Polymorphism In Plants Research Articles

Absence of long-term balancing selection on variation in EuMYB3, an R2R3-MYB gene responsible for the anther-color polymorphism in Erythronium umbilicatum

Balancing selection has been shown to be common in plants for several different types of traits, such as self-incompatibility and heterostyly. Generally, for these traits balancing selection is generated by interactions among individuals or between individuals and other species (e.g., pathogens or pollinators). However, there are phenotypic polymorphisms in plants that do not obviously involve types of interactions that generate balancing selection. Little is known about the extent to which balancing selection also acts to preserve these polymorphisms. Here we ask whether balancing selection preserves an anther-color polymorphism in Erythronium umbilicatum (Liliaceae). We identified a major gene underlying this polymorphism. We then attempted to detect signatures of balancing selection on that gene by developing a new coalescence test for balancing selection. We found that variation in anther color is in large part caused by variation in a paralog of EuMYB3, an anthocyanin-regulating R2R3-MYB transcription factor. However, we found little evidence for balancing selection having acted historically on EuMYB3. Our results thus suggest that plant polymorphisms, especially those not involved in interactions that are likely to generate negative frequency-dependent selection, may reflect a transient state in which one morph will eventually be fixed by either genetic drift or directional selection. Our results also suggest that regulation of the anthocyanin pathway is more evolutionarily labile than is generally believed.

Effects of fruit dimorphism on genetic structure and gene flow in the coastal shrub Scaevola taccada.

Plant propagules often possess specialized morphologies that facilitate dispersal across specific landscapes. In the fruit dimorphism of a coastal shrub, Scaevola taccada, individual plants produce either cork-morph or pulp-morph fruits. The former is buoyant and common on sandy beaches, whereas the latter does not float, is bird-dispersed, and is common on elevated sites such as slopes on sea cliffs and behind rocky shores. We hypothesized that beach populations bridge the heterogeneous landscapes by serving as a source of both fruit types, while dispersal is biased for the pulp morph on elevated sites within the islands and for the cork morph between beaches of different islands. Based on this hypothesis, we predicted that populations in elevated sites would diverge genetically over time due to isolation by distance, whereas beach populations would maintain high genetic similarity via current gene flow. The genetic structure and gene flow in S. taccada were evaluated by investigating genome-wide single nucleotide polymorphisms in plants from 17 sampling sites on six islands (belonging to the Ryukyu, Daito and Ogasawara Islands) in Japan. Geographical isolation was detected among the three distant island groups. Analyses within the Ryukyu Islands suggested that sandy beach populations were characterized by genetic admixture, whereas populations in elevated sites were relatively isolated between the islands. Pairwise FST values between islands were lowest between sandy beaches, intermediate between sandy beaches and elevated sites, and highest between elevated sites. Dispersal across the ocean by cork morphs is sufficiently frequent to prevent genetic divergence between beaches of different islands. Stronger genetic isolation of elevated sites between islands suggests that bird dispersal by pulp morphs is restricted mainly within islands. These contrasting patterns of gene flow realized by fruit dimorphism provide evidence that fruit characteristics can strongly mediate genetic structure.

Investigation of genetic polymorphism of Russian rape and turnip rape varieties using SSR and SRAP markers.

Rapeseed (Brassica napus L.) and turnip rape (B. rapa L. subsp. campestris (L.)) are important agricultural plants widely used for food, fodder and technical purposes and as green manure. Over the past decades, a large number of perspective varieties that are being currently cultivated in every region of Russia have been developed. To increase the breeding eff iciency and facilitate the seed production, modern molecular-genetic techniques should be introduced as means to estimate species and varietal diversity. The objective of the presented research study was to investigate DNA polymorphism of the rapeseed and turnip rape varieties developed at Federal Williams Research Center of Forage Production and Agroecology and detect informative markers for varietal identif ication and genetic certif ication. To genotype 18 gDNA samples, 42 and 25 combinations of respective SSR and SRAP primers were used. The results obtained demonstrate that SRAP markers were more effective for polymorphism analysis: 36 % of the tested markers revealed genetic polymorphism compared with only 16.7 % of microsatellite loci. Molecular markers to detect differences at interspecif ic and intervarietal levels have also been found. For the investigated set, such microsatellite loci as Na12A02, Ni2C12, Ni02-D08a, Ra02-E01, Ni03H07а and SRAP-marker combinations as F13-R9, Me4- R7, F11-Em2, F10-R7, F9-Em2 and F9-R8 proved to be informative. Application of the two marker techniques made it possible to detect a higher level of DNA polymorphism in plants of different types (spring and winter varieties) if compared against the intervarietal differences within a species or a group. According to Nei’s genetic diversity index, in the cluster of winter rapeseed, VIK 2 and Gorizont varieties had the longest genetic distance, and in the spring cluster, these were Novosel and Veles. A high level of similarity was found between Vikros and Bizon winter rapeseed varieties. The results obtained have a high practical value for varietal specif ication of seed material and genetic certif ication of rapeseed and turnip rape varieties.

Applications of Molecular Markers in Fruit Crops for Breeding Programs—A Review

Selection and use of molecular markers for evaluation of DNA polymorphism in plants are couple of the most important approaches in the field of molecular genetics. The assessment of genetic diversity using morphological markers is not sufficient due to little differentiating traits among the species, genera or their individuals. Morphological markers are not only highly influenced by environmental factors but skilled assessment is also prerequisite to find the variations in plant genetic resources. Therefore, molecular markers are considered as efficient tools for detailed DNA based characterization of fruit crops. Molecular markers provide new directions to the efforts of plant breeders particularly in genetic variability, gene tags, gene localization, taxonomy, genetic diversity, phylogenetic analysis and also play an important role to decrease the time required for development of new and excellent cultivars. The success of molecular markers technology in genetic improvement programs depends on the close relationship among the plant breeders, biotechnologists, skilled manpower and good financial support. The present review describes application and success of molecular markers technology used for genetic improvement in different fruit crops.

High resolution melting curve analysis revealed SNPs in major cannabinoid genes associated with drug and non-drug types of cannabis

ABSTRACTCannabis sativa L. has a long history of cultivation as food, fibre, medicine and recreational drugs. Production of high tetrahydrocannabinol (THC) plants for narcotic use (drug type) is illegal and under control in most countries. In contrast, cultivation of low THC plants (fibre type, also known as ‘industrial hemp’) is promoted in many countries. The determination of C. sativa L. chemotypes is based on the major cannabinoids content, THC, cannabidiol (CBD) and cannabinol (CBN). The THC:CBD content ratio is a candidate marker for differentiation of the fibre and drug type of cannabis. The ability to accurately characterize the cannabinoid chemical phenotype (chemotype) is crucial for the development of specific C. sativa cultivars for pharmacological, hemp fibre or seed end-use. High resolution melting (HRM) curve analysis is used as a rapid and effective mechanism for detection of single-nucleotide polymorphisms in plants. In this report, we developed a HRM protocol for differentiation of drug and non-drug cannabis plants. According to the results, HRM analysis based on single-nucleotide polymorphisms in THCAS gene is an accurate method to differentiate the drug type of cannabis which could be used for the control of legal and illegal cannabis cultivation.

Dimorphism in trichome production of Persicaria lapathifolia var. lapathifolia and Its multiple effects on a leaf beetle

Polymorphisms in plants are main factors that determine the diversity of associated animal communities and their population dynamics. Typically, Persicaria lapathifolia var. lapathifolia (Polygonaceae) has no trichomes on leaf surfaces (glabrous type), but a hairy type does sometimes occur. Based on a cultivation experiment, the presence or absence of trichomes is clarified to be under genetic control. To reveal the defensive function of trichomes against herbivores, laboratory experiments were conducted using a major herbivore, Galerucella grisescens (Coleoptera: Chrysomelidae). In both choice and no-choice feeding tests, the glabrous type was significantly more consumed by G. grisescens adults, while the hairy type was not consumed. In the hairy leaf treatment, larval duration tended to become longer, the adult body weight became significantly lower, and adults laid significantly more eggs than in the glabrous leaf treatment. Hairy leaves contained significantly more total phenolics and condensed tannins than glabrous leaves, suggesting that the hairy type allocates more resources for physical and chemical defence. Because no significant differences in leaf consumption were detected in the feeding experiment using powdered host leaves, G. grisescens seems to have adapted to the chemical defences of P. lapathifolia var. lapathifolia. These results clearly indicate that leaf trichomes of P. lapathifolia var. lapathifolia effectively act as a physical defence against G. grisescens.

POLYMORPHISM OF SIMPLE SEQUENCE REPEAT REGIONS OF SULAWESI EBONY (DIOSPHYROS CELEBICA BAKH.) IN EXPERIMENTAL FOREST OF HASANUDDIN UNIVERSITY PROVENANCE

Polymerase Chain Reaction (PCR)-based molecular techniques have been used to detect the polymorphism in plants. The utilization of molecular markers plays essential role in germplasm characterization and plant breeding since the information of DNA marker technology can be exchanged between laboratories and should have standard method to be reproducible. The molecular aspect has been commonly linked to DNA isolation protocol and polymorphic molecular marker, thus can be used for molecular research recommendation purposes. The objectives of this study were to evaluate the capability of microsatellite marker of Ebenaceae Family for amplifying Ebony DNA, and to determine the appropriate PCR annealing temperatures. The DNA isolation of Ebony leaves from Experimental Forest of Hasanuddin University Provenance was carried out using Genomic DNA Mini Kit (Plant) Geneaid protocol. Nine of seventeen selected primers from the Genus Diospyros were able to amplify Ebony DNA. Amplification products produced polymorphic bands with different annealing temperatures (ranged from 53 to 56°C). These nine polymorphic primers will be recommended to use for future studies in genetic diversity as well as pollen dispersal pattern analyses.

Genetic structure of Hepatica nobilis var. japonica, focusing on within population flower color polymorphism.

How phenotypic or genetic diversity is maintained in a natural habitat is a fundamental question in evolutionary biology. Flower color polymorphism in plants is a common polymorphism. Hepatica nobilis var. japonica on the Sea of Japan (SJ) side of the Japanese mainland exhibits within population flower color polymorphism (e.g., white, pink, and purple), while only white flowers are observed on the Pacific Ocean (PO) side. To determine the relationships between flower color polymorphism, within and among populations, and the genetic structure of H. nobilis var. japonica, we estimated the genetic variation using simple sequence repeat (SSR) markers. First, we examined whether cryptic lineages corresponding to distinct flower colors contribute to the flower color polymorphisms in H. nobilis var. japonica. In our field observations, no bias in color frequency was observed among populations on Sado Island, a region with high variation in flower color. Simple sequence repeat (SSR) analyses revealed that 18% of the genetic variance was explained by differences among populations, whereas no genetic variation was explained by flower color hue or intensity (0% for both components). These results indicate that the flower color polymorphism is likely not explained by cryptic lineages that have different flower colors. In contrast, populations in the SJ and PO regions were genetically distinguishable. As with the other plant species in these regions, refugial isolation and subsequent migration history may have caused the genetic structure as well as the spatially heterogeneous patterns of flower color polymorphisms in H. nobilis var. japonica.

Experimental insights on the function of ancillary pollen and stigma polymorphisms in plants with heteromorphic incompatibility.

Most heterostylous plants possess a reciprocal arrangement of stigmas and anthers (reciprocal herkogamy), heteromorphic self-incompatibility, and ancillary polymorphisms of pollen and stigmas. The topographical complementarity hypothesis proposes that ancillary polymorphisms function in the rejection of incompatible pollen thus promoting disassortative pollination. Here, we test this hypothesis by investigating patterns of pollen transfer and capture in populations of dimorphic Armeria maritima and A. pubigera and distylous Limonium vulgare (Plumbaginaceae), and by studying pollen adherence and germination patterns in A. maritima following controlled hand-pollinations. Armeria lacks reciprocal herkogamy allowing the evaluation of the extent to which ancillary polymorphisms affect the composition of pollen loads. We compared the amounts of compatible and incompatible pollen on stigmas in natural populations and calculated the proficiencies of pollen transfer for each mating type. We detected disassortative pollination in each species, and mating types did not differ in compatible pollen capture, although cob stigmas captured more incompatible pollen. Controlled hand-pollinations revealed the failure of incompatible pollen to adhere and germinate on stigmas. Our results provided evidence that, while structural in nature, pollen-stigma dimorphisms are tightly associated with heteromorphic incompatibility and likely function to promote disassortative pollination, especially in the absence of reciprocal herkogamy.

Neutral processes contribute to patterns of spatial variation for flower colour in the Mediterranean Iris lutescens (Iridaceae).

Flower colour polymorphism in plants has been used as a classic model for understanding the importance of neutral processes vs. natural selection in population differentiation. However, current explanations for the maintenance of flower colour polymorphism mainly rely on balancing selection, while neutral processes have seldom been championed. Iris lutescens (Iridaceae) is a widespread species in the northern Mediterranean basin, which shows a stable and striking purple-yellow flower colour polymorphism. To evaluate the roles of neutral processes in the spatial variation for flower colour in this species, patterns of neutral genetic variation across its distribution range were quantified, and phenotypic differentiation was compared with neutral genetic differentiation. Genetic diversity levels and population genetic structure were investigated through the genotyping of a collection of 1120 individuals in 41 populations ranging from Spain to France, using a set of eight newly developed microsatellite markers. In addition, phenotypic differentiation for flower colour was also quantified by counting colour morph frequency in each population, and measuring the reflectance spectra of sampled individuals. Populations in Spain present a sharp colour transition from solely purple to solely yellow. The results provide evidence that genetic drift through limited gene flow is important in the evolution of monomorphic populations. In contrast, most populations in France are polymorphic with both phenotypes, and the colour frequencies vary geographically without any spatial gradients observed. A pattern of isolation by distance is detected in France, and gene flow between adjacent populations seems to be an important factor maintaining populations polymorphic. Overall, neutral processes contribute to patterns of spatial variation for flower colour in I. lutescens, but it cannot be excluded that natural selection is also operating. An interaction between neutral processes and natural selection is suggested to explain the spatial variation for flower colour in I. lutescens.

Heterostilia, qué sabemos hasta el momento?

Ferrero, V. 2014. Heterostyly, what do we currently know?. Ecosistemas 23(3): 23-30. Doi.: 10.7818/ECOS.2014.23-3.04 Sexual polymorphisms are common in nature, both in animals and plants, and have been interpreted as mechanisms that promote outcrossing. Heterostyly is a floral polymorphism in plants that has interested biologists since Darwin described it and proposed its functional significance. As it happens with other polymorphisms, the study of heterostyly allows understanding how the different forms are maintained in the populations and why phenotypes are selected. However, as the number of studies on this floral polymorphism increases, new variations and exceptions suggest caution when generalizing the typical characteristics of the heterostylous syndrome and its adaptive significance. In this review the morphological and physiological characteristics in heterostyly and its evolutionary context are described. Additionally, recent studies showing the morphological, physiological and adaptive complexity of heterostyly are reported, and future study questions are also proposed.

Characterization of vine varieties by SSR markers

Abstract Nowadays, there are well known PCR-based techniques to detect polymorphism in plants. Marker technologies which are applied in breeding and varietal characterization can be exchanged across laboratories with standardization to yield reproducible results. This article describes applying of simple sequence repeats (SSR) markers in European laboratories, in which the reproducibility of these popular markers are examined. One of the European Union interests is characterization of grape varieties and preparation of International Vitis catalogue for comparison global varieties as Chardonnay, Sauvignon, Pinot noir with the domestic for each other country. Grape genome contains of many SSR, which are highly polymorphic. This review provides the latest information in SSR research including novels in research, comparison, development and application of microsatellites.

Disentangling the causes of heterogeneity in male fecundity in gynodioecious Beta vulgaris ssp. maritima

Variation among individuals in reproductive success is advocated as a major process driving evolution of sexual polymorphisms in plants, such as gynodioecy where females and hermaphrodites coexist. In gynodioecious Beta vulgaris ssp. maritima, sex determination involves cytoplasmic male sterility (CMS) genes and nuclear restorers of male fertility. Both restored CMS and non-CMS hermaphrodites co-occur. Genotype-specific differences in male fitness are theoretically expected to explain the maintenance of cytonuclear polymorphism. Using genotypic information on seedlings and flowering plants within two metapopulations, we investigated whether male fecundity was influenced by ecological, phenotypic and genetic factors, while taking into account the shape and scale of pollen dispersal. Along with spatially restricted pollen flow, we showed that male fecundity was affected by flowering synchrony, investment in reproduction, pollen production and cytoplasmic identity of potential fathers. Siring success of non-CMS hermaphrodites was higher than that of restored CMS hermaphrodites. However, the magnitude of the difference in fecundity depended on the likelihood of carrying restorer alleles for non-CMS hermaphrodites. Our results suggest the occurrence of a cost of silent restorers, a condition supported by scarce empirical evidence, but theoretically required to maintain a stable sexual polymorphism in gynodioecious species.

Plant secondary chemistry mediates the performance of a nutritional symbiont associated with a tree‐killing herbivore

Many herbivores consume microbial food sources in addition to plant tissues for nutrition. Despite the ubiquity of herbivore-microbe feeding associations, few studies examine how host plant phenotypes affect microbial symbionts of herbivores. We tested the hypothesis that chemical polymorphism in a plant population mediates the performance of nutritional microbial symbionts. We surveyed the composition of ponderosa pine resin in northern Arizona, USA, for variation in six monoterpenes, and we approximated four chemical phenotypes. We reared populations of an herbivorous tree-killing beetle (Dendroctonus brevicomis) in ponderosa pine host material, controlling for three monoterpene compositions representing an alpha-pinene to delta-3-carene gradient. Beetles were reared in host material where the dominant monoterpene was alpha-pinene, delta-3-carene, or a phenotype that was intermediate between the two. We isolated nutritional fungal symbionts (Entomocorticium sp. B) from beetle populations reared in each phenotype and performed reciprocal growth experiments in media amended to represent four "average" monoterpene compositions. This allowed us to test the effects of natal host phenotype, chemical polymorphism, and the interaction between natal host phenotype and chemical polymorphism on a nutritional symbiont. Three important findings emerged: (1) fungal isolates grew 25-32% faster when acquired from beetles reared in the intermediate phenotype; (2) the mean growth rate of nutritional fungi varied up to 44% depending on which monoterpene composition media was amended with; and (3) fungal isolates uniformly performed best in the intermediate phenotype regardless of the chemical composition of their natal host. The performance of nutritional fungi related to both the chemical "history" of their associated herbivore and the chemical phenotypes they are exposed to. However, all fungal isolates appeared adapted to a common chemical phenotype. These experiments argue in favor of the hypothesis that chemical polymorphism in plant populations mediates growth of nutritional symbionts of herbivores. Intraspecific chemical polymorphism in plants contributes indirectly to the regulation of herbivore populations, and our experiments demonstrate that the ecological effects of plant secondary chemistry extend beyond the trophic scale of the herbivore-plant interaction.

A Cost‐effective High‐resolution Melting Approach using the EvaGreen Dye for DNA Polymorphism Detection and Genotyping in Plants

High-resolution melting (HRM) analysis relies on the use of fluorescent dyes, such as LCGreen, ResoLight, and SYTO9, which bind in a saturated manner to the double-stranded DNAs. These dyes are expensive in use and may not be affordable when dealing with a large quantity of samples. EvaGreen is a much cheaper DNA helix intercalating dye and has been used in quantitative real-time polymerase chain reaction (PCR) and post-PCR DNA melt curve analysis. Here we report on the development of an EvaGreen-based HRM analysis and its performance, in comparison with the popular LCGreen-based HRM analysis, in detection of DNA polymorphism in plants. We found that various polymorphisms ranged from single nucleotide polymorphisms (SNPs) to Indels were equally detected by using EvaGreen- or LCGreen-based HRM. EvaGreen dye was sensitive enough in discovery of SNPs in fivefold pooled samples. Using this economical dye we successfully identified multiple novel mutant alleles of Gln1-3 gene, which produces a cytosolic glutamine synthetase isoenzyme (GS1), in a maize ethyl methanesulfonate (EMS)-mutagenized library, and genotyped rice mapping populations with SNP markers. The current results suggest that EvaGreen is a promising dye for HRM analysis for its ease to use and cost effectiveness.

Polymorphism of a new Ty1-copia retrotransposon in durum wheat under salt and light stresses

Long terminal repeat retrotransposons are the most abundant mobile elements in the plant genome and play an important role in the genome reorganization induced by environmental challenges. Their success depends on the ability of their promoters to respond to different signaling pathways that regulate plant adaptation to biotic and abiotic stresses. We have isolated a new Ty1-copia-like retrotransposon, named Ttd1a from the Triticum durum L. genome. To get insight into stress activation pathways in Ttd1a, we investigated the effect of salt and light stresses by RT-PCR and S-SAP profiling. We screened for Ttd1a insertion polymorphisms in plants grown to stress and showed that one new insertion was located near the resistance gene. Our analysis showed that the activation and mobilization of Ttd1a was controlled by salt and light stresses, which strengthened the hypothesis that stress mobilization of this element might play a role in the defense response to environmental stresses.

Somaclonal variation in rye (Secale cereale L.) analyzed using polymorphic and sequenced AFLP markers

Amplified fragment length polymorphism (AFLP) analysis of 24 in vitro regenerated rye plants was performed in order to evaluate the somaclonal variation rate in this species and to identify rye genomic regions where mutations are preferentially promoted by in vitro culture processes. Regenerated plants were obtained from cell lines derived from immature embryos and plants were regenerated by somatic embryogenesis. Twenty-three regenerants showed variation when compared against sibling plants obtained from the same cell line. A total number of 887 AFLP markers were scored, and 8.8% identified the same polymorphism in plants obtained independently from different cell lines, revealing putative mutational hot spots. Using controlled crossings and analysis of the corresponding progenies, we were able to verify the genetic stability in the next generation for only five of these polymorphisms. The nucleotide sequence of the AFLP amplicon of four of the polymorphic markers was obtained, but only the sequence of two markers was clearly identified in the databases. The sequence of marker A1-303 was identified as part of a tandemly repeated sequence, the 120-bp family, which is located at telomeric regions and is widely distributed among rye chromosomes. The marker A5-375 showed high similarity with regions of Angela retrotransposons.

Molecular evolution of the Li/li chemical defence polymorphism in white clover (Trifolium repens L.)

White clover (Trifolium repens) is naturally polymorphic for cyanogenesis (hydrogen cyanide release following tissue damage). The ecological factors favouring cyanogenic and acyanogenic plants have been examined in numerous studies over the last half century, making this one of the best-documented examples of an adaptive polymorphism in plants. White clover cyanogenesis is controlled by two, independently segregating Mendelian genes: Ac/ac controls the presence/absence of cyanogenic glucosides; and Li/li controls the presence/absence of their hydrolysing enzyme, linamarase. In this study, we examine the molecular evolution and population genetics of Li as it relates to the cyanogenesis polymorphism. We report here that Li exists as a single-copy gene in plants possessing linamarase activity, and that the absence of enzyme activity in li/li plants is correlated with the absence of much or all of the gene from the white clover genome. Consistent with this finding, we confirm by reverse transcription-polymerase chain reaction that Li gene expression is absent in plants lacking enzyme activity. In a molecular population genetic analysis of Li and three unlinked genes using a worldwide sample of clover plants, we find an absence of nucleotide variation and statistically significant deviations from neutrality at Li; these findings are consistent with recent positive directional selection at this cyanogenesis locus.

Reproducibility testing of RAPD and SSR markers in Tomato

Large no of PCR-based techniques can be used to detect polymorphisms in plants. For their wide-scale usage in germplasm characterization and breeding it is important that these marker technologies can be exchanged between laboratories, which in turn require that they can be standardized to yield reproducible results, so that direct collation and comparison of the data are possible. This article describes a network experiment involving screening of 60 RAPD primers and was used to confirm the reproducibility. Every experiment was performed 50 times by 10 candidates in which the reproducibility of two popular molecular marker techniques was examined: random-amplified fragment length polymorphism (RAPD), and sequence-tagged micro satellites (SSR). For each technique, an optimal system was chosen, which had been standardized and routinely used by one individual. This system (genetic screening package) was distributed to different candidates in the laboratory and the results obtained were compared with those of the original generator or sender. Different experiences were gained in this exchange experiment with the different techniques. RAPDs proved difficult to reproduce. Whilst SSR alleles were amplified by all candidates, but small differences in their sizing were obtained.

Efficient validation of single nucleotide polymorphisms in plants by allele-specific PCR, with an example from barley

Although the increasing number of expressed sequence tags (ESTs) from the public domain has facilitated the detection of single nucleotide polymorphisms (SNPs), further validation is needed before they can be used as markers. For SNP validation, we have compared 2 independent methods: (1) the primer extension method followed by capillary electrophoresis on an ABI PRISM 3100 Genetic Analyzer and (2) nested PCR followed by agarose-based visualization. We present an assessment of the efficiency and costs associated with these methods, based on a sample of barley cultivars.