Tetraploid Counterparts Research Articles

Enhanced Na+ and Cl− sequestration and secretion selectivity contribute to high salt tolerance in the tetraploid recretohalophyte Plumbago auriculata Lam.

Enhanced secretion of Na+ and Cl- in leaf glands and leaf vacuolar sequestration of Na+ or root retention of Cl-, combined with K+ retention, contribute to the improved salt tolerance of tetraploid recretohalophyte P. auriculata. Salt stress is one of the major abiotic factors threatening plant growth and development, and polyploids generally exhibit higher salt stress resistance than diploids. In recretohalophytes, which secrete ions from the salt gland in leaf epidermal cells, the effects of polyploidization on ion homeostasis and secretion remain unknown. In this study, we compared the morphology, physiology, and ion homeostasis regulation of diploid and autotetraploid accessions of the recretohalophyte Plumbago auriculata Lam. after treatment with 300mM NaCl for 0, 2, 4, 6, and 8 days. The results showed that salt stress altered the morphology, photosynthetic efficiency, and chloroplast structure of diploid P. auriculata to a greater extent than those of its tetraploid counterpart. Moreover, the contents of organic osmoregulatory substances (proline and soluble sugars) were significantly higher in the tetraploid than in the diploid, while those of H2O2 and malondialdehyde (MDA) were significantly lower. Analysis of ion homeostasis revealed that the tetraploid cytotype accumulated more Na+ in stems and leaves and more Cl- in roots but less K+ loss in roots compared with diploid P. auriculata. Additionally, the rate of Na+ and Cl- secretion from the leaf surface was higher, while that of K+, Mg2+, and Ca2+ secretion was lower in tetraploid plants. X-ray microanalysis of mesophyll cells revealed that Na+ mainly accumulated in different cellular compartments in the tetraploid (vacuole) and diploid (cytoplasm) plants. Our results suggest that polyploid recretohalophytes require the ability to sequester Na+ and Cl-(via accumulation in leaf cell vacuoles or unloading by roots) and selectively secrete these ions (through salt glands) together with the ability to prevent K+ loss (by roots). This mechanism required to maintain K+/Na+ homeostasis in polyploid recretohalophytes under high salinity provides new insights in the improved maintenance of ion homeostasis in polyploids under salt stress.

In Vitro Induction and Primary Evaluation of Octoploid Plants in Saskatoon Berry (Amelanchier alnifolia Nutt.)

Polyploids of many species of horticultural plants are widely used in breeding programs since they are characterized by vigorous growth, larger organs, and greater resistance to biotic and abiotic stress. Saskatoon berry (Amelanchier alnifolia Nutt.) is in a form of diploid (2n = 2x = 34) and tetraploid. So far, no reports on induction of polyploidy in this species have been found. The aim of the study was to obtain Saskatoon berry octoploids with the use of in vitro shoot cultures. Shoots of the tetraploid of Canadian cultivars ‘Martin’ and ‘Smoky’ derived from in vitro cultures were used for induction of chromosome doubling. The shoot explants were incubated in a multiplication medium with the addition of one of the following antimitotics: colchicine, trifluralin, oryzalin or amiprophos methyl (APM) for two weeks (six days in the darkness and then eight days under a 16-h photoperiod). Then, the shoots were transferred to the multiplication medium without antimitotics and propagated in vitro by two subcultures. Octoploids were selected based on flow cytometry. Trifluralin showed the most phytotoxic effect. Six octoploids were obtained, four for ‘Martin’ after treatment with 250 mg L−1 of colchicine and two for ‘Smoky’ following the treatment with 5 mg L−1 of APM. All obtained octoploid individuals were multiplied and the level of ploidy was re-examined by flow cytometry and chromosome counting, which confirmed their octoploid genotype (2n = 8x = 136). They are probably the first octoploids of the genus Amelanchier in the world. The newly obtained octoploids were rooted in vitro, acclimatized to ex vitro conditions and grown in a greenhouse. Octoploids grew very slowly and showed a tendency to premature dormancy, which was manifested by inhibition of growth. In octoploid plants, the stomata were significantly larger and the number of stomata per 1 mm2 of leaf area was lower, compared to the tetraploid counterparts.

Influence of Rootstock Genotype and Ploidy Level on Common Clementine (Citrus clementina Hort. ex Tan) Tolerance to Nutrient Deficiency.

Nutrient deficiency, in particular when this involves a major macronutrient (N, P, and K), is a limiting factor on the performance of plants in their natural habitat and agricultural environment. In the citrus industry, one of the eco-friendliest techniques for improving tolerance to biotic and abiotic stress is based on the grafting of a rootstock and a scion of economic interest. Scion tolerance may be improved by a tetraploid rootstock. The purpose of this study was to highlight if tolerance of a common clementine scion (C) (Citrus clementina Hort. ex Tan) to nutrient deficiency could be improved by several diploid (2×) and their tetraploid (4×) counterparts citrus genotypes commonly used as rootstocks: Trifoliate orange × Cleopatra mandarin (C/PMC2x and C/PMC4x), Carrizo citrange (C/CC2x and C/CC4x), Citrumelo 4475 (C/CM2x and C/CM4x). The allotetraploid FlhorAG1 (C/FL4x) was also included in the experimental design. The impact of nutrient deficiency on these seven scion/rootstock combinations was evaluated at root and leaf levels by investigating anatomical parameters, photosynthetic properties and oxidative and antioxidant metabolism. Nutrient deficiency affects foliar tissues, physiological parameters and oxidative metabolism in leaves and roots in different ways depending on the rootstock genotype and ploidy level. The best known nutrient deficiency-tolerant common clementine scions were grafted with the doubled diploid Citrumelo 4475 (C/CM4x) and the allotetraploid FlhorAG1 (C/FL4x). These combinations were found to have less foliar damage, fewer changes of photosynthetic processes [leaf net photosynthetic rate (Pnet), stomatal conductance (gs), transpiration (E), maximum quantum efficiency of PSII (Fv/Fm), electron transport rate (ETR), ETR/Pnet], and effective quantum yield of PSII [Y(II)], less malondialdehyde accumulation in leaves and better functional enzymatic and non-enzymatic antioxidant systems. Common clementine scions grafted on other 4× rootstocks did not show better tolerance than those grafted on their 2× counterparts. Chromosome doubling of rootstocks did not systematically improve the tolerance of the common clementine scion to nutrient deficiency.

SYNTHESIS OF NEW INTERSPECIFIC TRIPLOID HYBRIDS FROM NATURAL AB GERMPLASM IN BANANA (MUSA SP.)

The release of new sweet-acid banana cultivars resistant to black leaf streak, Sigatoka leaf spot and Fusarium wilt is important for domestic markets in tropical and subtropical countries. Common current breeding strategies consist of selecting tetraploid AAAB hybrids directly from crosses between AAB sweet-acid cultivars pollinated with AA clones carrying disease-resistance genes. However, this crossing pathway is hampered by low gamete fertility and the rare occurrence of desired 2N gametes on the AAB female side (N=3X=33 chromosomes). We propose an alternative pathway which aims to create new triploid hybrids directly from AB landraces. Natural AB clones are sterile but their AABB tetraploid counterparts obtained by colchicine treatment are fertile. This gamete fertility was made profitable in crosses with AA and BB accessions to generate AAB and ABB hybrids. We present here agronomical results of various progenies involving an in vitro synthesized tetraploid 'Kunnan' (AABB) and several AA and BB clones. These first results suggest the very high potential of this new strategy for the release of wellperforming new hybrids combining higher productivity, disease resistance and better fruit quality. Hybrids with a high added-value, produced in this way, could be made available for evaluation within the ProMusa network. (Resume d'auteur)

First record of the natural occurrence of pentaploid loach, Misgurnus anguillicaudatus in Hubei Province, China

Natural occurrence of pentaploid loach, Misgurnus anguillicaudatus discovered from Liangzi Lake area, Hubei Province, central China, is reported here for the first time. The evidences from karyotyping, DNA content analysis and nuclear volume measurements were described to confirm the pentaploid nature of the identified individual. This individual was phenotypically indistinguishable from its sympatric diploid and tetraploid counterparts. The chromosome number was 5n = 125, the mean erythrocyte nuclear DNA content was 2.62 and 1.25 times of their diploid and tetraploid counterparts, respectively. An origin of such polyploidy form either from genome addition of normal sperm nuclei to unreduced egg and the mating of tetraploid individual and hexaploid individual, respectively, is hypothesized.

Involvement of p38α in the mitotic progression of p53-/- tetraploid cells

The tumor suppressor protein p53 plays a major role in preserving genomic stability. p53 suppresses a pathway leading from normal diploidy to neoplastic aneuploidy (via an intermediate metastable stage of tetraploidy) at two levels: first by preventing the generation/survival of tetraploid cells, and second by repressing their aberrant multipolar division. Here, we report the characterization of p53-/- tetraploid cells, which - at difference with both their p53-/- diploid and their p53+/+ tetraploid counterparts - manifest a marked hyperphosporylation of the mitogen-activated protein kinase MAPK14 (best known as p38α) that is particularly strong during mitosis. In p53-/- tetraploid cells, phosphorylated p38α accumulated at centrosomes during the metaphase and at midbodies during the telophase. Selective knockdown or pharmacological inhibition of p38α had a dramatic effect on p53-/- (but not p53+/+) tetraploids, causing the activation of the spindle assembly checkpoint, an arrest during the metaphase, a major increase in abnormal bipolar and monopolar mitoses, as well as an increment in the generation of multinuclear cells. We conclude that the mitotic progression of p53-/- (but not p53+/+) tetraploids heavily relies on p38α, revealing a novel function for this protein in the context of aneuploidizing cell divisions.

Plant origin and ploidy influence gene expression and life cycle characteristics in an invasive weed

BackgroundEcological, evolutionary and physiological studies have thus far provided an incomplete picture of why some plants become invasive; therefore we used genomic resources to complement and advance this field. In order to gain insight into the invasive mechanism of Centaurea stoebe we compared plants of three geo-cytotypes, native Eurasian diploids, native Eurasian tetraploids and introduced North American tetraploids, grown in a common greenhouse environment. We monitored plant performance characteristics and life cycle habits and characterized the expression of genes related to constitutive defense and genome stability using quantitative PCR.ResultsPlant origin and ploidy were found to have a significant effect on both life cycle characteristics and gene expression, highlighting the importance of comparing appropriate taxonomic groups in studies of native and introduced plant species. We found that introduced populations of C. stoebe exhibit reduced expression of transcripts related to constitutive defense relative to their native tetraploid counterparts, as might be expected based on ideas of enemy release and rapid evolution. Measurements of several vegetative traits were similar for all geo-cytotypes; however, fecundity of tetraploids was significantly greater than diploids, due in part to their polycarpic nature. A simulation of seed production over time predicts that introduced tetraploids have the highest fecundity of the three geo-cytotypes.ConclusionOur results suggest that characterizing gene expression in an invasive species using populations from both its native and introduced range can provide insight into the biology of plant invasion that can complement traditional measurements of plant performance. In addition, these results highlight the importance of using appropriate taxonomic units in ecological genomics investigations.

In vitro induction, regeneration and analysis of autotetraploids derived from protoplasts and callus treated with colchicine in Citrus

In the present paper attempts were made to induce chromosome doubling of ‘Meiwa’ kumquat (Fortunella crassifolia) protoplasts and ‘Frost’ navel orange (Citrus sinensis Osbeck) embryogenic callus via colchicine treatment. Colchicine decreased protoplast viability, delayed protoplast division and inhibited callus growth, indicating presence of toxicity to cells. Cell lines established from ‘Meiwa’ protoplasts treated with 0.01 and 0.1% colchicine for 8, 16 and 24 h at each concentration showed different responses when they were cultured on embryoid-induction medium. Flow cytometry (FCM) demonstrated that tetraploids were detected in cell lines and embryoids from all of the treatments, with the highest frequency being 19.23%. As for ‘Frost’, tetraploid cells were only detected when the callus was treated with 0.1% colchicine for either 4 or 8 days, from which plantlets were regenerated. FCM and chromosome counting confirmed them as true tetraploids. The diploid cells were more active in mitotic division during a 12-day culture and smaller in size than their tetraploid counterpart. Potential applications of the novel tetraploid germplasms obtained through in vitro chromosome doubling to citrus cultivar improvement are discussed.

Tetraploidía en Ceratophrys (Anura, Leptodactylidae), análisis del registro fósil

It has been confirmed the existence of the tetraploid counterpart of the genus Ceratophrys Wied, 1824 (extint at the present time) in Argentina and Brazil since the Pliocene, and the apparition of the octaploidy in the Upper Pleistocene - Holocene. Fossil material of the diploid form, distinctive of the Chacoan District, has been reported from the horizons of the Montehermosense Formation in south-east of the province of Buenos Aires, Argentina. The coexistence of diploid-tetraploid and tetraploid-octaploid forms in the same province was documented.

Feulgen-DNA densitometry of embryo sacs permits discrimination between sexual and apomictic plants in Paspalum simplex

The chromosomes of sexual diploid plants of Paspalum simplex were colchicine-doubled and the plant obtained were crossed with their aposporous natural tetraploid counterparts to generate a F1 population segregating for apomixis. Analysis of the DNA content during megagametogenesis indicated that although the nuclei of nucellus and developing embryo sacs were in both the G1 and G2 phases, polar nuclei and egg cells of mature embryo sacs tended to remain in the G1 phase. Because both meiotic and aposporous mature embryo sacs are of the 8-nucleated-type in P. simplex and are barely distinguishable, nuclear DNA content of polar nuclei was used to distinguish apomictic and sexual phenotypes and confirmation obtained by progeny testing.

A comparison of performance of tetraploid progenies produced by diploid and their vegetatively doubled (tetraploid) counterpart parents

Two diploid potato parents, W5295.7 and W5337.3, and their colchicine-doubled tetraploids were used as male to cross with three cultivars: ‘Raritan’, ‘Shepody’ and ‘Superior’. Both diploids produced 2n gametes via first division restitution (FDR) during meiosis. Field experiments were carried out on seedlings of the 12 hybrid progenies in 1991 and 1992. Six of the progenies represented the tetraploid-diploid (4x-2x) hybrids and the other six tetraploid-tetraploid (4x-4x) progenies. The genetic consequence of FDR gametes was examined by comparing the mean and genotypic variance of six tuber traits of 4x-2x and 4x-4x progenies. Tuber appearance, eye depth, specific gravity and tuber size showed significant differences between the means of 4x-2x and 4x-4x progenies from either both or one of the diploid parents. The genotypic variances of these traits in 4x-2x progenies were reduced in various degrees from those observed in 4x-4x progenies. Major genes responsible for the inheritance of these traits are probably located close to the centromere. The two diploid parents may also carry alleles of different size and direction of non-additive genic effects in their 2n gametes. The 4x-2x progenies also outperformed the 4x-4x progenies with respect to the means of total and marketable yield. There were thus heterotic gene loci for high yield located close to the centromere. No marked reduction of genotypic variance, however, was observed. The results suggest that genes for yield would be scattered between the centromere and the site for maximum recombination.

Genetic diversity evaluation of some elite cotton varieties by RAPD analysis.

Random amplified polymorphic DNA (RAPD) analysis was used to evaluate the genetic diversity of elite commercial cotton varieties. Twenty two varieties belonging to Gossypium hirsutum L. and one to G. arboreum L. were analyzed with 50 random decamer primers using the polymerase chain reaction (PCR). Forty nine primers detected polymorphism in all 23 cotton varieties, while one produced monomorphic amplification profiles. A total of 349 bands were amplified, 89.1% of which were polymorphic. Cluster analysis by the unweighted pair group method of arithmetic means (UPGMA) showed that 17 varieties can be placed in two groups with a similarity ranging from 81.51% to 93.41%. G. hirsutum L. varieties S-12, V3 and MNH-93 showed a similarity of 78.12, 74.46 and 69.56% respectively with rest of the varieties. One variety, CIM-1100, showed 57.02% similarity and was quite distinct. The diploid cotton G. arboreum L. var. Ravi was also very distinct from rest of its tetraploid counterparts and showed only 55.7% similarity. The analysis revealed that the intervarietal genetic relationships of several varieties is related to their center of origin. As expected, most of the varieties have a narrow genetic base. The results obtained can be used for the selection of possible parents to generate a mapping population. The results also reveal the genetic relationship of elite commercial cotton varieties with some standard "Coker" varieties and the diploid G. arboreum L. var. Ravi (old world cotton).

Comparison of diploid and tetraploid potato families derived from Solanum phureja x dihaploid S. tuberosum hybrids and their vegetatively doubled counterparts

To determine whether in potatoes the tetraploid level is preferable to the diploid level, especially regarding tuber yield, four diploid (2n=2×=24) Solanum phureja x dihaploid S. tuberosum hybrid parents and their vegetatively doubled, tetraploid (2n=4×=48) counterparts were intermated, which resulted in two F1 hybrid families at both levels of ploidy. The parents and clones of the F1 families and their offspring were used in crosses in such a way that in addition Sib1, Sib2, F1×Sib1, BC1 and Sib1×Sib1 families were produced. Of the first clonal generation of the 12 2 x families and their 12 counterpart 4 x families two tubers per clone were planted in three replications in a field experiment at Sturgeon Bay in 1969; of the parents six tubers were planted in each replication. Data were recorded on 16 characters, including plant height at four different times.

Chromosome elimination and chromosome pairing in tetraploid hybrids of Hordeum vulgare � H. bulbosum

The C0 tetraploid counterparts of diploid hybrids of Hordeum vulgare × H. bulbosum were meiotically analysed, and were found to be chromosomally less stable than the same genotypes had been as diploids. The 14 bulbosum chromosomes present in the tetraploid cytotypes were probably eliminated as pairs rather than randomly or one genome at the time. Development of the vulgare and bulbosum genomes was asynchronous in some hybrids, the bulbosum chromosomes appearing less advanced than the vulgare chromosomes in the same cell. This appeared to reduce pairing between bulbosum homologues and also suppressed homoeologous pairing.

Diploid‐Tetraploid Comparisons in Rye. III. Temperature Effects on Seedling Development1

The late fall planting season for rye in the southeastern USA is typically characterized by an extreme range of temperature conditions. The potential for selection of diploid and tetraploid rye (Secale cereale L.) populations possessing superior seedling growth characters over a broad range of temperatures was assessed by examining the effect of genetic background on various seeding growth characters at three temperatures (12, 20, 28°C). Diploid populations, and their colchicine‐derived tetraploid counterparts from five genetic backgrounds (‘Elbon’, ‘Explorer’, ‘Florida Black’, ‘Gator’, ‘Wrens Abruzzi’) were compared. The seedling characters included shoot and primary root growth day−1, root cross‐sectional area at the meristematic region, and primary root bluntness ratio (distance from the plane of maximum root diameter at the meristematic region to the root tip divided by the maximum root diameter at the meristematic region). Highly significant ploidy level (PL), genetic background (GB), and temperature (T) main effects were obtained for both shoot and primary root growth day−1. A highly significant T✕GB✕PL interaction was found for shoot growth day−1, but this effect was not significant for root growth day−1. The T ✕ PL interaction was significant for primary root growth day−1 at the 0.05 level. Highly significant ploidy level (PL) and temperature (T) main effects were obtained for root cross‐sectional area. A highly significant ploidy level (PL) main effect was found for the bluntness ratio. The lower ratios obtained for the tetraploid populations indicated that the tetraploid root tips were blunter than those of diploids, which may be a factor in soil penetration and root proliferation. The results indicated that substantial genetic variation for seedling growth characters was present among genetic backgrounds as well as within populations at both ploidy levels over a wide temperature range. It was concluded that selection for diploid as well as tetraploid rye populations possessing superior seedling growth characters over a broad range of temperatures would be successful.

Single cross alfalfa (Medicago sativa L.) hybrids produced via 2n gametes and somatic chromosome doubling: experimental and theoretical comparisons

The potential breeding value of 2n gametes from diploid alfalfa (2n = 2x = 16) was tested by comparing single cross alfalfa hybrids produced via 2n = 2x gametes from diploids versus n = 2x gametes from somatic-chromosome-doubled, tetraploid counterparts. Three diploid clones, designated 2x-(rprp), homozygous for the gene rp (conditions 2n gamete formation by a first division restitution mechanism) were colchicine-doubled to produce their tetraploid counterparts, designated 4x-(SCD). These six clones were crossed as males to the same cytoplasmic male sterile clone. Yield comparisons of progeny from the six clones demonstrated a significant yield increase of the hybrid progeny from 2n = 2x gametes from the diploids over the hybrid progeny from n = 2x gametes from the chromosome doubled tetraploid counterparts. The yield gain ranged from a 12% increase to a 32% increase. Theoretical comparisons indicated the 2n = 2x gametes from diploids would have 12.5 to 50% more heterozygous loci, on average, than the n = 2x gametes derived from somatic doubling. These results confirm the importance of heterozygosity on alfalfa yield, and the results demonstrate that 2n gametes formed by first division restitution offer a unique method for producing highly heterotic alfalfa hybrids.

The effects of colchicine doubling of potato dihaploid X wild species hybrids on eelworm resistance and pollen ‘stainability’

Diploid potato clones (dihaploid x wild species hybrids) and their colchicine doubled, tetraploid counterparts were compared for resistance to eelworm (Globodera rostochiensis andG. pallida) and for pollen ‘stainability’ as an indicator of viability. The responses due to doubling were not uniform. For eelworm resistance, three of the five clone pairs showed no differences; in one pair the tetraploid was significantly more susceptible to one pathotype but not to two others and in the remaining pair, the tetraploid was significantly more susceptible to both pathotypes used. In eleven of fourteen pairs tested for pollen ‘stainability’, the tetraploid had significantly less ‘good’ pollen. In one case, the tetraploid had significantly more ‘good’ pollen. Generally there was sufficient ‘good’ pollen to allow further crossing at the tetraploid level.

Changes in the lignin and hemicellulose concentrations of ten varieties of temperate grasses with increasing maturity

AbstractThe lignin and total hemicellulose concentrations of ten varieties of temperate grasses (five species) were determined with increasing maturity. A strong correlation existed between the lignin and hemicellulose concentrations. Lignin and hemicellulose concentrations tended to be higher in cocksfoot and timothy than in perennial ryegrass and diploid varieties also had higher lignin and hemicellulose concentrations than their tetraploid counterparts. The hemicellulose fractions were further separated into their linear and branched components whose compositions were determined. The varieties with higher lignin concentrations showed a higher linear:branched ratio and the ‘linear’ components also had higher xylose: arabinose ratios. These findings were consistent for both leaf and stem tissue although the effects from stem tissue were more marked.

Heterosis and Ploidy Effects on the Growth of Alfalfa Callus1

The influence of heterosis and ploidy level on callus fresh weight production in alfalfa (Medicago sativa L.) was studied using diploids (2x), their isogenic tetraploid (4xCD) counterparts derived by chromosome doubling, hybrids at 2x and 4x levels, and 4x and isogenlc 8xCD materials Chromosome doubling has an inbreeding fect equivalento sewing (2 to 3 generations in 4x), thus 4xCD and 8xCD materials were partial inbreds. Both the 4xCD and 8xCD materials produced less callus than their non‐inbred 2x and 4x counterparts, respectively. A set of 4x partial inbreds obtained by two generations of conventional self‐pollination (S2) also produced less callus than the non‐inbred (S0). Among hybrids evaluated, there was no heterosis for callus production among 2x hybrids which were derived from non‐inbred parents, but there was appreciable heterosis among most 4x hybrids which were derived from partially inbred 4xCD parents. Midparent heterosis for callus production was highly significant (p = 0.01) in three of four hybrids on one medium and in fottr of six 4x hybrids on the other medium. Ploidy effects which were not confounded with the inbreeding associated with chromosome doubling were evaluated using three sets of equivalent 2x and 4x hybrids. Two of the three 4x hybrids produced several fold more callus than equivalent 2x ybrids and the differences were highly significant (p = 0.01). Heterosis and ploidy effects were found to be more pronounced on one of two media used in several experiments. The potential use of heterosis as a selective mechanism in tissue culture experiments was discussed.

Cytogenetics of cultivated Bougainvilleas. V. Induced tetraploidy and restoration of fertility in sterile cultivars

Induced tetraploidy restores fertility in sterile cultivars of Bougainvillea. However, like the fertile diploid cultivars induced tetraploids are self-incompatible, but set seeds readily on crossing. Diploid progenitors show irregular meiosis, while in tetraploid counterparts there is predominant bivalent pairing. Low frequency of quadrivalents at 4x level implies that pairing at diploid level is between chromosomes heterozygous for cryptic structural changes. This also accounts for lower number of chiasmata per chromosome in diploids in comparison with their tetraploid counterparts. Sterility at 2x level appears to be due to recombination of cryptic structural differences and/or unequal distribution or aberrant behaviour of univalents. Predominant bivalent pairing accompanted by high fertility is a strong pointer towards preferential pairing in induced tetraploids.