Molecular-marker-facilitated investigation of host-plant response to Exserohilum turcicum in maize (Zea mays L.): components of resistance

RFLPs were used to investigate components of host-plant response to Exserohilum turcicum in 150 unselected F₂₃ lines of a B52/Mo17 maize population. Following inoculation with spore suspensions of the pathogen (race 0), components of disease development were measured and then quantitative trait mapping was performed to identify the location and effects of quantitative trait loci (QTLs) determining host-plant response. Components of interest were the average number of lesions per leaf, the average percent leaf tissue diseased (severity) and the average size of lesions (cm²). Based on a LOD threshold of 2.31 (P<0.05), the number of lesions appears to be associated with QTLs on chromosomes 1S, 3L, 5S. Severity was associated with analogous regions and, in addition, QTLs on chromosomes 7L and 8L. Most QTLs, for either of these two components, involve additive gene action and partial dominance or overdominance. In contrast, lesion size was associated with QTLs on chromosomes 7L and 5L; recessive gene action may be involved at 7L.Journal Paper No. J-15178 of the Iowa Agriculture and Home Economic Experiment Station, Ames, Iowa. Project No. 3134     

Genetic variation at bx1 controls DIMBOA content in maize

The main hydroxamic acid in maize (Zea mays L.) is 2-4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA). DIMBOA confers resistance to leaf-feeding by several corn borers. Most genes involved in the DIMBOA metabolic pathway are located on the short arm of chromosome 4, and quantitative trait loci (QTLs) involved in maize resistance to leaf-feeding by corn borers have been localized to that region. However, the low resolution of QTL linkage mapping does not allow convincing proof that genetic variation at bx loci was responsible for the variability for resistance. This study addressed the following objectives: to determine the QTLs involved in DIMBOA synthesis across genetically divergent maize inbreds using eight RIL families from the nested association mapping population, to check the stability of QTLs for DIMBOA content across years by evaluating two of those RIL families in 2 years, and to test the involvement of bx1 by performing association mapping with a panel of 281 diverse inbred lines. QTLs were stable across different environments. A genetic model including eight markers explained approximately 34% of phenotypic variability across eight RIL families and the position of the largest QTL co-localizes with the majority of structural genes of the DIMBOA pathway. Candidate association analysis determined that sequence polymorphisms at bx1 greatly affects variation of DIMBOA content in a diverse panel of maize inbreds, but the specific causal polymorphism or polymorphisms responsible for the QTL detected in the region 4.01 were not identified. This result may be because the causal polymorphism(s) were not sequenced, identity is masked by linkage disequilibrium, adjustments for population structure reduce significance of causal polymorphisms or multiple causal polymorphisms affecting bx1 segregate among inbred lines.     

Genetic mapping of <Emphasis Type="Italic">psl</Emphasis> locus and quantitative trait loci for angular leaf spot resistance in cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.)

One of the most important cucumber diseases is bacterial angular leaf spot (ALS), whose increased occurrence in open-field production has been observed over the last years. To map ALS resistance genes, a recombinant inbred line (RIL) mapping population was developed from a narrow cross of cucumber line Gy14 carrying psl resistance gene and susceptible B10 line. Parental lines and RILs were tested under growth chamber conditions as well as in the field for angular leaf spot symptoms. Based on simple sequence repeat and DArTseq, genotyping a genetic map was constructed, which contained 717 loci in seven linkage groups, spanning 599.7 cM with 0.84 cM on average between markers. Monogenic inheritance of the lack of chlorotic halo around the lesions, which is typical for ALS resistance and related with the presence of recessive psl resistance gene, was confirmed. The psl locus was mapped on cucumber chromosome 5. Two major quantitative trait loci (QTL) psl5.1 and psl5.2 related to disease severity were found and located next to each other on chromosome 5; moreover, psl5.1 was co-located with psl locus. Identified QTL were validated in the field experiment. Constructed genetic map and markers linked to ALS resistance loci are novel resources that can contribute to cucumber breeding programs.     

Resistance to <Emphasis Type="Italic">Rhynchosporium commune</Emphasis> in a collection of European spring barley germplasm

Key message

Association analyses of resistance to Rhynchosporium commune in a collection of European spring barley germplasm detected 17 significant resistance quantitative trait loci. The most significant association was confirmed as Rrs1.

Abstract

Rhynchosporium commune is a fungal pathogen of barley which causes a highly destructive and economically important disease known as rhynchosporium. Genome-wide association mapping was used to investigate the genetic control of host resistance to R. commune in a collection of predominantly European spring barley accessions. Multi-year disease nursery field trials revealed 8 significant resistance quantitative trait loci (QTL), whilst a separate association mapping analysis using historical data from UK national and recommended list trials identified 9 significant associations. The most significant association identified in both current and historical data sources, collocated with the known position of the major resistance gene Rrs1. Seedling assays with R. commune single-spore isolates expressing the corresponding avirulence protein NIP1 confirmed that this locus is Rrs1. These results highlight the significant and continuing contribution of Rrs1 to host resistance in current elite spring barley germplasm. Varietal height was shown to be negatively correlated with disease severity, and a resistance QTL was identified that co-localised with the semi-dwarfing gene sdw1, previously shown to contribute to disease escape. The remaining QTL represent novel resistances that are present within European spring barley accessions. Associated markers to Rrs1 and other resistance loci, identified in this study, represent a set of tools that can be exploited by breeders for the sustainable deployment of varietal resistance in new cultivars.

     

Feline polycystic kidney disease is linked to the PKD1 region

Autosomal dominant polycystic kidney disease (ADPKD) is a commonly inherited disorder (1/1000) in humans characterized by fluid-filled cysts in the kidneys. Defects in the PKD genes, PKD1 and PKD2, cause 85% and 15% of human ADPKD cases, respectively. Mutations in the PKHD1 gene cause autosomal recessive PKD (ARPKD). Mutations in several genes, including Nek8, cause PKD in mice. Although PKD affects 38% of Persian cats worldwide, making it the most prominent inherited feline disease, a causative gene has not been identified. Feline PKD is an autosomal dominant disease with clinical presentations similar to human ADPKD. Forty-three microsatellites were chosen from the feline genetic maps based on known homology with human chromosomal regions containing the PKD1, PKD2, PKHD1, and Nek8 genes. Linkage analysis using seven Persian cat pedigrees segregating for PKD has shown significant linkage and no recombinants (Z=5.83, =0) between the PKD disease phenotype and marker FCA476, which is within 10 cR of the feline PKD1 gene on Chromosome E3. This suggests that the PKD1 gene or another gene within this region may cause feline PKD. Further investigation into the cause of PKD will be valuable for feline health and provide insights into human ADPKD.     

Molecular marker analysis of genes controlling morphological variation in Brassica rapa (syn. campestris)

Construction of a detailed RFLP linkage map of B. rapa (syn. campestris) made it possible, for the first time, to study individual genes controlling quantitative traits in this species. Ninety-five F₂ individuals from a cross of Chinese cabbage cv Michihili by Spring broccoli were analyzed for segregation at 220 RFLP loci and for variation in leaf, stem, and flowering characteristics. The number, location, and magnitude of genes underlying 28 traits were determined by using an interval mapping method. Zero to five putative quantitative trait loci (QTL) were detected for each of the traits examined. There were unequal gene effects on the expression of many traits, and the inheritance patterns of traits ranged from those controlled by a single major gene plus minor genes to those controlled by polygenes with small and similar effects. The effect of marker locus density on detection of QTL was analyzed, and the results showed that the number of QTL detected did not change when the number of marker loci used for QTL mapping was decreased from 220 to 126; however, a further reduction from 126 to 56 caused more than 15% loss of the total QTL detected. The detection of putative minor QTL by removing the masking effects of major QTL was explored.     

RFLP mapping of quantitative trait loci controlling seed aliphatic-glucosinolate content in oilseed rape (Brassica napus L)

We report the RFLP mapping of quantitative trait loci (QTLs) which regulate the total seed aliphaticglucosinolate content in Brassica napus L. A population of 99 F₁-derived doubled-haploid (DH) recombinant lines from a cross between the cultivars Stellar (low-glucosinolate) and Major (high-glucosinolate) was used for singlemarker analysis and the interval mapping of QTLs associated with total seed glucosinolates. Two major loci, GSL-1 and GSL-2, with the largest influence on total seed aliphatic-glucosinolates, were mapped onto LG 20 and LG 1, respectively. Three loci with smaller effects, GSL-3, GSL-4 and GSL-5, were tentatively mapped to LG 18, LG 4 and LG 13, respectively. The QTLs acted in an additive manner and accounted for 71 % of the variation in total seed glucosinolates, with GSL-1 and GSL-2 accounting for 33% and 17%, respectively. The recombinant population had aliphatic-glucosinolate levels of between 6 and 160 moles per g^-1 dry wt of seed. Transgressive segregation for high seed glucosinolate content was apparent in 25 individuals. These phenotypes possessed Stellar alleles at GSL-3 and Major alleles at the four other GSL loci demonstrating that low-glucosinolate genotypes (i.e. Stellar) may possess alleles for high glucosinolates which are only expressed in particular genetic backgrounds. Gsl-elong and Gsl-alk, loci which regulate the ratio of individual aliphatic glucosinolates, were also mapped. Gsl-elong-1 and Gsl-elong-2, which control elongation of the -amino-acid precursors, mapped to LG 18 and LG 20 and were coincident with GSL loci which regulate total seed aliphatic glucosinolates. A third tentative QTL, which regulates side-chain elongation, was tentatively mapped to LG 12. Gsl-alk, which regulates H₃CS-removal and side-chain de-saturation, mapped to LG 20.     

Molecular mapping of k2 Mdh1-n y20, an unstable chromosomal region in soybean [Glycine max (L.) Merr.]

In the soybean genome, a chromosomal region covering three tightly linked genes, k2, Mdh1-n, and y20, was found very unstable. It was suspected that the instability of the k2 Mdh1-n y20 chromosomal region was caused by a non-autonomous transposable element residing adjacent to or in this region. In this study, we located and mapped this region with simple sequence repeat (SSR) markers on the soybean integrated map using five mapping populations. The k2 Mdh1-n y20 chromosomal region was located on molecular linkage group H. The integrated map from five mapping populations consisted of 13 loci in the order Satt541, Satt469, Sat_122, Satt279, Satt253, Satt314, Mdh1-n,y20, k2, Satt302, Satt142, Satt181, and Satt434. The k2 Mdh1-n y20 chromosomal region was very close to Satt314, Satt253, and Satt279. The genetic distance between the Mdh1-n gene and Satt314 was less than 1 cM. The results of the mapping study were consistent with the results from previous studies that the Mdh1-n mutation in T261 (k2 Mdh1-n) and the Mdh1-n y20 mutation in T317 (Mdh1-n y20) were caused by deletions. In addition, another putative deletion was found in the genome of T261 which covered three SSR markers (Satt314, Satt253, and Satt279). This is a joint contribution of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa, Project No. 3769, and from the USDA, Agricultural Research Service, Corn Insects and Crop Genetics Research Unit, and supported by the Hatch Act and the State of Iowa. The mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by Iowa State University or the USDA, and the use of the name by Iowa State University or the USDA implies no approval of the product to the exclusion of others that may also be suitable.     

Molecular-marker-facilitated studies of morphological traits in maize. II: Determination of QTLs for grain yield and yield components

Genetic factors controlling quantitative inheritance of grain yield and its components have not previously been investigated by using replicated lines of an elite maize (Zea mays L.) population. The present study was conducted to identify quantitative trait loci (QTLs) associated with grain yield and grain-yield components by using restriction fragment length polymorphism (RFLP) markers. A population of 150 random F₂₃ lines was derived from the single cross of inbreds Mo17 and H99, which are considered to belong to the Lancaster heterotic group. Trait values were measured in a replicated trial near Ames, Iowa, in 1989. QTLs were located on a linkage map constructed with one morphological and 103 RFLP loci. QTLs were found for grain yield and all yield components. Partial dominance to overdominance was the primary mode of gene action. Only one QTL, accounting for 35% of the phenotypic variation, was identified for grain yield. Two to six QTLs were identified for the other traits. Several regions with pleiotropic or linked effects on several of the yield components were detected.     

An analysis of precursors accumulated by several chlorophyll biosynthetic mutants of maize

Summary Several mutants of maize defective in chlorophyll synthesis are analysed. By feeding shoots of dark-grown seedlings -aminolevulinic acid, the regulatory step in chlorophyll biosynthesis is bypassed and chlorophyll precursors accumulate. In normal plants this results in a buildup of protoporphyrin IX and protochlorophyllide, while mutants accumulate precursors, depending on the site of the mutant-induced lesion. Mutants at three loci, l ^*-Blandy4, 113, and oy, are defective in conversion of protoporphyrin IX to Mg-protoporphyrin. Mutants at the oro and oro2 loci are defective in conversion of Mg-protoporphyrin monomethyl ester to protochlorophyllide. A dominant modifier gene, Orom, which allows oro seedlings to bypass their lesion is also described.Journal Paper No J-9076 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa Project No. 2035     

Interval mapping of genes for quantitative resistance of maize to Setosphaeria turcica,cause of northern leaf blight,in a tropical environment

Quantitative trait loci (QTL) involved in the resistance of maize to Setosphaeria turcica, the causal agent of northern leaf blight, were located by interval mapping analysis of 121 F_2:3 lines derived from a cross between Mo17 (moderately resistant) and B52 (susceptible). A linkage map spanning 112 RFLP loci with 15 cM mean interval length was constructed, based on marker data recorded in a previous study. Field tests with artificial inoculation were conducted at three sites in tropical mid- to high-altitude regions of Kenya, East Africa. Host-plant response was measured in terms of incubation period, disease severity (five scoring dates), and the area under the disease progress curve (AUDPC). Heritability of all traits was high (around 0.75). QTL associated with the incubation period were located on chromosomes 2S and 8L. For disease severity and AUDPC, significant QTL were detected in the putative centromeric region of chromosome 1 and on 2S, 3L, 5S, 6L, 7L, 8L and 9S. On 2S the same marker interval which carried a gene enhancing latent period was also associated with reduced disease severity of juvenile plants. QTL on chromosomes 3L, 5S, 7L and 8L were significant across environments but all other QTL were affected by a large genotype x environment interaction. Partially dominant gene action for resistance as well as for susceptibility was prevailing. Single QTL explained 10 to 38% of the phenotypic variation of the traits. All but the QTL on chromosomes 1, 6 and 9 were contributed by the resistant parent Mo17. On chromosome 8L a QTL mapped to the same region as the major race-specific gene Ht2, supporting the hypothesis that some qualitative and quantitative resistance genes may be allelic.Abbreviations AUDPC area under the disease progress curve - CIMMYT International Maize and Wheat Improvement Center - KARI Kenya Agricultural Research Institute - NCLB northern corn leaf blight - QTL quantitative trait locus/loci     

Comparative mapping in F2∶3 and F6∶7 generations of quantitative trait loci for grain yield and yield components in maize

This study was conducted to compare maize quantitative trait loci (QTL) detection for grain yield and yield components in F₂₃ and F₆₇ recombinant inbred (RI) lines from the same population. One hundred and eighty-six F₆₇ RIs from a Mo17×H99 population were grown in a replicated field experiment and analyzed at 101 loci detected by restriction fragment length polymorphisms (RFLPs). Single-factor analysis of variance was conducted for each locus-trait combination to identify QTL. For grain yield, 6 QTL were detected accounting for 22% of the phenotypic variation. A total of 63 QTL were identified for the seven grain yield components with alleles from both parents contributing to increased trait values. Several genetic regions were associated with more than one trait, indicating possible linked and/or pleiotropic effects. In a comparison with 150 F₂₃ lines from the same population, the same genetic regions and parental effects were detected across generations despite being evaluated under diverse environmental conditions. Some of the QTL detected in the F₂₃ seem to be dissected into multiple, linked QTL in the F₆₇ generation, indicating better genetic resolution for QTL detection with RIs. Also, genetic effects at QTL are smaller in the F₆₇ generation for all traits.Abbreviations RFLPs Restriction fragment length polymorphisms - QTL quantitative trait loci - RIs recombinant inbreds Journal Paper no. J-16261 of the Iowa Agric and Home Economics Exp Stn Project no. 3134     

Theobroma cacao L.: a genetic linkage map and quantitative trait loci analysis

A genetic linkage map of Theobroma cacao (cocoa) has been constructed from 131 backcross trees derived from a cross between a single tree of the variety Catongo and an F1 tree from the cross of Catongo by Pound 12. The map comprises 138 markers: 104 RAPD loci, 32 RFLP loci and two morphologic loci. Ten linkage groups were found which cover 1068 centimorgans (cM). Only six (4%) molecular-marker loci show a significant deviation from the expected 11 segregation ratio.The average distance between two adjacent markers is 8.3 cM. The final genome-size estimates based on two-point linkage data ranged from 1078 to 1112 cM for the cocoa genome. This backcross progeny segregates for two apparently single gene loci controlling (1) anthocyanidin synthesis (Anth) in seeds, leaves and flowers and (2) self-compatibility (Autoc). The Anth locus was found to be 25 cM from Autoc and two molecular markers co-segregate with Anth. The genetic linkage map was used to localize QTLs for early flowering, trunk diameter, jorquette height and ovule number in the BC₁ generation using both single-point ANOVA and interval mapping. A minimum number of 2–4 QTLs (P<0.01) involved in the genetic expression of the traits studied was detected. Coincident map locations of a QTL for jorquette height and trunk diameter suggests the possibility of pleiotropic effects in cocoa for these traits. The combined estimated effects of the different mapped QTLs explained between 11.2% and 25.8% of the phenotypic variance observed in the BC₁ population.     

Life-prolonging effect of immunocell BAK (BRM-activated killer) therapy for advanced solid cancer patients: prognostic significance of serum immunosuppressive acidic protein levels

We devised an innovative type of immunocell therapy called BRM (biological response modifier)-activated killer (BAK) therapy, which utilizes most of non-MHC (major histocompatibility complex) restricted lymphocytes, CD56⁺ cells including T cells and NK cells. Peripheral blood lymphocytes were selected by immobilizing them with anti-CD3 monoclonal antibody, cultured for 2 weeks with serum-free medium containing IL-2, and then were reactivated by 1,000 U/ml of IFN- for 15 min. The patients were infused with about 6×10⁹ BAK cells by intravenous drip infusion at 1-month intervals. All advanced solid cancer patients who had received chemotherapy but for whom it was not effective or have refused chemotherapy were included in the present study. A good marker of impairment of host immune response by chemotherapy is an immunosuppressive acidic protein (IAP) level in serum above 580 g/ml; survival rates were compared with the high (>580 g/ml) and the low (580 g/ml) serum IAP groups. We enrolled in this study 23 immunosuppressed patients whose IAP levels in serum were over 580 g/ml, and 42 immunoreactive solid cancer outpatients whose IAP level in serum were under 580 g/ml and whose performance statuses were over 80% on the Karnofsky scale. After giving informed consent, patients were treated with BAK therapy on an outpatient basis at our hospital. The ethical review board of the Miyagi Cancer Center approved this pilot study. Treated with BAK therapy, the mean survival of immunosuppressed patients was 4.6 months. On the other hand, survival for one of immunoreactive advanced postoperative patients (stage IV) and inoperable lung cancer patients (stage IIIb) was 24.7 months. The difference in survival between the 2 groups was significant (P<0.01). This shows that BAK therapy is not indicated for an advanced cancer patient whose serum IAP is over 580 g/ml, perhaps due to extensive chemotherapy. Overall response to BAK therapy was complete response (CR) in 5 cases, partial response (PR) in 1 case, and prolonged no change (NC) in 26 cases, with an effectiveness rate at 76.2% in 42 advanced stage IIIb and IV cancer patients. BAK therapy has a life-prolonging effect without any adverse effects and maintains satisfactory quality of life (QOL) for advanced cancer patients.     

Genetic mapping of the Gy4 and Gy5 glycinin genes in soybean and the analysis of a variant of Gy4

The predominant storage protein of soybean [Glycine max (L.) Merr.] seed is a globulin called glycinin. Thus far five genes encoding glycinin subunits have been described, and these are denoted by the gene symbols Gy1 to Gy5. The objectives of this study were to map two of these genes, Gy4 and Gy5, and to conduct a genetic analysis of a subunit size-variant from an allele of Gy4. For this purpose a population was formed with an interspecific cross between PI 468916 (G. soja) and A81-356022 (G. max). The two size forms of G4, the subunit from Gy4, segregated codominantly in the mapping population, and were due to a short insertion in the hypervariable region of the mutant protein. The biochemical and molecular characteristics of the two subunits indicate that they are produced from alternate alleles of the same gene. The gene symbols Gy ^a and Gy ^b have been assigned to the normal and variant genes, respectively. When genomic DNA from the two parents was probed with a Gy4 cDNA, RFLPs were identified for both Gy4 and Gy5. Using these genetic markers, the Gy4 and Gy5 glycinin genes were mapped in linkage group O and F on the public soybean genomic map.Joint contribution of North Central Region, USDA-ARS and Journal Paper No. J-14736 of the Iowa Agric. and Home Economics Exp. Stn., Ames, IA 50011; Project 2763. This work was supported, in part, with grants from the Iowa State Biotechnology Program (No. 480-46-09) and the Iowa Soybean Promotion Board to RCS, and the American Soybean Association to NCN     

Genetic mapping of QTLs controlling horticultural traits in diploid roses

A segregating progeny set of 96 F₁ diploid hybrids (2n=2x=14) between Blush Noisette (D10), one of the first seedlings from the original Champneys Pink Cluster, and Rosa wichurana (E15), was used to construct a genetic linkage map of the rose genome following a pseudo-testcross mapping strategy. A total of 133 markers (130 RAPD, one morphological and two microsatellites) were located on the 14 linkage groups (LGs) of the D10 and E15 maps, covering total map lengths of 388 and 260 cM, respectively. Due to the presence of common biparental markers the homology of four LGs between parental maps (D10-1/E15-1 to D10-4/E15-4) could be inferred. Four horticulturally interesting quantitative traits, flower size (FS), days to flowering (DF), leaf size (LS), and resistance to powdery mildew (PM) were analysed in the progeny in order to map quantitative trait loci (QTLs) controlling these traits. A total of 13 putative QTLs (LOD>3.0) were identified, four for FS, two for flowering time, five for LS, and two for resistance to PM. Possible homologies between QTLs detected in the D10 and E15 maps could be established between Fs1 and Fs3, Fs2 and Fs4, and Ls1 and Ls3. Screening for pairwise epistatic interactions between loci revealed additional, epistatic QTLs (EQTLs) for DF and LS that were not detected in the original QTL analysis. The genetic maps developed in this study will be useful to add new markers and locate genes for important traits in the genus providing a practical resource for marker-assisted selection programs in roses.     

Marker-assisted congenic screening (MACS): A database tool for the efficient production and characterization of congenic lines

Over the past decades, genetic studies in rodent models of human multifactorial disorders have led to the detection of numerous chromosomal regions associated with disease phenotypes. Owing to the complex control of these phenotypes and the size of the disease loci, identifying the underlying genes requires further analyses in new original models, including chromosome substitution (consomic) and congenic lines, derived to evaluate the phenotypic effects of disease susceptibility loci and fine-map the disease genes. We have developed a relational database (MACS) specifically designed for the genetic marker-assisted production of large series of rodent consomic and congenic lines (speed congenics), the organization of their genetic and phenotypic characterizations, and the acquisition and archiving of both genetic and phenotypic data. This database, originally optimized for the production of rat congenics, can also be applied to mouse mapping projects. MACS represents an essential system for significantly improving efficiency and accuracy in investigations of multiple consomic and congenic lines simultaneously derived for different disease loci, and ultimately cloning genes underlying complex phenotypes.     

An in vitro approach to study chromosomal DNA damage

In this study a simple electrophoresis approach has been proposed for assessing DNA damage per chromosome in vitro. Novel procedures of gel casting, sample loading, electrophoresis and quantification of damage have been suggested. Sets of Saccharomyces cerevisiae chromosomes subjected to DNA damage by Bleomycin, Co⁶⁰--radiation alone and in combination with Hoechst were studied in detail. Statistical analyses showed that damage induced by Bleomycin bore linear positive correlation with %GA (r=0.97) and %GT (r=0.61) contents of chromosomes. Samples pre-treated with Hoechst showed much less damage by Co⁶⁰--irradiation as compared to samples not treated with Hoechst but exposed to Co⁶⁰--irradiation. The `protective effect of Hoechst' bore linear positive correlation (r=0.8) with %TAT content of chromosomes.