Cross-species Transferability of Rice Microsatellites in its Wild Relatives and the Potential for Conservation Genetic Studies

Here, we investigated the transferability of 60 microsatellite markers characterized for cultivated rice Oryza sativa L. in three wild Oryza species representing different genome types: O. rufipogon Griff. (AA), O. officinalis Wall. et Watt. (CC), and O. granulate Nees et Arn. ex Watt. (G). The results indicate the 60 rice SSR loci tested produced homologous amplification products to different extents in O. rufipogon (100%), O. officinalis (90%) and O. granulata (73.3%). Proportions of polymorphism for successfully amplified loci ranged from 0.983 via 0.667 to 0.364 in O. rufipogon, O. officinalis and O. granulata, respectively. The utility of these microsatellite markers was tested for the characterization of genetic diversity in 117 genotypes of these four Oryza species. The values of genetic diversity in cultivated rice are higher than the other two wild species O. officinalis and O. granulata, suggesting microsatellites tend to have more variability in the focal species than in non-focal species to which they are applied. However, much lower levels of genetic variation were observed in rice than in its wild progenitor O. rufipogon, which indicates severe loss of genetic variation may reflect the ‘domestication bottleneck’ through which rice passed. The observation that most of the rice microsatellites are able to detect allelic polymorphisms at different extent in Oryza species suggest that rice microsatellite loci should be useful for the analysis of genetic diversity and inter- and intra-specific relationships in the genus. Therefore, high rates of successful cross-amplification of rice microsatellites among Oryza species with different genome types will offer excellent opportunities to investigate the population genetic structure of wild rice species and explore their conservation genetics.     

Diversity of native rice (Oryza Poaceae:) species of Costa Rica

We found several populations of wild Oryza species in the lowlands of Costa Rica. The plants showed extensive morphological variation, suggesting the presence of several species. In the morphologic study, 33 traits were scored for plants of all the species. A principal component analysis revealed the significant morphological separation of the different species. The analyses indicated that there are three species, O. grandi­glumis, O. latifolia and O. glumaepatula. Two putative hybrid types were found, both significantly differing in their morphology from the known species and intermediate at several traits. O. grandi­glumis is a new record for Costa Rican flora. Its main population is located in Caño Negro Wildlife Refuge, Los Chiles, Alajuela. O. latifolia is distributed throughout the lowlands of the country and the plants of the Atlantic slope are significantly bigger in general habit than those of the Guanacaste area. During this study a population of O. glumaepatula of hundreds of thousands of plants was discovered in the Medio Queso River wetland, Los Chiles, Alajuela. This population is the most important source of genes for cultivar's improvement from the primary gene pool of rice in Costa Rica. The small ligule and the wide flag leaf characteristic of the two CCDD species separated them from the AA diploid O. glumaepatula. Seed size, ligule size, number of branches in the panicle, plant height and sterile lemma length are all bigger in O. grandi­glumis, and influenced the second factor that separated the CCDD species in two discrete clusters. The species found offer great possibilities for the improvement of rice cultivars and they should be thoroughly studied and appropriately protected.     

Exploring genetic diversity and potential novel disease resistance genes in a collection of rice (<Emphasis Type="Italic">Oryza</Emphasis> spp.) wild relatives

Wild relatives of rice (Oryza spp.) are an important source of novel resistance (R-)genes for rice improvement. Rice sheath blight, caused by Rhizotonia solani, and leaf blast, caused by Magnaportha oryzae, are major fungal diseases of rice worldwide. To identify novel R-genes, a group of Oryza spp. accessions represented by O. alta, O. australiensis, O. barthii, O. glaberrima, O. glumaepatula, O. latifolia, O. meridionolis, O. nivara, O. officinalis, and O. rufipogon, were evaluated for their reaction to leaf blast and sheath blight disease, and genotyped with 176 microsatellite (SSR) markers. Selected rice (O. sativa) accessions were included as reference. Cluster analysis performed with PowerMarker software using Rogers genetic distance and UPGMA, revealed most Oryza spp. accessions clustered with the same species or a closely related Oryza spp. Only a few Oryza spp. accessions grouped with the O. sativa accessions included as a reference. Analysis of this genotypic data in the software Structure revealed that the Oryza spp. accessions were assigned into eight different subpopulations and fit well into eight different backgrounds. Marker-trait associations between the SSR markers and disease reactions to blast and sheath blight were ascertained using the software TASSEL. Associations with blast disease were identified in ten different chromosomal regions and five of the ten were not located near known blast R-genes. Three associations were discovered with sheath blight disease and one was not near previously reported sheath blight QTL. These newly identified regions may represent novel R-genes that will be the basis future fine mapping studies. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Electrophoretic variation in seed proteins and interrelationships of species in the genus <Emphasis Type="Italic">Oryza</Emphasis>

Comparative studies of two cultivated and sixteen wild species of the genus Oryza were carried out using one- and two-dimensional gel electrophoresis for variation in their seed proteins for interrelationships of these species. A number of polypeptides in the range of molecular weight 13–110 kDa were seen to vary. Under reducing conditions, polypeptides spread over the regions of mol. wt. 33–40.5, 25–27 and 19–21.5 kDa exhibited maximum variation in their patterns. Two-dimensional gel electrophoresis revealed the occurrence of disulphide-linked glutelin polypeptide pairs of mol. wt. 60, 58, 52 and 25 kDa breaking into a large and a small subunit each in the range of mol. wt. 18–40.5 and 16–25 kDa respectively in Oryza sativa. The number of such polypeptide pairs varied from 2 to 6 in different species and also in O. sativa showed variation in mol. wts. of their constituent subunits. The UPGMA dendrogram revealed that most of the Oryza species occurred in different clusters and subclusters and thus did not share very close relationships. The undisputed and closest relationship observed was that of cultivated rice O. sativa with the O. rufipogon followed by that with O. nivara. The African cultivated O. glaberrima occurring on the nearest branch of the same subcluster, thereby, supporting the phylogenetic of these species suggested in earlier studies. Eight diploid species and seven tetraploid species were included in one part of the dendrogram while the remaining two species with AA genome i.e. O. glumaepatula and O. meridionalis and one with FF i.e. O. brachyantha stood separately from these as scattered in the group of seven tetraploid species with BBCC, CCDD and HHJJ genomes. The tetraploids O. alta, O. latifolia and O. grandiglumis with CCDD genomes which occurred on the farthest part were distantly related with O. sativa. The cyanogen bromide peptide maps and two dimensional gel electrophoresis also supported the closest relationship between O. sativa and O. rufipogon.     

Phosphorus Response of Oryza sativa,O. glaberrima,and Hybrid Rice Cultivars on an Ultisol

Phosphorus (P) deficiency is a major constraint to upland rice production on Ultisols in the humid zone of West Africa. Integrated use of P-efficient cultivars and P nutrition is needed for enhanced sustainable productivity on these soils. This article reports on the P responsiveness of interspecific rice hybrids (crosses from Oryza sativa and O. glaberrima) along with O. sativa and O. glaberrima cultivars grown on an acidic Ultisol, low in available P. The cultivars differed in yield and P-uptake response to fresh and residual P. Two interspecific cultivars gave a linear response to P and produced the greatest grain yield under direct and residual P. The O. glaberrima cultivar CG 14 did not respond to the applied P, whereas the O. sativa cultivar was moderate in its performance. Our results show that the interspecific rice cultivars have the potential to adapt and perform well on acidic upland soils.     

Genetic diversity of cultivated rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) and wild rice (<Emphasis Type="Italic">Oryza rufipogon</Emphasis> Griff.) in Asia,especially in Myanmar,as revealed by organelle markers

Asian rice (Oryza sativa L.) is widely cultivated in Asia, where it has been classified into Indica and Japonica Group, the latter is further classified into Tropical and Temperate Japonica Subgroup. O. rufipogon is believed to be the closest ancestor to O. sativa, but it remains unclear whether the two groups arose from a single ancestor or different ancestors. Therefore, here, we investigated the matrilineal ancestors of O. sativa using markers for organelle (chloroplast and mitochondrial) genomes, and 119 O. sativa landraces, 10 O. glaberrima Steud., 115 O. rufipogon Griff. from Asia, and 39 accessions from other wild rice species with AA genomes. We screened 18 organelle markers developed based on polymorphic loci in the organelle genomes. In addition, we used the open reading frame 100 of a chloroplast marker. The results indicated that O. rufipogon first differentiated into two lineages and then further differentiated into Indica and Japonica Group, respectively. Accessions of O. rufipogon (R-1f and R-2d types) from Myanmar appear to be the closest ancestors of Tropical Japonica Subgroup and Indica Group, respectively. Therefore, these wild strains may have made a strong contribution to the domestication of rice landraces in Myanmar.     

Genetic Diversity in AA and CC Genome Oryza Species in Southern South Asia

The CC genome of Oryza is found in nine species of Oryza that are distributed on all continents having a tropical climate. Three diploid Oryza species with CC genome are found in Africa and Asia to Papua New Guinea. In southern South Asia these three CC genome diploid species can be found, O. eichingeri and O. rhizomatis in Sri Lanka and O. officinalis in India. AA genome wild relatives of rice are also found in the same geographic region. Germplasm of both diploid CC and AA genome Oryza germplasm has recently been collected from Sri Lanka. AFLP analysis was used to compare the genetic diversity of the two Oryza genomes from a similar geographic region in southern South Asia. In addition, the diploid CC Oryza germplasm was also analyzed by RAPD and SSR methodologies and the combined results were analyzed. The results show that in southern South Asia the diploid CC genome species have a high level of genetic diversity compared to the diploid AA genome species. Molecular marker analysis revealed that populations of O. rhizomatis from northern and southeastern Sri Lanka are genetically differentiated. One accession of O. rhizomatis was aligned with O. eichingeri. This accession was collected from the site of O. rhizomatis that is the closest to a population of O. eichingeri. O. eichingeri showed lower genetic diversity than the other two diploid Oryza CC genome species. O. officinalis accessions from Assam, India, and China were genetically less diverged from O. eichingeri and O. rhizomatis than two accessions of O. officinalis from Kerala state, India. The first two authors contributed equally to this research     

Dual origin of the cultivated rice based on molecular markers of newly collected annual and perennial strains of wild rice species, Oryza nivara and O. rufipogon

The direct ancestor of rice (Oryza sativa L.) is believed to be AA genome wild relatives of rice in Asia. However, the AA genome wild relatives involve both annual and perennial forms. The distribution of the retrotransposon p-SINE1-r2, a short interspersed nuclear element (SINE) at the waxy locus was analyzed in diverse accessions of the AA genome wild relatives of rice (O. rufipogon sensu lato). Most annual wild rice accessions had this retrotransposon, while most perennial types lacked this element, contradicting results to the previous studies. Results presented here suggest that O. sativa has dual origin that lead to indica-japonica differentiation. Results suggest the indica line of rice varieties evolved from the annual genepool of AA genome and the japonica varieties from the perennial genepool of AA genome wild rice.     

The fragrance (fgr) gene in natural populations of wild rice (Oryza rufipogon Griff.)

The Asian cultivated rice, Oryza sativa L. (spp. indica or japonica), is assumed to have originated from one or both of the two wild Asian species, O. rufipogon Griff. and O. nivara Sharma and Shastry. They occur throughout the monsoon Asia and west Oceania. Fragrance is the most important trait among the domesticated characters of basmati and jasmine rice of Asia. The gene for fragrance in a scented rice shows the presence of a mutated portion (i.e., an eight base pair deletion in exon 7) that result in its loss of fragrance. In the present study, 229 wild rice O. rufipogon accessions were genotyped for this locus using a PCR assay. The wild rice species contained the mutated allele of the fgr gene at a low frequencies of 0.23. The surveyed populations were in Hardy–Weinberg equilibrium. This observation supports the hypothesis that the allele for fragrance was already present in the wild rice, and that this trait appeared in scented rice cultivars because of selection by the farmers of genotypes possessing this character during the process of domestication.     

Identification of <Emphasis Type="Italic">SUB1A</Emphasis> alleles from wild rice <Emphasis Type="Italic">Oryza rufipogon</Emphasis> Griff.

Submergence stress is a major constraint to rice production in South and Southeast Asia. Most rice (Oryza sativa L.) cultivars die within a week of complete submergence, while a small number of accessions are submergence-tolerant for up to 2 weeks or more. These cultivars have the tolerant allele of the SUB1A gene, one of three ERF genes at this locus on rice chromosome 9. In all O. sativa varieties studied, the SUB1A gene is limited to a subset of indica accessions of O. sativa. Thus far, there has been no published report of the SUB1A gene in wild rice species. Here we report evidence of the SUB1A gene found in wild species of O. rufipogon Griff. accessions by the use of degenerate primers corresponding to the most highly conserved regions of the SUB1 locus. The results indicated that two SUB1A-like alleles, e.g. OrSub1A-1 and OrSub1A-2, were identified from two O. rufipogon accessions. Submergence treatment shows that both of the accessions with SUB1A-like genes were submergence-intolerant. This preliminary study provides insight into the origin and allelic variation of SUB1A, an agronomically important gene that is rapidly being introduced into widely-grown rice cultivars.     

Population structure of the primary gene pool of Oryza sativa in Thailand

The gene pool of cultivated Asian rice consists of wild rice (Oryza rufipogon Griff.), cultivated rice (O. sativa L.) and a weedy form (O. sativa f. spontanea). All three components are widespread in Thailand, frequently co-occurring within fields and providing the opportunity for gene flow and introgression. The purpose to this study is to understand the on-going evolutionary processes that affect the gene pool of rice by analysis of microsatellite variation. Results indicate that O. rufipogon, the wild ancestor of rice, has high levels of genetic variation both within and among populations. Moreover, the variation is structured predominantly by annual and perennial life history. High levels of variation are detected among cultivars indicating Thai cultivated rice has a broad genetic base with only a 20 % reduction in diversity from its wild ancestor. The weedy rice populations reveal varying levels of genetic variation, from nearly as high as wild rice to near zero. Weedy rice is genetically structured into 2 groups. Some populations of invasive weedy rice are the result of hybridization and gene flow between local wild rice and local cultivated rice in the regions of co-occurrence. Other populations of weedy rice are genetically nearly identical to the local cultivated rice. The diversity analysis indicates that the rice gene pool in Thailand is a dynamic genetic system. Gene flow is ongoing among its three main components, first between cultivated and wild rice resulting in weedy rice. Weedy rice in turn crosses with both cultivated varieties and wild rice.     

Evaluation of Antioxidant,Lipid, and Protein Fractions of Accessions of Oryza Species

The genus Oryza has given rise to rice (Oryza sativa L.), a major source of food for much of the human population. The Oryza genus is small, including only 23 species, but it is remarkably diverse in terms of its ecological adaptation. This diversity may not be only restricted to ecological characteristics but also to kernel end‐use quality characteristics. This study was undertaken to evaluate Oryza species as a gene pool for improving the properties of rice bran for human consumption. Several accessions of 13 Oryza species were grown in a greenhouse, along with eight rice (Oryza sativa L.) accessions displaying low and high bran oil content, as well as low and high palmitic acid content. The total lipid content of the Oryza species was within the range found for Oryza sativa accessions. However, the level of palmitic acid in the O. species was as high as that reported for soybean high‐palmitic acid mutants. Oryza species also contained higher levels of the γ‐oryzanol and phenolic fractions compared to cultivated rice. Low or not significant phenotypic correlations between lipid, palmitic acid, and γ‐oryzanol suggest that these fractions in cultivated rice could be increased simultaneously using several of the Oryza species accessions identified in this study. A cultivar with enhanced levels of these fractions would be suitable for use in the production of high γ‐oryzanol margarine, shortening, and frying oils.     

Genetic diversity,population structure and differentiation of rice species from Niger and their potential for rice genetic resources conservation and enhancement

Rice genetic resources conservation and evaluation is crucial to ensure germplasm sources for further crop breeding. We conducted a wide collection of Oryza species in Niger and characterize its diversity with microsatellites (or simple sequence repeats, SSR). The aims of this research were to get a better understanding of the extent of genetic diversity, its structure and partition within rice eco-geographical zones of Niger. There were 264 accessions found in farmers’ and other fields: 173 O. sativa (Asia’s rice), 65 O. glaberrima (Africa’s rice), 25 O. barthii, and 1 O. longistaminata (weedy perennial rice), which were genotyped with 18 SSR. A total of 178 alleles were detected, with a mean of 9.89 alleles per locus. The polymorphism information content was 0.65 and heterozygosity was estimated as 0.14. Two main well-differentiate genotypic groups, which correspond to Asian and African rice species, were identified. The SSR set divided the Asia’s rice group (solely indica) into irrigated and floating rice, with rainfed lowland rice in between. The African rice species group was composed of O. glaberrima, O. longistaminata and O. barthii accessions, but without any clear genetic differentiation among them likely due admixtures within the samples of O. barthii. Five accessions that could be natural interspecific hybrids were too admixed for assigning them to any of the two well-differentiated groups. The partitioning of the overall diversity showed that maximum variation was within genotypic groups and subgroups or cropping ecologies, rather than between eco-geographical zones. The eco-geographical distribution of the diversity suggests germplasm exchange in Niger. Next-steps for conserving rice and crop wild relatives in Niger could be taken using the findings of this research.     

Diversity and differentiation of <Emphasis Type="Italic">Oryza sativa</Emphasis> and <Emphasis Type="Italic">O. rufipogon</Emphasis> in Indonesia

Analysis of the genetic structure of Indonesian Oryza sativa and O. rufipogon using neighbour-joining trees based on single nucleotide polymorphism (SNP) and simple sequence repeat (SSR) markers revealed that O. sativa in Indonesia is separated from O. rufipogon. Accessions of O. sativa in this study were differentiated into two major groups, indica and tropical japonica, excluding some varieties. SSR and SNP markers revealed the high value of differentiation (F _ST) and genetic distance (D) between indica and tropical japonica and we discovered four loci by SNP markers and one locus by SSR markers that play a role in differentiation between indica and tropical japonica. Interestingly, genetic diversity (H) in O. rufipogon was lower than that in O. sativa, however H in O. rufipogon was the highest and H in tropical japonica was the lowest when O. sativa was divided into two groups. Inbreeding coefficient (Fst) showed evidences that gene flow (Nm) between species and within species might be one of the mechanisms related to the diversification and differentiation of Indonesian rice germplasm by asymmetric pattern between species and within O. sativa as revealed by SSR and SNP markers. In addition, we found evidences on stabilizing selection in Indonesian rice germplasm and they might be the reasons why Indonesian rice germplasm did not differentiate due to source location of landrace. However, we found a weak relation between SSR and SNP markers probably due to highly polymorphic in SSR and the different properties of both markers.     

Spatial and temporal dynamics in genetic diversity in upland rice and late millet (<Emphasis Type="Italic">Pennisetum glaucum</Emphasis> (L.) R. Br.) in The Gambia

Ambiguity exists about the level of genetic diversity represented by farmer crop varieties, how it develops over time and how it relates to the diversity comprised by formal varieties. As part of an interdisciplinary technological/sociological study on farmer management of gene flow, upland rice (Oryza sativa L.) and late millet (Pennisetum glaucum (L.) R.Br.) from The Gambia were investigated for morphological and molecular variation. The goal of these analyses was to obtain insight into the level of crop genetic diversity of farmer’s materials planted in several case study villages in The Gambia. For both crops, samples were collected from villages and various research institutes. Based on variety names, different rice and millet varieties were expected to be used in different villages. In fact, there was a large overlap in genetic diversity for both crops, masked by the use of synonyms. The considerable similarity in rice genetic diversity between villages most likely results from the exchange of varieties between farmers. For millet this seems the result of development of varieties from the same gene pool. Some farmer varieties of rice, however, are apparent hybrid forms between the species O. sativa and O. glaberrima Steud., and farmer varieties in general displayed higher levels of genetic diversity than formal varieties. This indicates that, for rice, genetic diversity develops in farmers’ fields and may have potential use in formal breeding programs.     

Genetic diversity associated with conservation of endangered Dongxiang wild rice (<Emphasis Type="Italic">Oryza rufipogon</Emphasis>)

The wild progenitor species (Oryza rufipogon) of Asian cultivated rice (O. sativa L.) is located in Dongxiang county, China which is considered its the northernmost range worldwide. Nine ex situ and three in situ populations of the Dongxiang wild rice (DXWR) and four groups of modern cultivars were genotyped using 21 SSR markers for study of population structure, conservation efficiency and genetic relationship. We demonstrated that the ex situ conservation of the DXWR failed to maintain the genetic identity and reduced genetic diversity. Therefore, in situ conservation is absolutely necessary to maintain the genetic identity, diversity and heterozygosity. Also, in situ conservation is urgently needed because natural populations in DXWR have decreased from nine to three at present due to farming activity and urban expansion. In DXWR, the three surviving in situ populations had greater expected heterozygosity than any cultivated rice, and were genetically closer to japonica than either the male-sterile maintainer or restorer lines, or indica. Japonica has the lowest genetic diversity of cultivated rice. As a result, DXWR is a rich gene pool and is especially valuable for genetic improvement of japonica rice because these O. rufipogon accessions are most closely related to the japonica as compared to O. rufipogon collected anywhere else in the world.     

Evaluation of Genetic Diversity of Wild Rice (Oryza rufipogon Griff.) in Myanmar using Simple Sequence Repeats (SSRs)

As a part of an in situ survey of wild rice (Oryza rufipogon Griff.) in Myanmar (Burma), 16 strains of wild rice were collected, and analyzed for allelic diversity over 74 loci with simple sequence repeat (SSR) markers to obtain a basic information for their conservation. Three each of indica and japonica cultivars were added for a comparison. In the six cultivars and 16 strains of wild rice, three to 15 alleles were detected per locus with an average of 7.9. The wild rice revealed a large number of unique alleles throughout their chromosomes with much wider ranges of variation than those detected in the six cultivars of O. sativa L.. The alleles found in the wild rice were classified into those specific to wild rice, common to wild rice and cultivars, and those similar to indica or japonica cultivars. According to the classification, the genotype of each of the 16 strains of wild rice was schematically depicted. The genetic variation among individual strains within a collection site was larger than the variation among the collection sites.     

Cloning of genome-specific repetitive DNA sequences in wild rice (O. rufipogon Griff.), and the development of Ty3-gypsy retrotransposon-based SSAP marker for distinguishing rice (O. sativa L.) indica and japonica subspecies

In the rice genome, insertions and eliminations of transposable elements have generated numerous transposon insertion polymorphisms (TIPs). Common wild rice (O. rufipogon Griff.), the ancestor of Asian cultivated rice (O. sativa L.), carries abundant genetic variations. To find subspecies-specific (SS) markers that can distinguish O. sativa ssp. indica and ssp. japonica, some long terminal repeat (LTR) sequences (sc1-14) of AA genome-specific RIRE retrotransposon were isolated from O. rufipogon genome. Sequences sc1 and sc12 were successfully utilized to develop the SS marker system based on retrotransposon inserted position polymorphisms. Twenty-two SS markers (ssi1-9, ssj1-13) were developed, where ssi1-9 are the indica-specific types, and ssj1-13 the japonica-specific types. The average accuracy of these markers in distinguishing the two subspecies is over 85%. SS marker ssj-10 can distinguish the two subspecies at 100% accuracy. Principal component analysis (PCA) showed that these markers could successfully distinguish indica from japonica varieties, regardless of their geographical origin.     

Identification of genomic constitutions of <Emphasis Type="Italic">Oryza</Emphasis> species with the B and C genomes by the PCR-RFLP method

Oryza officinalis complex is the largest and the most complicated group in the genus Oryza L., consisting of about ten species with the B, C, BC, CD, and E genomes. Taxonomy and identification of the species, particularly those with the B, C and BC genomes, are difficult due to the similar morphology and overlapping distribution of some species. The difference in ploidy levels of some species adds more complexity. In the present study, we surveyed 64 accessions of rice germplasm in the O. officinalis complex using RFLP analysis of PCR-amplified Adh genes in addition to chromosome counting. The results confirmed that all O. rhizomatis accessions are diploids with the C genome, whereas all O. minuta accessions are tetraploids having the BC genome. However, both diploid and tetraploid forms were found for the accessions identified in the genebank as O. officinalis, O. punctata and O. eichingeri. The tetraploid form of O. officinalis with the BC genome is exclusively distributed in India and has been described as O. malampuzhaensis. The tetraploid form of O. punctata which has been called O. schweinfurthiana by some workers was found to be as widely distributed as its diploid form in Africa. It is noteworthy that two accessions that had been determined as tetraploid O. officinalis were actually belonging to a species with the CD genome (O. latifolia). Our results promote a better understanding of the genomic constitutions of many accessions in the O. officinalis complex and correct determination of the genebank material, which serves as an important basis of germplasm cataloguing for further research and utilization.