the Pistacia species was obtained from the extracts of flower, while the lowest germination rate was recorded in the mixture of all parts of N. oleander mentioned above. Moreover, the treatment time was found to reduce the germination ratio. It was also found that the extracts from the stem of N. oleander were the most effective on the stem height of the Pistacia species studied. It was followed by the extracts from branches, leaves and flower, respectively. Furthermore, the findings indicated that the extracts from the stem of N. oleander had pronounced effect on the stem diameter of the Pistacia species studied. It was followed by the extracts from flower, leaves, and branches, respectively. In general, the effects of the extracts from several parts of N. oleander on the germination and other growing parameters were found to be almost comparable to those of synthetic promoter, Gibberellic Acid (GA3).

The contribution of leaf life span to sexual dimorphism in deciduous and evergreen Pistacia species under Mediterranean conditions

Gender differentiation may be quite different between species with different strategies to cope with environmental stresses. In this study, we tested the hypothesis that sexual dimorphism is stronger in plant species under summer drought stresses and long leaf longevity. For that, we selected two Mediterranean dioecious species: the winter-deciduous Pistacia atlantica Desf. from Iran and the evergreen-sclerophyllous Pistacia lentiscus L. from Portugal. Our results indicated that there were no morphological differences between genders except for leaf mass-per-area (LMA) in P. lentiscus, where females displayed higher values than males. Leaves from female P. lentiscus presented a higher C/N ratio and lower carbon isotope composition (δ13C), which is related with higher stomatal conductance during photosynthesis; whereas males had higher nitrogen concentration and more enriched leaf δ13C values. Pistacia atlantica, which experiences a shorter growing season, did not show any sexual dimorphism. Accordingly to our initial hypothesis, environmental conditions with the particular stressful conditions and leaf life strategy play a crucial role in gender dimorphism, both in physiological and morphological responses. Thus, a long longevity of P. lentiscus leaves was crucial for the observation of differences in gender-related physiological differences.

The sex expression and sex determining mechanism in Pistacia species.

Generally, Pistacia species are dioecious, but monoecious strains in several populations have been found, providing excellent models for studying sex differentiation and sex determination mechanisms. Although the mechanisms of sex determination and sex evolution have been extensively studied, related research on heterozygous woody plants is limited. Here, we discuss the expressions of various sex types, which showed broad diversity and complex instability. We have also reviewed the sex determination systems in the plant kingdom and the morphological, cytological, physiological, and molecular aspects of the sex-linked markers in Pistacia trees. Moreover, hypotheses to explain the origin of monoecy are discussed, which is more likely to be the interaction between sex-related genes and environment factors in female plants. Besides, further prospects for the utilization of monoecious resources and the research directions of sex determination mechanism are proposed. This study provides important information on sex expression and provides more insights into sex differentiation and determination.

Linking functional traits and climate-growth relationships in Mediterranean species through wood density

ABSTRACTFunctional traits are considered to influence the performance of woody plants. However, few field studies have tested this idea by using radial-growth data. Here, I test if five major traits of the leafand wood-economics spectra (height, leaf area, specific leaf area – SLA, wood density – WD and hydraulic diameter) explain climate-growth relationships in 14 Mediterranean trees and shrubs. Traits were measured for both gymnosperm (four Juniperus species plus three Pinus species) and angiosperm species (two Quercus species, two Pistacia species, Arbutus unedo, Celtis australis, and one Tamarix species). Climategrowth relationships were calculated relating ring-width indices (RWIs) and local climate data. Leaf area and SLA were high in broadleaf deciduous species (e.g., C. australis), and low in junipers. WD reached minimum and maximum values in pine and oak species, respectively. WD explained 45 % of the variation of the association observed between RWI and April precipitation, one of the main climatic variables driving radial growth. Therefore, WD is a relevant functional trait useful to predict the performance of Mediterranean woody plant species, specifically concerning their growth responses to climate. Functional traits as WD should be further explored to explain growth variability between and within woody species, and to link this variability with responsiveness to climate and ecosystem productivity.

Parameters Influencing Phenolic compounds extraction from Pistacia palaestina fruits during different stages of Maturity

Pistacia species are used in food industry and as medicinal for the remedy for various diseases. Chemical studies on Pistacia genus have led to discovering diverse secondary metabolites in addition to high level of vitamins and minerals. The present study aimed to investigate the effects of experimental variables, such as solvent composition, time and temperature on total phenolic content and DPPH activity of P. palaestina fruits cultivated in Syria at three maturity stages. Antioxidant activities and total phenolic content values were estimated using (2, 2’-diphenyl-1-picrylhydrazyl) DPPH reagent and Folin Ciocalteu reagent methods respectively. The highest total phenolic compounds and antioxidant activity were found in unripe fruits with 6g/100g and 85.67% respectively. The optimum extraction solvents for phenolic recovery and antioxidant capacity were methanol 60% and acetone 60% respectively. Changes in both temperature (25, 40, 60, 80°C) and time (15, 30, 45, 60 min) had no significant influence on TPC and AA% of Pistacia extracts. Therefore, phenolic compounds could be extracted in a short time and low temperature and could be used as antioxidant agents.

GC-MS analysis of terpenes from Sicilian Pistacia vera L. oleoresin. A source of biologically active compounds.

AbstractThe genus Pistacia includes different species diffused worldwide able to produce oleoresins used in several folk medicines. Oleoresins are phytocomplexes exudate from the trunk of plants and possess several biological properties thanks to the simultaneous presence of essential oils, di‐ and triterpenes and phenols. Pistacia vera L. oleoresin chemical composition is not well known owing a lack of studies compared with other Pistacia species. The aim of this work was to report the terpenes profile of this phytocomplex collected in Sicily (Italy) by means of GC–FID and GC–MS analysis of its essential oil and less volatile fractions. Results show that this profile is peculiar and different from the other Pistacia species mainly in terms of relative percentages of its components. α‐Pinene is the main compound of the essential oil, while dammarane and tirucallene derivatives are the main triterpenes identified in the less volatile fractions. This work represents the first complete terpenoidic chemical profile of the oleoresin of P. vera L. and should be considered as a first step for a deeper evaluation of biological properties and future application of this phytocomplex.

LC-ESI-MS/MS and cytotoxic activity of three Pistacia species

LC-ESI-MS/MS was used for a comprehensive characterisation of ethanol extract from the leaves of three Pistacia species. After optimisation of the method and the use of the negative ionisation mode, a total of 42 different compounds were identified, of which 22 were tentatively characterised in P. chinensis Bunge, 33 in P. khinjuk stocks and 25 in P. lentiscus L. leaves. Flavonoids, phenolic acids, and their derivatives were the most abundant identified compounds. LC-ESI-MS/MS revealed identification of 15, 18 and 6 not previously detected compounds in P. chinensis Bunge, P. khinjuk Stocks and P. lentiscus L., respectively. The three extracts were also tested for their cytotoxic activities against human PC3 prostate cancer, A549 lung cancer, MCF7 breast cancer and HepG2 liver cancer. Generally, all the extracts have a moderate cytotoxic activity against lung, breast and prostate cancer, with different IC50. However, only P. lentiscus L. showed moderate activity against liver cancer.

Retraction Note to: Potential Start Codon Targeted (SCoT) and Interretrotransposon Amplified Polymorphism (IRAP) Markers for Evaluation of Genetic Diversity and Conservation of Wild Pistacia Species Population.

"This article has been retracted by the Publisher in agreement with the Editor-in-Chief, because it contains portions of writings on the same topic already published and without sufficient attribution to these earlier works being given. The principal authors of the paper acknowledged that text from background sources was mistakenly used in this article without proper reference to the original source. Upon investigation carried out according to the Committee on Publication Ethics guidelines, it has been found that the authors have duplicated or rephrased parts from other articles of which the main sources.

Clonal propagation of mastic tree (Pistacia lentiscus var. chia Duham.) in outdoor beds using different rootstock and grafting techniques

We assessed different rootstocks and grafting techniques to clonally propagate mastic tree (Pistacia lentiscus var. chia). Mastic tree is a dioecious tree but the quantity and quality of the resin secreted by female trees are very low; therefore, male trees that are clonally propagated are used to establish commercial plantations for mastic production. Conventional long branch cuttings for clonal production requires too much materials and has a very low rooting rate. Rooting with tissue culture and green cuttings has also failed outdoors. Grafting of mastic on other Pistacia species has not been tried so far; therefore, this study was aimed at developing grafting methods for the clonal propagation of mastic. P. atlantica and P. lentiscus rootstocks were grafted from 15 February to 15 October every 15 days using three grafting methods and a two-factor randomized block trial design. Early spring (15 February–15 March) proved to be the best time for grafting using either of the rootstocks. The highest grafting success was attained using the scions grafted on P. atlantica from 15 February to 15 March. The best bud-burst percentage was obtained using P. atlantica and P. lentiscus rootstocks.

Genetic diversity and population structure analysis of Pistacia species revealed by phenylalanine ammonia-lyase gene markers and implications for conservation

Information on population genetic structure and crop genetic diversity is important for genetically improving crop species and conserving threatened species. The PAL gene sequence is part of a multigene family that can be utilized to design DNA marker systems for genetic diversity and population structure investigation. In the current study, genetic diversity and population structure of 100 accessions of wild Pistacia species were investigated with 78 PAL markers. A protocol for using PAL sequences as DNA markers was developed. A total of 313 PAL loci were recognized, showing 100% polymorphism for PAL markers. The PAL markers produced relatively more observed and effective alleles in Pistacia falcata and Pistacia atlantica, with a higher Shannon’s information index and expected heterozygosity in P. atlantica, Pistacia vera and Pistacia mutica. Pairwise assessment of Nei’s genetic distance and genetic identity between populations revealed a close association between geographically isolated populations of Pistacia khinjuk and Pistacia chinensis. The accessions of wild Pistacia species had more genetic relationship among studied groups of species. Analysis of molecular variance indicated 19% among-population variation and 81% within-population variation for the PAL gene based DNA marker. Population structure analysis based on PAL revealed four groups with high genetic admixture among populations. The results establish PAL markers as a functional DNA marker system and provide important genetic information about accessions from wild populations of Pistacia species.

Chemical and biomolecular analyses to discriminate three taxa of Pistacia genus from Sardinia Island (Italy) and their antifungal activity

This work reports the results and the comparison concerning the chemical and biomolecular analyses and the antifungal activity of three wild Pistacia species (Anacardiaceae) from Sardinia. Volatile oils from leaves and twigs of Pistacia x saportae, Pistacia lentiscus and Pistacia terebinthus were characterised using GC-FID and GC-MS techniques and tested against some fungal strains. Two DNA nuclear regions (ITS and 5S-rRNA-NTS) were amplified through PCR technique and sequenced. The three **Pistacia have similar chemical profile, although there are some important quantitative differences. The analysis of ITS and 5S-rRNA-NTS regions, reveals a species-specific nucleotide variation among the three **taxa. This method could emerge as a powerful tool for the species identification, especially because the discrimination of these three **taxa appears difficult for non-expert botanists. Concerning the antifungal activity, P. lentiscus and P. x saportae show the highest activity against Cryptococcus neoformans, with a MIC value of 0.32 μL/mL.