Phenotypic diversity of barley powdery mildew resistance sources

One hundred and twenty-one of 123 previously detected new sources of wild barley (Hordeum vulgare ssp. spontaneum) resistant to powdery mildew (Blumeria graminis f. sp. hordei), and originating from Mediterranean area, and 20 standards were tested at the seedling stage for reaction to 38 selected Israeli powdery mildew isolates. The obtained resistance spectrum (a set of resistant and susceptible reactions) of each accession was divided into triplets and converted to octal numbers. This concentrates information on the resistance phenotype of each accession and makes their comparison much easier. One hundred and thirty-four different resistance spectra were determined. No compatible reaction was found in standards possessing genes mlo and Mlhb2 and in the accession PI 466634. The results demonstrate large diversity in new resistance sources, their distinction from the sources described till now as well as diversity in powdery mildew resistance of wild barley in the Mediterranean area. The results facilitate more effective further studies of such a large set of new resistance sources, and contribute to speeding up their use in barley breeding.     

Resistance to powdery mildew in populations of barley landraces from Morocco

Forty-eight populations of barley landraces collected from Morocco were screened for resistance to powdery mildew and a number of different resistance genes were detected. Landraces originated from the collection of the International Center for Agricultural Research in the Dry Areas – ICARDA, Aleppo, Syria. Twenty populations of tested landraces (about 42%) showed resistance reactions and 46 single plant lines were selected. Fourteen of these lines were tested in the seedling stage with 17 and another 32 lines with 23 differential isolates of powdery mildew, respectively. The isolates were chosen according to their virulence spectra observed on the Pallas isolines differential set. Five lines originating from five populations of landraces showed resistance to all prevalent in Europe powdery mildew virulence genes. Thirty-five lines (76%) showed resistance reaction type 2. The distribution of reaction type scores indicated that about 81% of all reaction types observed were classified as powdery mildew resistance (scores 0, 1 and 2). In forty-one lines (89%) the presence of unknown genes alone or in combination with a specific one was detected. Four different resistance alleles (Mlat, Mla6, Mla14 and Mla22) were postulated to be present in the tested lines alone or in combination. Among specific resistance alleles the most common was allele Mlat (resistance Atlas). This allele was postulated to be present in twenty-three (50%) tested lines. The use of new identified sources of resistance to powdery mildew in barley breeding is discussed.     

Sources of powdery mildew resistance in a wild barley collection

A total of 1,383 accessions of wild barley (Hordeum vulgare ssp. spontaneum) held in the USDA-National Small Grains Collection, Aberdeen were screened for resistance to powdery mildew (Blumeria graminis f. sp. hordei) in the Czech Republic. They were subsequently tested at the seedling stage for reaction to selected powdery mildew pathotypes and simultaneously for their adult plant resistance in the field. One hundred and twenty-three accessions exhibited resistance to 22 pathotypes in the greenhouse tests (reaction type 2–3 or lower) as well as resistance to the natural population of this pathogen in the field in the Czech Republic during at least 2-year testing. These accessions represent large amount of promising sources in breeding barley for resistance to powdery mildew.     

Genetic studies on the powdery mildew resistance of winter barley lines derived from Hordeum spontaneum accessions collected in Israel

The genetics of the powdery mildew resistance of 15 Hordeum spontaneum-derived winter barley lines were studied in testcrosses with selected cultivars carrying the MIa12 and MIa13 resistance alleles, in order to identify the inheritance of the resistance and to test for linkage with the MIa locus on chromosome 1H (5). A total of 27 genes were identified. Only one of these genes (in line 1–12 x Dura) could eventually be allelic to a gene previously identified in H. spontaneum Koch. A monofactorial dominant inheritance was identified in the majority of the lines. Three of the tested lines possessed recessively inherited genes and in one line a dominant and a recessive gene were identified. In respect to the reaction of the lines against European mildew cultures, lines with a bifactorially inherited resistance had a broader resistance spectrum than those with a monofactorial inheritance, while lines carrying recessive factors showed a moderately expressed resistance. In three cases a linkage with the MIa locus was found, while in two further lines a possible allelism to this locus was indicated by the absence of recombinant progeny plants. By using more than two isolates for linkage analysis, it was possible to identify additional genes for mildew resistance, reaching a maximum number of four genes in the same line. In some cases, a different inheritance was observed for the resistance of the same line against different isolates. It was assumed that different genes exist, each being effective against a different isolate.     

Ploidic and Molecular Analysis of ‘Morado de Huetor’ Asparagus (Asparagus officinale L.) Population; A Spanish Tetraploid Landrace

A study has been conducted into the genetic variation analysis of the tetraploid asparagus landrace ‘Morado de Huetor’ and its relationship with current commercial cultivars using Random Amplified Polymorphic DNA (RAPD) molecular markers. The presence of different ploidic levels in the ‘Morado de Huetor’ landrace has been also studied using cytogenetic analysis. Ten decamer oligonucleotides were used to obtain RAPD markers and to characterise 52 individuals of ‘Morado de Huetor’, 7 of the tetraploid cultivar ‘Purple Passion’, and 55 of 5 diploid hybrid commercial cultivars. Jaccard similarity index was calculated and a cluster analysis using UPGMA (Unweighted Pair-Group Method using Arithmetic Average) was performed. Tetraploid asparagus, ‘Morado de Huetor’ and ‘Purple Passion’, were well differentiated from the diploid ones showing a higher genetic variability. This result suggests a different origin for tetraploid and diploid asparagus varieties. Within the diploid cultivars a variety distribution was obtained. A specific monomorphic band (OPB20₈₈₃) was found in tetraploid varieties. Within the diploid cultivars, two bands (OPB20₈₃₀, OPC15₇₀₅) can be used to distinguish between the hybrid cultivars used in this study. Tri-, penta-, hexa-, and octoploid plants were found in ‘Morado de Huetor’. The origin of these ploidic levels is discussed and their use in the development of new varieties is proposed. In conclusion, ‘Morado de Huetor’ is a genetic resource that could be used to increase the narrow genetic background reported in diploid asparagus cultivars.     

Powdery mildew resistance in Aegilops tauschii coss. and synthetic hexaploid wheats

Summary A collection of 400 Ae. tauschii (syn. Ae. squarrosa) Coss. accessions were screened for powdery mildew resistance based on the response patterns of 13 wheat cultivars/lines possessing major resistance genes to nine differential mildew isolates. 106 accessions showed complete resistance to all isolates, and 174 accessions revealed isolate-specific resistance, among which were 40 accessions exhibiting an identical response pattern as wheat cultivar Ulka/^*8Cc which is known to possess resistance gene Pm2. Expression of both complete and isolate-specific resistance from Ae. tauschii was observed in some synthetic hexaploid wheats derived from four mildew susceptible T. durum Desf. parents, each crossed with five to 38 resistant diploid Ae. tauschii accessions. Synthetic amphiploids involving different combinations of T. durum and Ae. tauschii generally showed a decrease in resistance compared with that expressed by the Ae. tauschii parental lines.     

Sources of Resistance to Stem Rust (<Emphasis Type="Italic">Puccinia graminis</Emphasis> f. sp. <Emphasis Type="Italic">tritici</Emphasis>) in Ethiopian Tetraploid Wheat Accessions

Seedlings of 41 emmer (Triticum dicoccon Schrank) and 56 durum (T. durum Desf.) wheat accessions were evaluated for their response to stem rust (Puccinia graminis f. sp. tritici) infection under greenhouse condition at Kulumsa Agricultural Research Center, Ethiopia. The objectives were to identify tetraploid wheat accessions that could serve as sources of resistance to stem rust, and postulate the stem rust (Sr) resistance genes through multipatotype testing. The test included screening of accessions for stem rust resistance and multipatotype testing. To ensure vigorous screening, a mixture of six isolates (Si-1a, Am-2, Ku-3, Dz-4a, Ro-4 and Na-22) that were collected from severely infected emmer, durum, and bread wheat (Triticum aestivum L.) varieties of major wheat growing areas of Ethiopia was used as inocula. Out of the tested accessions, 18 emmer and 6 durum accessions exhibited low infection types (0–2) response and hence selected as a source of resistance to stem rust infection. Multipatotype testing was done to postulate Sr genes in the selected accessions. In the test, 10 different stem rust races (A2, A9, A11, A14, A16, A17, B3, B7, B15, and B21), 33 stem rust differential lines, and a universal susceptible check variety, Morocco were used, The high (3–4) and low infection type reaction patterns of the tested accessions and differential lines were used to postulate the genes that exhibit gene-for-gene relationship. The presence of Sr 7b, 8b, 9a, 9b, 10, 14, 24, 27, 28, 29, 30, 31, 32 and Tt-3+10 genes were postulated in 16 selected emmer and 5 durum wheat accessions. Efforts to transfer these valuable Sr genes from cultivated tetraploid wheats could be rewarding to get stem rust resistant varieties and boost wheat production.     

Evaluation ofFusarium wilt-resistance in primitive races ofGossypium hirsutum L.

Seven primitive races ofGossypium hirsutum L.punctatum, marie-galante, richmondi, latifolium, morrilli, palmeri, mexicanum and five F₁ crosses were inoculated withFusarium f. sp.vasinfectum physiological strain I. in the wilt field of Cixi Cotton Institute in 1987. There were highly significant differences among these primitive races.Punctatum andpalmeri were highly resistant. The resistances ofmarie-galante andmexicanum was similar with that of cultivar ‘Simian2’;richmondi, latifolium andmorrilli were susceptible. The disease incidence of F₁ in five primitive races hybridized with cultivar ‘Simian2’ was generally higher than that of mid-parent value. Inheritance of wilt resistance was partially dominant. In addition, 178 accessions of eight primitive races were evaluated for their resistance as before in 1989. There were significant differences in resistance among races and intra-race accessions. There were wilt resistance accessions among primitive races in which disease incidence was below 10%–25%. Inpalmeri, marie-galante, richmondi andpunctatum, one accession was immune with 0% disease incidence, respectively; 22.7–77.4% accessions were susceptible with 50% disease incidence. It showed that primitive races were mixed populations. They should be evaluated and screened for their utilization in cotton breeding program. Some best plants were selected from F₂ of two crosses, ‘Simian2’ ×palmeri and ‘Simian2’ ×mexicanum, based on the resistance toFusarium wilt and the response to photoperiod. 11 lines had been obtained with good fiber quality and relatively high potential production by backcrossing improvement. These lines are now being tested.     

Genetic Nature of Yams (Dioscorea sp.) Domesticated by Farmers in Benin (West Africa)

‘Domestication’ is a traditional farmers’ practice reported for yams (Dioscorea sp.) in Benin (West Africa). It involves introducing ‘spontaneous’ (naturally occurring) yams, supposedly wild (D. abyssinica and D. praehensilis), in varieties of the D. cayenensis–D. rotundata cultivated species complex. In this study, we established the genetic nature of ‘predomesticated’ yam plants using the amplified fragment length polymorphism (AFLP) technique. A total of 213 accessions, consisting of 32 predomesticated yams, 70 D. cayenensis–D. rotundata, 86 D. abyssinica and 25 D. praehensilis yams were analysed. Using 91 AFLP markers, three groups of accessions were distinguished, broadly corresponding to the above botanical species. Of the 32 predomesticated accessions, 16% were clustered with D. praehensilis, 37% with D. abyssinica and the remaining 47% with D. cayenensis–D. rotundata. These results demonstrated the use of wild plants by farmers in their domestication process, and suggested that plants derived from intervarietal and interspecific hybridisation may also be subject to this process. This study has shown that through domestication farmers influence and increase the genetic diversity in yam by using sexual reproduction of wild and possibly cultivated yams.     

On the Taxonomy of the Members of ‘Saccharum Complex’

The difficulty and problems encountered in the study of cultivated plants, in general and sugarcane, in particular has been indicated. In order to understand these problems, a brief review on the taxonomy of Saccharum and closely related taxa, namely, Erianthus, Sclerostachya, Narenga and Miscanthus (generally known as ‘Saccharum complex’) has been given. A short account on the important morphological features that are specific to sugarcane has also been stated as classification is commonly based on such morphological characters. A note has been added on the chromosome number, origin, and distribution of the species of ‘Saccharum complex’ members. Taxonomic keys have been devised for identification of the genera of Saccharinae and for the species of Saccharum and Erianthus occurring in India. A new combination, Sclerostachya fallax (Balansa) Amalraj et Balasundaram, has also been proposed.     

Sources of resistance to Hessian fly (Diptera: Cecidomyiidae) in Syria identified among Aegilops species and synthetic derived bread wheat lines

The Hessian fly, Mayetiola destructor (Say), is a major pest of wheat in North Africa, southern Europe, North America, and northern Kazakhstan. It is believed this pest (like wheat) originated in West Asia. The Syrian Hessian fly biotype has been found to be the most virulent worldwide, and has been used at the International Center for Agricultural Research in the Dry Areas (ICARDA) for screening wheat and its wild relatives to identify new sources of resistance. The screening was conducted in an insect rearing room set at 20°C and 70% RH using a Hessian fly population collected from Lattakia region, Syria. The experimental design was a randomized complete block with four replications. ‘Nasma’ (bread wheat) and ‘Cando’ (durum wheat) were used as susceptible and resistant checks, respectively. A total of 623 lines/accessions of wheat and its wild relatives (Aegilops and Triticum) were evaluated. Twenty-nine Aegilops accessions and four synthetic derived bread wheat lines were found resistant. The presence of dead first instars confirmed the resistance reaction and also showed that antibiosis is the major mechanism of resistance in these materials. These sources of resistance are used in ICARDA’s wheat breeding programs for the development of Hessian fly-resistant germplasm/varieties.     

Identification of new sources for resistance to powdery mildew in H. spontaneum-derived winter barley lines

Twenty four barley lines derived from the F ₇generation of crosses between two winter barley cultivars and different accessions of Hordeum spontaneum Koch collected in Israel were tested against a set of ten European and five Israeli powdery mildew cultures, possessing virulence genes which completely match the spectrum of known mildew resistance genes. The comparison of reaction patterns justified the conclusion that new genes for resistance have been conferred from H. spontaneum which also differ from genes previously identified in other wild barley accessions from Israel. Participation in the expression of the resistance reaction of one of the two barley cultivars used in the development of the lines is well documented in some cases. The majority of the lines was found to be highly resistant against all or almost all European isolates, while various kinds of reactions were observed against the Israeli cultures. The effective transfer of novel mildew resistance from H. spontaneum into winter barley confirms similar results with spring barley, indicating natural wild barley populations in Israel as a significant gene pool for yet unexploited mildew resistance in barley.     

Genetic Variation Among Portuguese Landraces of ‘Arrancada’ Wheat and <Emphasis Type="Italic">Triticum petropavlovskyi</Emphasis> by AFLP-based Assessment

Portuguese wheat landraces, ‘Arrancada’ were collected from the Aveiro region, Portugal before the 1950s. We found in eight accessions of `Arrancada' hexaploid wheat with the long glume phenotype. We assessed the comparative genetic diversity among Portuguese `Arrancada' wheat and Triticum petropavlovskyi Udacz. et Migusch. using AFLP assays and discuss the origin of long glumed `Arrancada' wheat. With the four primer pairs a total of 4885 visible bands were scored corresponding to 99 AFLP markers as putative loci, of which 55 markers (54%) were polymorphic. UPGMA clustering and PCO grouping showed that long glumed ‘Arrancada’ wheat and T. petropavlovskyi were genetically diverse. Long glumed ‘Arrancada’ hexaploid wheat separated into two clusters (groups) in both the UPGMA dendrogram and in PCO analysis. Four long glumed accessions fell in the cluster of tetraploid wheat. A similar argument could be made for another four accessions which belong to the cluster of hexaploid wheat. The substantial level of genetic variation indicated that long glumed ‘Arrancada’ wheat and T. petropavlovskyi originated independently. It is most likely that the P-gene of long glumed ‘Arrancada’ hexaploid wheat was introduced from T. turgidum ssp. polonicum (L.) Thell. to T. aestivum via natural introgression or breeding. We suggest that the long glumed ‘Arrancada’ hexaploid wheat did not originate from T. aestivum through spontaneous mutation at the P locus     

Exploring the Serbian GenBank barley (Hordeum vulgare L. subsp. vulgare) collection for powdery mildew resistance

Unlocking resistance genes in genbank collections are of prime importance for securing sustainable crop production. In this regard, the Serbian GenBank barley collection, comprising 93 local landraces and 36 commercial cultivars and elite barley breeding lines, was screened for novel resistances to powdery mildew (Blumeria graminis f. sp. hordei) using a set of 28 isolates with a wide spectrum of virulences/avirulences. No line was resistant to all the isolates, but one and three accessions showed resistance to 27 and 26 isolates, respectively. Twenty landraces (21.51 %) and ten cultivars (27.78 %) exhibited resistance to 50 % of the isolates. Infection type 2 was most frequent among resistant accessions. Nine B. graminis isolates were sufficient for gene postulation in 73 barley lines. In total, thirty-five different resistance spectra were recorded and the following known resistance genes were postulated namely, Mlra,Mlh,Mla12, Mla7(Mlu), Mlg, MlLa, Mla6, Mla7, Mlt, Mla22, Mlat, Mla1, Mlk. The majority of resistance profiles was constituted by only one line. Unidentified genes alone or in combination were proposed for twenty landraces and six cultivars. This report demonstrated that the barley collection held at the Serbian GenBank could be exploited as a new source for powdery mildew resistance.     

A note on the evolution of kabuli chickpeas as shown by induced mutations in <Emphasis Type="Italic">Cicer reticulatum</Emphasis> Ladizinsky

Two distinct chickpeas of the domestic chickpea, C. arietinum L., exist and are referred to as ‘desi’ or microsperma and ‘kabuli’ or macrosperma. Cicer reticulatum Ladiz. is considered to be the wild progenitor of the domestic chickpea. However, the morphological variation in 18 original accessions of C. reticulatum is narrower than those of the domestic chickpeas. The aim of the study is to increase the variability in C. reticulatum. In M₂ generation, a mutant with white flower color was isolated despite of the fact that the parent has the pink flower. Although seed coat color of the parent was dark brown, the mutant was cream like ‘kabuli’ chickpea. It is commonly accepted that the large seeded domestic ‘kabuli’ chickpeas originated from the small seeded ‘desi’ chickpeas, but the induced mutants (white flower and cream seed coat color) of C. reticulatum may suggest an additional path for the evolution of ‘kabuli’ chickpea. ‘Kabuli’ chickpeas could have originated from spontaneous mutants of C. reticulatum. In M₃ generation, multipinnate leaf, erect growth habit, green seed and double-podded chickpeas were isolated. Among these progenies, morphologic variability increased and approached domesticated chickpea. Based on historical records and the induced mutants obtained from this study, the domestic ‘kabuli’ chickpea could have directly emerged from C. reticulatum in ancient Eastern Turkey.     

Evaluation of cultivated and wild barley for resistance to pathotypes ofPuccinia hordei with wide virulence

An isolate ofPuccinia hordei (ND89-3) originally collected in Morocco is virulent on most barley genotypes reported to possess resistance, except cultivar Estate (CI 3410), which possesses theRph3 gene and exhibits a low to intermediate level of resistance (infection type 12). Isolate ND89-3 possesses one of the widest virulence spectrums reported forP. hordei. Accessions ofHordeum vulgare (1,997 in total) andH. spontaneum (885 in total), mostly originating from the Mediterranean region and parts of North Africa, were evaluated with isolate ND89-3 at the seedling stage to identify new sources of leaf rust resistance. Fifty-eight accessions ofH. vulgare, and 222 accessions ofH. spontaneum exhibited low infection types to this isolate. Further evaluations of these resistant accessions with isolates ofP. hordei virulent forRph3,Rph7, andRph12 suggested that most of the resistantH. vulgare accessions possess theRph3 gene. Data suggested additional sources of effective resistance inH. vulgare are rather limited. FiveH. vulgare accessions and 167H. spontaneum accessions were identified as possible sources of new genes for leaf rust resistance. These accessions likely possess resistance genes that are different fromRph1 toRph12, or gene combinations thereof based on their reaction to four leaf rust isolates. Utilization of these accessions in barley breeding will broaden the germplasm resources available for genetic control ofP. hordei. North Dakota Agricultural Experiment Station Journal Series No. 2123. The U.S. Government right to retain a non-exclusive, royalty free licence in and to any copyright is acknowledged.     

<Emphasis Type="Italic">Zizania latifolia</Emphasis> Turcz. cultivated in China

Zizania latifolia, which belongs to the tribe Oryzeae, has been cultivated for more than 2000 years and has historically been used in China mainly as an aquatic vegetable. In China, the largest area under cultivation of this plant is located in the regions surrounding Tai Lake, in the provinces of Jiangsu and Zhejiang. Two main types of cultivars have been developed, in one the shoots can be harvested only once, in the fall; in the other, the shoots may be harvested twice in the fall and once the following summer. Z. latifolia can be bred and conserved through clonal propagation as a means of preventing infection of flowering stems by the epiphyte Ustilago esculenta. At the end of the harvest period, healthy individuals and the plants with black spots in the shoots are removed, and those with a healthy clone will be identified and selected as ‘seed’ for the next year.     

Assessing genetic diversity of Polish wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis>) varieties using microsatellite markers

Fifty-three wheat cultivars have been genotyped using 24 SSR (simple sequence repeat) markers in order to evaluate genetic similarities among Polish wheats, i.e. 53 spring and winter cultivars; ‘Chinese Spring’ was taken as reference. ll but one SSR marker allowed to identify DNA polymorphisms, giving in total 166 alleles (including nulls), from 3 to 13 alleles per marker with mean of 7.22. Based on marker data, genetic similarities were calculated and a dendrogram was created. ‘Spring’ cultivars were less diverse than winter ones, showing the biggest similarity to ‘Chinese Spring’. Four sister cultivars (Nutka, Tonacja, Zyta and Sukces), formed a cluster of very similar materials, of which Zyta and Sukces had the highest similarity indices. Parental lines Jubilatka and SMH 2182 were more distant from each other (genetic similarity of 0.227). It was possible to differentiate all the wheats using only four SSR markers: Xgwm186, Xgwm389, Xgwm459 and Xgwm577.     

Molecular Markers for the Classification of Switchgrass (Panicum virgatum L.) Germplasm and to Assess Genetic Diversity in Three Synthetic Switchgrass Populations

Information regarding the amount of genetic diversity is necessary to enhance the effectiveness of breeding programs and germplasm conservation efforts. Genetic variation between 21 switchgrass genotypes randomly selected from two lowland (‘Alamo’ and ‘Kanlow’) and one upland (‘Summer’) synthetic cultivars were estimated using restriction fragment length polymorphism (RFLP) markers. Comparison of 85 RFLP loci revealed 92% polymorphism between at least two genotypes from the upland and lowland ecotypes. Within ecotypes, the upland genotypes showed higher polymorphism than lowland genotypes (64% vs. 56%). ‘Kanlow’ had a lower percent of polymorphic loci than ‘Alamo’ (52% vs. 60%). Jaccard distances revealed higher genetic diversity between upland and lowland ecotypes than between genotypes within each ecotype. Hierarchical cluster analysis using Ward's minimum variance grouped the genotypes into two major clusters, one representing the upland group and the other the lowland group. Phylogenetic analysis of chloroplast non-coding region trnL (UAA) intron sequences from 34 switchgrass accessions (6 upland cultivars, 2 lowland cultivars, and 26 accessions of unknown affiliation) produced a neighbor-joining dendrogram comprised of two major clusters with 99% bootstrap support. All accessions grouped in the same cluster with the lowland cultivars (‘Alamo’ and ‘Kanlow’) had a deletion of 49 nucleotides. Phenotypic identification of greenhouse-grown plants showed that all accessions with the deletion are of the lowland type. The deletion in trnL (UAA) sequences appears to be specific to lowland accessions and should be useful as a DNA marker for the classification of upland and lowland germplasm.     

Genetic Analysis of Egyptian Date (<Emphasis Type="Italic">Phoenix dactylifera</Emphasis> L.) Accessions Using AFLP Markers

Forty-seven samples of date palm (Phoenix dactylifera L.) collected from eight locations in Egypt were studied using four sets of amplified fragment length polymorphism (AFLP) markers with near infrared fluorescence labeled primers. These samples belonged to 21 named accessions and 9 of unknown pedigrees. A total of 350 bands were scored and 233 (66.6%) were polymorphic. Twenty-seven Egyptian accessions and ‘Medjool’and ‘Deglet Noor’accessions from California could beclassified into the major cluster. This major cluster may represent a major group of date palm germplasm in North Africa. There were four other clusters, each containing one or two accessions. The variety ‘Halawy’and one accession of unknown provenance were most likely from hybridization between two clusters. Six groups of accessions of which had the same names, revealed similar but not identical AFLP profiles suggesting these accessions might derive from seedlings rather thanthrough clonal offshoot propagation.