Mycorrhizal is very popular and important symbiosis phenomena between higher plant roots and mycorrhizal fungi in the rhizosphere soil, and the arbuscular mycorrhizal (Arbuscular Mycorrhizal, AM) is one of the most prevalent type of mycorrhizal fungi. Arbuscular Mycorrhizal Fungi(Arbuscular Mycorrhizal Fungi, AMF or AM fungi) is a kind of the beneficial microbial, which can form symbionts with the majority of higher plant roots. AM fungi can form a dense filament network with the rhizosphere soil and root cortical cells. It not only influence the primary metabolism of the host plant, but also influence the plant secondary metabolism by enlarging the absorption area of the plant roots, improving the water use efficiency, promoting the absorption and utilization of the mineral nutrition elements, improving the Carbon and Nitrogen cycle.This review introduced the effects, research situation of arbuscular mycorrhizal fungi on plant secondary metabolism, and the role and application potential of the ecological system.

Resistin (RETN) is a specifically produced hormone by fat cells, which plays an important role in glucose and lipid metabolism and also is one of popular candidate genes in animal fat deposition and metabolism. Aiming at study the expression pattern of RETN gene in rump-fat deposition and metabolism of sheep (Ovis aires), long fragment PCR, bioinformatics method and semi-quantitative RT-PCR were used to amplify the full-length sequences, analyze the sequences and detect gene expression profiling of sheep RETN gene, respectively. Fluorescence quantitative Real-time PCR (qRT-PCR) method was used to analyze the expression change of RETN gene between starving and its control groups based on starving model in Altay sheep. The results of cloning and sequence analysis showed that RETN gene encoded 109 amino acids (GenBank No. KJ704841), which was different from the published RETN sequence in GenBank database. RETN mRNA expression profiling showed that RETN expressed in liver, spleen, lung and rump fat differently, especially highly in the liver, significantly higher than that of other groups (P＜0.01), which indicated that RETN gene might play important physiological and biochemical functions in fat synthesis and metabolism in the liver. The result of qRT-PCR showed that RETN mRNA significantly differentially expressed in between starving group and its control groups, with a expression level in persistent starvation group of 5.2 times than that of control group, suggesting that RETN gene might play an important role in fat mobilization. The above findings provide fundamental data for further study on RETN biological function in fat-rump sheep.

Sugar apple (Annona squamosa L.) is a significant fruit in tropics due to its abundant nutrition and high commercial value. In order to explore the effects of pruning and illumination on the flowering of axillary bud, new and mature branches growing in the same year were selected as experimental materials treated by different pruning ways with different colors of plastic film bagging. Nature growing branches were served as control. The contents of N, P, K and carbohydrates in inducing flowering parts and flower buds in different periods and developmental stage were determined. The results showed that if the branches were not removed the leaves and petiole, axillary bud of these branches failed to flower. Axillary bud could flower only under the condition of leaf and petiole removed. qRT-PCR analysis showed that the expression of LYF (LEAFY) and AP2 (APETALA2) were up-regulated at high level (P＜0.05) in tissues with proper pruning. When shoots with tipping and leaf and petiole removal were bagged with different color plastic films, the flowering was comparable among the treatments. This result indicated that flowering was not affected by light wavelength. In addition, axillary bud under proper pruning could flower at different seasons. Meanwhile, the content of N, P, K, starch and total soluble sugars in the branches of treatment 1 decreased in the first phase and then increased in the later compared with the control. This experiment demonstrated that the presence of petiole inhibited the flowering of axillary bud, removal of the petiole induced flowering genes expression, which switched from vegetative to reproductive growth and then led to blossoming and bearing fruit. In the process of floral induction, processing branches could not carry out photosynthesis in the short term, which resulted in the main nutrient contents presenting the increase after decline. The flowering process was not affected by the influence of illumination and light quality differences. While the completion of the floral induction, normal light was needed to promote the normal growth of the new germination branches, which provided the guarantee to blossom. The results preliminarily reveal the mechanism of the flowering by the unique pruning way from the physiological and molecular levels, and provide theory basis and practical guidance to regulate flowering period and off-season production of sweetsop tree.

In order to reveal the genetic basis of market weight trait and scan molecular markers and candidate genes which can be used to improve the market trait, market weight of 400 female Jinghai yellow chicken (Gallus gallus) in core group was measured at 16 weeks of age. Genome-wide association study (GWAS) was carried out using specific-locus amplified fragment sequencing technology to detect nucleotide polymorphisms (SNPs) associated with market weight. Finally, 15 single SNPs that associated with market weight were detected using General liner model (GLM). Four SNPs reached 5% Bonferroni genome-wide significance (P＜1.87E-06) and 11 SNPs reached “suggestive” genome-wide significance (P＜3.73E-05). 6 SNPs that associated with market weight were detected using mixed linear model (MLM). 2 SNPs reached 5% Bonferroni genome-wide significance (P＜1.87E-06) and 4 SNPs reached “suggestive” genome-wide significance (P＜3.73E-05). 6 SNPs of rs1169337928, rs475641139, rs31633105, rs475856030, rs476878211 and rs477915563 were detected both using GLM and MLM. Genes in the candidate regions with 1-Mb windows (SNP position±0.5 Mb) surrounding each significant SNP were screened. Finally, 14 candidate genes were obtained, among which 6 genes of FAM124A, QDPR, BCL11A, LDB2, BOD1L1 and WDR1 might be important candidate genes influencing market weight. Furthermore, It was also found that 9 out of 14 SNPs were distributed in the region of 75.641~79.592 Mb on chromosome 4, and this region was important candidate region which influenced the market weight of Jinghai yellow chicken. This study provides theory material for marker assisted selection of Jinghai yellow chicken.

WRKY transcription factors are novel transcriptional regulatory factors, which were found to be involved in regulating plant development, metabolism and other physiological processes in recent years. In this study, a new Dendrobium officinale WRKY transcription factor, designated as DoWRKY2 was cloned(GenBank Accession No. KF953910). The full-length cDNA was of 1 309 bp with a 999 bp of open reading frame (ORF), encoding a protein of 332 amino acid residues, and its domain had typical structural features of WRKY family, with four β-sheets. Bioinformatics analysis demonstrated that DoWRKY2 shared the highest similarity with PdWRKY17 of Phoenix dactylifera, which achieved 66%. It had a special motif of HARF, in addition, cluster analysis by phylogenetic tree revealed that it belonged toⅡd subfamily, and shared closest evolutionary relationship with AtWRKY11 and AtWRKY17. It could be expressed in Escherichia coli stably by prokaryotic expression. The results of quantitative Real-time PCR (qRT-PCR) analysis suggested that DoWRKY2 expressed in root, stem, leaf and protocorm-like bodies, with a highest level in leaf and lowest in stem. Also, DoWRKY2 could be response to induction of methyl jasmonate (MeJA) and chitosan, and reached peak at 4 and 1 h, respectively. The research results provide basic materials for further function alanalysis of this gene in the future.

Calcium/calmodulin-dependent protein kinases (CaMKs) are important downstream targets of calmodulin. The activation of CaMKs can lead to the phosphorylation of various metabolic key enzymes or transcription factors, and complete the regulation of cell metabolic activity or the expression of numerous genes. In this research, 4 different CaMK gene fragments of Setosphaeria turcica named as CAK1, CAK2, CAK3 and CAK4 were obtained using degenerate primers. The full-length cDNAs of CAK1 (GenBank No. EU605885), CAK2 (GenBank No. EU605886) and CAK3 (GenBank No. EU605887) were cloned through 3'RACE and 5'RACE. The lengths of ORF, 3' UTR and 5' UTR of CAK1 were 1 218, 317 and 500 bp; those of CAK2 were 1 194, 400 and 563 bp; and those of CAK3 were 2 304, 405 and 207 bp, respectively. And the encoded products length of CAK1, CAK2 and CAK3 were 405, 397 and 767 amino acid residues (aa), respectively. The bioinformatics analysis revealed that these 4 CaMKs all belonged to multifunctional CaMKs and each contained 12 conserved kinase motifs. The conserved kinase domains of both CAK1 and CAK2 belonged to the catalytic domain of CaMK Ser/Thr protein kinase while that of CAK4 belonged to the RCK1-like Ser/Thr protein kinase. Unlikely, the conserved kinase domain of CAK3 was similar to the catalytic domain of liver kinase B1 (LB1) and calmodulin dependent protein kinase kinase (CaMKK). And they related to fungal CaMKs, CaMKKs and MAPK-activated protein kinases (MAPKAPKs) in the phylogenetic tree, respectively. Results of Real-time quantitative PCR (qRT-PCR) showed that during the conidial germination process, CAK2 and CAK3 were actively transcribed, and the transcriptional levels at every stage were higher than that at 0 h post induction. Especially, the expression levels of both CAK2 and CAK3 were significantly up-regulated at 6 and 24 h post induction (P＜0.05). On the contrary, the transcription levels of CAK1 and CAK4 at all stages were significantly lower than the mock (P＜0.05). In this research, we isolated 4 CaMK genes, preliminarily clarified their expression patterns during the conidial germination process, which provided informative references for the subsequent functional dissection of calcium signal pathway in S. turcica.

In order to reveal the genetic diversity and tissue expression pattern of Parathyroid hormone-related peptide gene (PTHrP) in Gaojiao chickens (Gallus), Gaojiao chickens in Guizhou province were used in the present study, Aijiao chickens and Baiyi black chickens as controls. Its polymorphic loci were detected with DNA pool, Single Strand Conformation Polymorphism (SSCP) and DNA sequencing techniques, and its differential expression levels in heart, liver, breast muscl and leg muscle were analyzed by using real-time quantitative PCR technology. The results showed that the C→T point mutation was detected in 209bp of exon 3 of PTHrP gene of Gaojiao chickens，which was a silent mutation, hree genotypes (CC, CT and TT) were found.The relationship between PTHrP gene and shank length in Gaojiao chickens was analyzed. The C209T polymorphism wad significantly associate with shank length of Gaojiao chicken, and the individuals with CT genotype had significant shank length than CC and TT (P＜0.05). Thefitness test indicated that Gaojiao chickens at the mutation locus were Hardy-weinberg equilibrium(P＞0.05), and itpresented moderate polymorphism (PIC=0.350).. The RQ-PCR detection showed that the expression level of PTHrP gene was the highest in leg muscle of Gaojiao chickens, and significant higher than other tissues (P＜0.05). In addition, compare with Aijiao chicken and Baiyi black chicken,the expression levels of various tissues of Gaojiao chickens were higher than that of Baiyi black chickens (P＜0.05)，but lower than Aijiao chickens (P＜0.05). The results indicate that polymorphisms of PTHrP gene are significantly correlated with shank length traits of Gaojiao chickens, and individuals with CT genotype can be used as molecular genetic marker to select shank length of Gaojiao chicken for reproduction traits, and the expression levels of PTHrP has an influence on chickens' shank length.

To reveal the molecular mechanism of the temperature/photoperiod sensitive male sterile lines of wheat (Triticum aestivum L.), the differently expressed genes in anthers at the tetrad and bicellular stage of the sterile and fertile lines of Bainong sterility (BNS) were analyzed using Affymetrix gene chip technology of wheat. The semi-quantitative RT-PCR was utilized to characterize some sterile candidate genes. The results showed that among the 61 127 probes transcripts, there were 418 valid transcripts presenting different expression between the sterile and fertile lines. Among these transcripts, 142 of them were down-regulated and 276 of them were up-regulated in the bicellular stage. Functional classification indicated that these genes participated in a series of significant life processes of metabolism process, cytological process, biological regulation and irritative reaction etc. Moreover, to verify chip data, 10 genes of leucine-rich repeats (Ta.17228.1), ATP-binding cassette transporter (Ta.21809.1), peptidoglycan binding protein (Ta.22570.1), Ta.37113.2(unknown), cytochrome P450 (Ta.19531.1), phosphoribulose kinase (Ta.18368.1), transcriptional regulator (Ta.9239.1), peptidase C1A subfamily (Ta.37113.1), thionin super family (Ta.21983.1) and V-type proton ATPase subunit D (Ta.18091.1), differentially expressed significantly, were identified by cDNA semi-quantitative RT-PCR. Result showed that the expression models of the 10 genes were concordant with the result of gene chip detection. Conservative structure domain analysis showed that Ta.18091.1, Ta.21809.1 and Ta.19531.1 most likely involved in the abortive anthers in the BNS sterile lines. The results provide important theoretical basis for sterility-related genes cloning and verification, and contribute to find the key gene which leads to pollen abortion in thermo-sensitive genic male sterile wheat (BNS).

In order to study the molecular mechanism of melanin deposition in White Silky Fowl (Gallus gallus domesticus Brisson), the quantitative Real-time PCR (qRT-PCR) was used to detect the expression level of tyrosinase (TYR), agouti signaling protein (ASIP) and microphthalmia-associated transcription factor(MITF) genes in the skin, muscle, liver, kidney and gizzard of White Silky Fowl and the ultraviolet spectrophotometry was adopted to measure the melanin content in tissues for exploring the relationship between mRNA expression of the 3 genes and melanin deposition in tissues of White Silky Fowl. Results showed that the mRNA of TYR, MITF and ASIP expressed very significantly differently in all test tissues of White Silky Fowl (P＜0.01) with an order of skin＞kidney＞gizzard＞liver＞muscle of MITF and TYR, which were consistent with the law of melanin deposition in White Silky Fowl. However, the mRNA expression of ASIP in tissues showed a contrary trend of muscle＞liver＞gizzard＞kidney＞skin. Correlation analysis indicated that the mRNA expression of TYR and MITF were significently positively correlated with the melanin content in tissues of White Silky Fowl (P＜0.05), while that of ASIP showed significently negative correlation (P＜0.05). It was speculated that the expression of TYR, MITF and ASIP had certain correlation with the melanin deposition in White Silky Fowl, respectively. High expression of TYR and MITF might promote melanin deposition while ASIP might inhibit melanin deposition in White Silky Fowl. The results would provide a theoretical basis for furtherly elucidating the genetic regulation mechanism of melanin deposition.

In wheat breeding, young ear development process and photosynthesis are two factors which must be considered. In this study, a complete set of wheat-Psathyrostachys huashanica disomic alien addition lines were studied on young ear development process and photosynthesis. The purpose of this article is to map associated genetic traits on chromosome, and comprehensive evaluation of the young ear development process and photosynthetic effect of Psathyrostachys huashanica alien chromosomes. The sequenced characterized amplified region (SCAR) maker identification showed that all of this wheat-Psathyrostachys huashanica disomic alien addition lines had Psathyrostachys huashanica chromosomes. Anatomic observation indicated that the young ear of the addition line of 6Ns developed fastest, and the mature period shortened 14~16 days compared with parents 7182; the development rate of the 5Ns and 2Ns addition line were behind, and the mature period of the 4Ns addition line were 6~7 days later compared with parents 7182. The photosynthetic indexes determination showed that the photosynthetic rate of the 1Ns addition line had reached at 23.73 μmol/(m2·s), made the largest contribution, with a significant difference (P＜0.05) from wheat 7182 (15.73 μmol/(m2·s)) and suggesting a significantly positive effect. However the 3Ns (11.10 μmol/(m2·s)) and 6Ns (11.64 μmol/(m2·s)) addition line made a least contribution, suggesting a significantly negative effect. All results showed that the 6Ns and 1Ns chromosomes might have the genes which can promote young ear development and high photosynthesis efficiency, and can play an important role in germplasm improvement of existing varieties.

The aim of this study was to explore the feasibility of rabbit bone marrow mesenchymal stem cells (BMSCs) into islet cells induced by growth factors in three phase induction method in vitro, and observe their dynamic change. BMSCs were obtained by red blood cell lysis, the CD90, CD44 and CD34 antigen expression were detected by immunocytochemistry technique. Growth factors three-step strategy was adopted to induce BMSCs to differentiate toward islet cells. The growth of BMSCs were observed under inverted microscope. The differentiated cells were stained with dithizone (DTZ). The express of inslet cells related gene(Pdx-1, Insulin, Nkx6.1) was detected by RT-PCR, and the expression of specific protein like Insulin in derived cells was investigated by immunocytochemistry technique. Insulin secretion was assayed by enzyme linked immunosorbent assay (ELISA). The result showed that the isolated BMSCs were shuttle or gathered into a vortex-like uniform growth, and positive for CD90、CD44 antigen, negative for CD34. At the end of the first-step strategy (3 d), the cell morphology had no obvious change, and part of cells become round, DTZ staining negative, any specific gene mRNA expression was detected. After inducted 7 d, BMSCs gradually merged to form cluster, DTZ staining positive, only Pdx-1 mRNA expression was observed, which was verified as insulin progenitor cells. At the end of second-step strategy (11 d), the number of differentiated cells cluster was incteased, RT-PCR showed that Pdx-1 and Insulin were expressed. After inducted 19 d, cell clusters looked like islet-like structure in terminal, DTZ staining positive and detected Pdx-1, Insulin and Nkx6.1 expression. Immunocytocheemistry analysis showed expression of Pdx-1 in the islet-like clusters. A lot of dark brown particles appeared in the cytoplasm of cells in the experimental group, the results showed positive reaction. The control group did not appear brown particles in the cytoplasm of cells, the results showed negative reaction. Insulin content in the culture supernatant detected by ELISA showed that there was no significant difference between the induced group and the control group (P＞0.05) on the 3rd day because there was no cell clusters like changes and insulin secretion, in addition, there was a significant difference on the 7th day, 11th day and 19th day(P＜0.05). Inducing BMSCs secrete insulin at the 3rd、7th、11th、15th、19th days, and the difference was significant (P＜0.05) between BMSCs and induced groups except the 3rd days. It illustrated that isolated and purified BMSCs in vitro could be inducted to differentiate into fuctional mature islet cells. The study provides basic data for transplanting autologous BMSCs to cure diabetes for diabetics.

In order to probe the factors which resultes in the different aroma among the fruits of different location of the cucumber (Cucumis sativus), the contents and components of fruit aroma, the contents of relative substrate and hydroperoxide lyase genes (HPL) expression in the fruits of different location were investigated in two inbred lines No. 26 (North China ecotype) and No. 14-1 (Europe type), which differred significantly in fruit aroma. Results showed that the amounts and compounds in two cucumber inbred lines were different. There were 42 kinds of volatile compounds in No. 14-1, including 36 kinds of aromatic substances in root cucumber, 32 types in waist cucumber and 39 species in host cucumber, meanwhile there were 40 types of volatile compounds in No. 26, including 36 kinds of aromatic substances in root cucumber, 34 types in waist cucumber and 32 species in host cucumber. Among all of these more than 65% aromatic substances were common. The contents of characteristic aromatic substances (E,Z)-2,6-nonadienal and (E)-2-nonenal were higher in root and host cucumber, respectively. The substrate contents varied in two cucumber fruits of different location, including the highest linoleic acid of root cucumber and the highest linolenic acid of waist cucumber of No. 14-1, the highest contents of both linoleic acid and linolenic acid of root cucumber of No. 26. However the ratio of both (E,Z)-2,6-nonadienal/(E)-2-nonenal and linolenic acid/linoleic acid had a highest proportion in waist cucumber than root and host cucumber of two cucumber fruits of different location. Relative expression of 9-HPL, 13-HPL, 9/13-HPL were different in both cucumber fruits of different location, all of these genes relative expressions of No.14-1 were waist cucumber＞ host cucumber＞ root cucumber, in No. 26 that was host cucumber＞root cucumber＞waist cucumber. The changes of HPL gene relative expressions were consistent with content changes of (E,Z)-2,6-nonadienal and (E)-2-nonenal in No.26, and the 9-HPL gene had the highest expression in both cucumber cultivars. The results suggested that the different location of cucumber fruits led to the great differences of the contents and species of aromatic substances, which would not only be affected by the relatively contents of linoleic acid and linolenic acid but also HPL gene expression. This study provides basic data for further study of the formation mechanism of cucumber aroma.

Aims of this research were to well identify chromosome and chromosome fragments from cabbage under background of Chinese cabbage, and to analyze the effect of cabbage (Brassica oleracea, CC) chromosome or chromosome fragments on Chinese cabbage (Brassica rapa, AA). A total of 797 expressed sequence tag SSR (EST- SSR) markers were used to screen polymorphism between Chinese cabbage and cabbage, 269 of them were polymorphic, and 12 of them were assigned on linkage group C01, 10 of them were assigned on linkage group C02, 22 of them were assigned on linkage group C03, 10 of them were assigned linkage group on C04, 13 of them were assigned on linkage group C05, 10 of them were assigned on linkage group C06, 16 of them were assigned on linkage group C07, 14 of them were assigned on linkage group C08, 13 of them were assigned on linkage group C09, and 156 of them were NOMATCH, Scaffold or Unknown. The EST sequences corresponding 176 EST-SSRs with clear polymorphism and stable amplification between Chinese cabbage and cabbage were blasted with databases of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). EST sequences annotations were obtained. Annotations about cell components had 124, accounting for 12.29% of the total, and mainly including organelle, membrane-enclosed lumen, cell junction, membrane, cell, macromolecular complex, extracellular region, cell part，membrance part, organelle part ect.. Annotations about molecular function had 184, representing 18.24% of the total, and mainly including catalytic activity, nucleic acid bingding transcription factor activity, protein binding transcription factor activity, structural molecule activity, transporter activity, binding, electron carrier activity etc.. Annotations about biological pathways had 688, representing 68.19% of the total, and mainly including cell metabolic processes, biological regulation and stimulus response systerm morphological structure, signal ransduction and signal transmission system, the immune system ect.. The sequences with unknown function had 13, representing 1.29% of the total. The obtained polymorphic EST-SSRs markers between cabbage and Chinese cabbage will be very useful for identification of exogenous cabbage chromosome and chromosome fragments in alien addition lines and translocation lines of Chinese Cabbage - Cabbage. Function annotation of EST-SSRs markers target gene were analyzed in this research, which will provide a reference for speculating the effect of DNA form cabbage on Chinese cabbage.

To explore the function of 1-aminocyclopropane-1-carboxylic acid oxidase gene (ACO) and ethylene biosynthesis pathway in Agaricus bisporus, an efficient genetic transformation approach was developed for constructing the ACO-RNAi mutants. Cationic liposome was mixed with the dsRNA of ACO expression plasmid pBHg-dsACO at the ratio of 1 μL∶2.5 μg, then diluted 1 000-fold and co-incubated with fruit body gill tissue pieces (4 mm of length) of Agaricus bisporus at room temperature for 100 min. The transformation solution and tissue pieces were mixed with regeneration complete medium (RCM) and plated and cultured at 25 ℃ for about 7 d. The germinating tissue pieces were transplanted on PDA plates containing hygromycin for screening and subculturing. The transformants were obtained by PCR identification of putative transformants. Results showed that the ACO-RNAi transformants had genetic stability with the mRNA expression of ACO reduced 47%~74% compared with wild type (WT) (P＜0.01), ACO activity and ethylene production reduced 68%~86% and 27%~48% compared with WT (P＜0.05), respectively, indicating that the ethylene biosynthesis pathway in the Agaricus bisporus was ACC pathway, same as higher plants. The results demonstrated that the method of liposome-mediated fruit body gill tissue transformation of Agaricus bisporus was convenient and facile, and it provides an effective molecular biology approach for genetic improvement of Agaricus bisporus.

The metallothionein (MT) of bivalves has been taken as a valid biomarker to indicate heavy metal pollution of aquatic environment. The present study was aimed to prepare the polyclonal antibodies against MT of Crassostrea hongkongensis which is an important oyster living largely at nearshore area and estuary of the South China Sea. The sufficient MT was required to immune a New Zealand rabbit (Oryctolagus cuniculus) to generate polyclonal anti-MT antibodies. Therefore, the plasmid pQE30/MT containing C. hongkongensis MT coding sequence and the prokaryotic expression vector pGEX-4T-1 were digested by both BamHⅠand SalⅠ, and the digested pGEX-4T-1 and MT DNA sequence were ligated together into an expression plasmid pGEX-4-1/MT. The constructed plasmid was transformed into the expression host Escherichia coli M15, and the soluble recombinant protein (glutathione S-transferase (GST)-tagged MT, GST-MT) was induced by 0.2 mmol/L IPTG for 16 h at 20 ℃. The GST-MT was purified by affinity chromatography column filled with Glutathione Sepharose 4B, subsequently digested in situ by thrombin, finally the recombinant MT with a molecular weight of 9 kD was purified and collected, and the GST-tag was removed from glutathione sepharose 4B. The purified MT was employed to immunize a New Zealand rabbit for 4 times at 3-week interval. The antiserum was collected from the immunized rabbit heart, and its antibody titer was 1∶128 000 through detection of indirect enzyme-linked immunosorbent assay (ELISA). The anti-MT antibodies were specific against the MT in tissues of C. hongkongensis, and will play an important role in research of heavy metal biomarker characteristics.