Mol Gen Genomics (2006) 276:427–435 DOI 10.1007/s00438-006-0149-1
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ORIGINAL PAPER
Molecular genetics of berry colour variation in table grape
Diego Lijavetzky · Leonor Ruiz-García · José A. Cabezas · María T. De Andrés · Gemma Bravo · Ana Ibá?ez · Juan Carre?o · Félix Cabello · Javier Ibá?ez · José M. Martínez-Zapater
Received: 17 April 2006 / Accepted: 27 June 2006 / Published online: 19 August 2006? Springer-Verlag 2006
Abstract The genetics and biochemistry of anthocya-nins and X avonol biosynthesis and their role in plant organ pigmentation is well established in model spe-cies. However, the genetic basis of colour variation is species speci W c and understanding this variation is very relevant in many fruit and X ower crop species. Among grape cultivars, there is a wide genetic variation for berry colour ranging from yellow-green (“white” culti-vars) to dark blue berries. Berry colour results from the synthesis and accumulation of anthocyanins in the berry skin, which in plants is commonly regulated by
transcription factors belonging to the MYB and bHLH families. In this work, we aimed to identify the major genetic determinants of berry colour variation in a large collection of table grape cultivars and somatic variants. The genetic analyses of berry colour in a few grape segregating progenies had previously identi W ed a single locus on linkage group 2 responsible for colour variation. Furthermore, somatic variation for berry skin colour in cultivar Italia had been associated with the presence of a Gret1 retrotransposon in the pro-moter region of VvmybA1, a Myb gene whose expres-sion is associated to skin colouration. The results show that VvmybA1 is the gene underlying the mapped locus controlling berry colour in grape. Additionally, the molecular analyses indicate that genetic and somatic berry colour variation can be associated to molecular variation at VvmybA1 in more than 95% of the ana-lyzed cultivars. Thus, VvmybA1 is a major determinant of berry colour variation in table grape and its instabil-ity is the major cause of somatic variation for this trait.Keywords Berry colour · MYB genes · Grapevine · Somatic variation · Colour variation
Introduction
Anthocyanin pigments are responsible for a large range of colours displayed by di V erent plant organs such as leaves, X owers, fruits and seeds. Colour has been shown to play a role in the attraction of verte-brate and invertebrate animals required for pollination and seed dispersal (Gautier-Hion et al. 1985; Kevan and Baker 1983). The genetics and biochemistry of the anthocyanin biosynthetic pathway, a major branch of
Communicated by R. Hagemann
Nucleotide sequence data reported are available in the GenBank databases under the accession numbers: DQ403722, DQ345541, DQ345539 and DQ403721.
Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at https://www.doczj.com/doc/16106924.html,/10.1007/s00438-006-0149-1.
D. Lijavetzky (&) · J. A. Cabezas · G. Bravo · A. Ibá?ez ·J. M. Martínez-Zapater
Departamento de Genética Molecular de Plantas, Centro Nacional de Biotecnología,
Consejo Superior de Investigaciones CientíW cas (CSIC),C/ Darwin 3, 28049 Madrid, Spain e-mail: dlijavetzky@cnb.uam.es
L. Ruiz-García · J. Carre?o
Instituto Murciano de Investigación y Desarrollo Agrario (IMIDA), La Alberca, Murcia, Spain
M. T. De Andrés · F. Cabello · J. Ibá?ez
Instituto Madrile?o de Investigación y Desarrollo Rural, Agrario y Alimentario (IMIDRA), Finca El Encín, Alcalá de Henares, Madrid, Spain
X avonoid metabolism, has been well characterized in maize, Petunia, snapdragon, and more recently in Ara-bidopsis (Holton and Cornish 1995; Winkel-Shirley 2001). Main regulatory genes associated with the con-trol of anthocyanin biosynthesis in plants have also been identi W ed as belonging to two major groups of transcription factors, the MYB and bHLH families (Borovsky et al. 2004; Holton and Cornish 1995; Kobayashi et al. 2002; Ozeki et al. 2003; Ramsay et al. 2003; Robbins et al. 2003; Sainz et al. 1997; Schwinn et al. 2006; Spelt et al. 2000). The genetic basis of col-our variation is species spci W c and di V erent genes in the pathway have been found responsible for colour varia-tion in di V erent species (Arakawa et al. 1999; Awad et al. 2000; Ben-Yehudah et al. 2005; Jaakola et al. 2002; James and Howard 1989; van Dyk et al. 2005). In this work we aimed to identify the major determinants for colour variation in table grape cultivars.
Berry colour in grapes (Vitis vinifera L.) displays a wide range of variation between yellow-green (“white”cultivars) and dark blue. Colour is used in some cases to di V erentiate cultivars being a relevant trait in X uenc-ing both table grape and wine qualities. Pigmentation of red/blue berry grapevine cultivars results from vacu-olar accumulation of anthocyanins in berry skin cells. Analysis of berry colour segregation in a few grape mapping populations have identi W ed a single locus as responsible for the presence or absence of skin colour (Doligez et al. 2002; Fisher et al. 2004) on the currently known linkage group 2 (LG2) (Adam-Blondon et al. 2004). Furthermore, molecular analyses of structural genes involved in the anthocyanin biosynthetic path-way showed that all structural genes tested, except the UDP-glucose:X avonoid 3-O-glucosyltransferase (UFGT) gene, were expressed in most berry tissues; while expression of UFGT was only detected in the berry skin and was always associated to anthocyanins accu-mulation (Boss et al. 1996a, b; Kobayashi et al. 2001). Thus, regulation of UFGT, encoding one of the latest steps of the pathway, was identi W ed as a key point in the control of berry colour although no di V erences were observed in either coding or promoter sequences of this gene between coloured and white cultivars (Kobayashi et al. 2002). Somatic variation for berry skin colour in cultivar Italia was later associated with the presence of the Gret1 retrotransposon in the pro-moter region of VvMybA1 (Kobayashi et al. 2004), a Myb gene whose expression is associated to skin col-ouration and which seems to be required to positively regulate UFGT expression (Kobayashi et al. 2002). These authors genotyped 22 grape cultivars of di V erent origins and showed the presence of homozygous Gret1 insertions in the promoter region of the VvmybA1gene in the 12 white berries bearing cultivars. The same insertion allele was also observed in the hemizy-gous state in the 10 coloured cultivars analyzed (Kobayashi et al. 2004). These results suggested that VvmybA1 could be a major determinant of berry colour variation in grapes.
To analyze the role of the VvMybA1 gene in berry colour variation in table grape we performed a coseg-regation analysis of berry colour and VvMybA1 geno-types in a selfcross progeny derived from the red table grape cultivar Ruby Seedless. Moreover, we genotyped this locus in a large collection of table grape cultivars to ascertain the existent allelic variation for this gene and its association with berry colour. Finally, we analyzed independent events of colour somatic variation in sev-eral cultivars to understand the molecular basis of this variation. The results show that VvMybA1 is likely the gene underlying the mapped locus controlling berry colour in grapes and that genetic and somatic colour variation analyzed is associated to molecular variation at VvMybA1 in over 95% of the analyzed cultivars. Materials and methods
Plant materials and colour analyses
The molecular basis of colour variation was analyzed in a collection of 189 grape cultivars used for table grape production or with double usage, table and wine, which are mostly maintained at the germplasm collection of “El Encín” (IMIDRA, Alcalá de Henares, Madrid, Spain). Their name and collection code are described in Table S1 of the Supplementary Materials. The molecular basis of colour somatic variation was ana-lyzed in a subgroup of 23 table grape cultivars corre-sponding to 9 original genotypes and 14 derived somatic variants. Most of them were also from the germplasm collection of “El Encín”. Their names and origin are also listed in Table S1. The genetic identity between the original cultivars and their derived somatic variants was veri W ed by genotyping 6 microsat-ellite loci: VVMD5, VVMD7, VVMD27, VrZAG62, VrZAG79 and VVS2 (This et al. 2004). Cosegregation between the colour phenotype and VvmybA1 was ana-lyzed in 19 plants derived from selfcross of the col-oured cultivar Ruby Seedless. This progeny was generated and maintained at the IMIDA (Murcia, Spain). In this progeny, colour was quanti W ed using a colourimetric value (CIRG) derived from the colour evaluation. CIRG=(180-H)L+C, where H=hue angle, L=lightness and C=chroma (Carre?o et al. 1995). This procedure assigns the following mean
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threshold values for the di V erent berry colours: yellow-green=1.55; pink=2.49; red=3.66; violet=4.75; dark violet=5.57. Statistical analyses were performed using the STATISTICA v7.0 software package (StatSoft).
DNA extraction and analysis
Total genomic DNA was isolated from young leaves using the DNeasy Plant Mini Kit (Qiagen). The VvmybA1 genotypes were W rst scored using a PCR assay originally based on the ampli W cation of the 5?-X anking region and coding sequences of the insertion allele of VvmybA1 and its functional reversion allele, named as VvmybA1a and VvmybA1b by (Kobayashi et al. 2004), with minor modi W cations. For simplicity in the use of the allele names, and given the number of natural alleles that can be found for VvmybA1, we will refer to them, from here on, as the gene followed by a three letter code representing the name of the cultivar were the allele was W rst described. In this way the two mentioned alleles will be named as VvmybA1ITA (for cultivar Italia insertion allele) and VvmybA1RUO (for cultivar Ruby Okuyama reversion allele). Primers used for VvmybA1ITA were a (5?-AAAAAGGGGGGCA ATGTAGGGACCC-3?) and d3 (5?-CCTGCAGCTT TTTCGGCATCT-3?), and those used for VvmybA1RUO were b (5?-GGACGTTAAAAAATGGTTGCACGT G-3?) and d3 (Fig.4b). Ampli W cation of the 5?-LTRs and 3?-LTR from Italia and Sugraone VvmybA1ITA alleles were performed with primers b and e1 (5?-GTCTTTCGCTTGCCAACTGT-3?) and a and d3, respectively, (Fig.4b). The solo-LTRs present in somatic variants Ruby Okuyama, Ralli Seedless and Super Red were ampli W ed with primers b and d3. The VvmybA1AFL, VvmybA1SUB and VvmybA1ROD alleles were PCR ampli W ed using primers b and cDNAs (5?-TCAGATCAAGTGATTTACTTGTGT G-3?) (Fig.4b), with the exception of VvmybA1ROD which required a 3? RACE approach for the 3? end characterization, as described below. PCR ampli W ca-tions were performed with an initial denaturing step of 95°C for 5min followed by 35 cycles of 94, 65 and 72°C for 1min each and a W nal extension step of 72°C for 10min. Ampli W cation products were separated by elec-trophoresis in 1% agarose gels. Cloning of PCR prod-ucts was carried out with the pGEM-T easy vector (Promega) and sequenced at the Genomic Unit of the Parque CientíW co de Madrid. Sequencing analysis and alignments were performed with BIOEDIT v7.0.5.3 (Hall 1999). Additional primers used in the somatic variants analysis were: 3s (5?-TCGAAAATCAGTGA GGGTAACAAAG-3?), 4as (5?-CGACACGTTTCT TATGGTGCATT-3?) and c (5?-GAACCTCCTTTT TGAAGTGGTGACT-3?). SSR analyses were per-formed according to previously described conditions (Cabezas et al. 2003).
DNA blot hybridization analyses were performed according to Sambrook et al. (1989). Brie X y, genomic DNA (10 g) was digested with the restriction enzyme Apa I and the digestion products were separated by electrophoresis in 0.7% agarose gels, transferred and immobilized into Hybond-N+(Amersham Biosci-ence) nylon membranes and hybridized with a 32P labeled probe. The VvmybA1 probe was generated by PCR ampli W cation with primers Ps (5?-TCACGGGG TTTAGAAAGTGG-3?) and Pas (5?-ATCAATTGG GGAATTGGTGA-3?) using Muscat Hamburg geno-mic DNA as template. The hybridized membranes were scanned with a STORM PhosphorImager (Molecular Dynamics).
RT-PCR and 3?-RACE
Total RNA extractions from berry skin tissues were performed according to Zeng and Yang (2002) with a W nal puri W cation step using the RNeasy Plant Mini Kit (Qiagen). RT-PCR assays were carried out using the conditions and primers described in Kobayashi et al. (2004). Isolation of 3?-end of Roditis cDNA was per-formed using the BD SMART RACE cDNA Ampli W-cation Kit (BD Bioscience, Clontech).
Results and discussion
VvmybA1 co-localizes with the locus responsible for berry colour in grape segregating progenies
The genetic determinism of grapevine berry colour has been analyzed in two F1 progenies ([Dattier de Beyrouth￡75 Pirovano]￡[Alphonse Lavallée￡Sultanine] and [Regent￡Lemberger]) segregating for this trait (Doligez et al. 2002; Fisher et al. 2004). In both cases the presence or absence of colour segre-gated as a monogenic trait determined by a locus on LG2. Independently, Kobayashi et al. (2004) showed that insertion of retrotransposon Gret1 upstream of the VvmybA1 gene was responsible for the lack of colour in berries of cultivar Italia. The results suggested that recombination between Gret1LTRs and lost of the retrotransposon in a VvmybA1 allele was responsible for berry colouration in the Italia somatic variant known as Ruby Okuyama. Moreover, homozygosity for the insertion of this retrotransposon upstream of VvmybA1 was shown to be associated with the lack of berry colour in 12 additional white berry cultivars
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(Kobayashi et al. 2004). Thus, it is possible that VvmybA1 could be the gene underlying the previously mapped colour locus. To con W rm this hypothesis, we analyzed the segregation of both, the VvmybA1 gene and the colour trait in an F1 progeny derived from the selfcross of the red cultivar Ruby Seedless. The prog-eny was selected because Ruby Seedless is hemizygous for the presence of the retrotransposon insertion (VvmybA1ITA/VvmybA1ALF?ALF=allele found in Alphonse Lavallé) and displayed berry colour segrega-tion. Nineteen F1 plants were phenotyped for berry colour and their CIRG values were scored as described in Materials and methods. The same plants were also genotyped for the presence of Gret1 in the VvmybA1 locus (Fig.1a) and for microsatellite loci VMC6B11, VMC5G7, VMC7G3 and VMC8C2 linked to the col-our locus on LG2 and found heterozygous in Ruby Seedless. Colour segregated as a monogenic dominant trait ( 2=1.9, df=1; n/s). Linkage analysis indicated a close linkage between the VvmybA1 sequence and SSR markers on LG2 [recombination frequency (r), r=0.026§0.025, r=0.026§0.025, r=0.052§0.036
and r=0.078§0.043], respectively, for VMC5G7, VMC6B11, VMC7G3 and VMC8C2 as previously described for the colour locus. There was a strong asso-ciation between berry colouration and the presence of the ALF allele at the VvmybA1 gene (P<0.00001). All plants bearing white-skinned berries presented CIGR values bellow 1.55 and were homozygous for the Gret1 insertion allele VvmybA1ITA, while all plants yielding coloured berries carried one or two copies of the func-tional VvmybA1ALF allele and presented CIRG values above 2.49. The ANOVA analysis of average CIRG values for the corresponding genotypic classes (ALF/ ALF, ITA/ALF and ITA/ITA) showed highly signi W-cant di V erences (P<0.0001) between white and col-oured berry plants (Fig.1b). Individuals homozygous for the VvmybA1ALF allele (ALF/ALF) showed slightly higher CIRG values than the heterozygous (ITA/ALF), which suggests the existence of certain level of haploinsu Y ciency. Altogether, the results indi-cate that variation at the VvmybA1 gene is responsible for the berry colour phenotypic variation observed in previous genetic analyses (Doligez et al. 2002; Fisher et al. 2004).
Berry colour is always associated to VvmybA1 functional alleles
To evaluate the extent of berry colour variation explained by VvmybA1 in table grape we analyzed the allelic composition for this locus in a representative sample of 189 table grape cultivars containing an equiv-alent number of coloured and white-skin accessions. The W rst step in the analysis consisted in the PCR detec-tion of the insertion VvmybA1ITA allele. This insertion allele was considered non-functional or null allele based on Kobayashi et al. (2004). Additionally, ampli W cation of PCR products with primers speci W c for the 5?upstream and coding regions of VvmybA1 was consid-ered an indication of the existence of a functional allele, as shown in Fig.2, where six di V erent genotypes for VvmybA1 could be identi W ed. Interestingly, in 91 out of 96 white cultivars analyzed (exempli W ed by Dominga in Fig.2) we only detected the presence of the null ITA allele (Table1). Furthermore, in 88 out of 93 coloured cultivars (exempli W ed by Muscat Hamburg, Sultanine Roseé, Alphonse Lavallée and Black Seedless in Fig.2) we found the presence of at least one putative func-tional allele (Table1 and Fig.2). This PCR assay allowed the detection of three putative functional alle-les VvmybA1ALF, VvmybA1SUB and VvmybA1RUO (see somatic mutation section), with VvmybA1ALF being the most frequent in the colour table grape cultivars ana-lyzed (present in 71 out of 93 cultivars). Two coloured cultivars (Black Monukka and Black Seedless) were heterozygous for the putative functional alleles VvmybA1ALF and VvmybA1SUB (i.e., Black Seedless in Fig.2). Thus, in 179 cultivars out of the 189 tested (95%) the berry colour phenotype can be explained on the basis of the PCR established VvmybA1 genotype, being white when no functional allele can be ampli W ed and coloured when at least one functional allele is detected. These results con W rm the original proposal of Fig.1
Association between VvmybA1 genotype and berry col-our in a selfcross progeny of Ruby seedless. a PCR analysis of VvmybA1 alleles for the Ruby Seedless selfcross population. Numbers on each lane represent the CIRG values for the corre-sponding samples. M molecular weight marker, RS Ruby Seed-less, ITA(VvmybA1ITA); ALF(VvmybA1ALF). b Graphical representation of mean CIRG values and their standard devia-tions calculated for each VvmybA1 genotypic class
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Kobayashi et al. (2004) suggesting that variation in grape berry colour could largely be due to VvmybA1variation. They show a strong parallelism with what has been already reported in plant species such as maize, pepper or petunia where variation at a puta-tive MYB orthologous gene is responsible for colour variation at seeds, fruits and X owers (Borovsky et al.2004; Quattrocchio et al. 1993; Spelt et al. 2000).
The existence of W ve coloured cultivars in which no functional VvmybA1 allele could be ampli W ed as well as W ve white cultivars, which apparently carry VvmybA1 functional alleles (Table 1), might suggest the existence of additional loci controlling grape berry colour and deserved further molecular analyses.
The results regarding the W ve white cultivars will be described in the next paragraph, while the W ve col-oured cultivars that were known somatic variants will be further described in the somatic variation section.The existence of W ve white cultivars carrying putative functional alleles VvmybA1ALF (Roditis) or VvmybA1SUB (Sultanine, Autumn Seedless, Kischmisch Ali Blanc and Pirovano 166A) (Table 1 and Fig.2)might provide support for the existence of a second locus responsible for berry colour variation. Interest-ingly, all these cultivars are hybrids derived directly or indirectly from Sultanine. To evaluate the involvement of VvmybA1 in this phenotype we W rst analyzed the expression of the VvmybA1 alleles present in those white cultivars. Cultivar Roditis carries a PCR typi W ed VvmybA1ALF allele (Fig.2a) and RT-PCR analyses on berry skin total RNA showed regular VvmybA1expression (Fig.3). On the other hand, Sultanine,consistently with its white phenotype, did not display any expression of VvmybA1 in the berry skin. Never-theless, two coloured somatic variants (Sultanine Roseé and Sultanine Rouge) did show expression of the VvmybA1SUB allele.
To obtain more information about the molecular nature of those alleles we PCR ampli W ed and sequenced the corresponding VvmybA1 genome region from Alphonse Lavallé, Sultanine, Sultanine Rosée and Roditis using primers b, cDNAas and a 3?RACE strategy as described in Materials and methods .Based on sequence di V erences the VvmybA1ALF from Roditis was renamed as VvmybA1ROD (Fig.4). Nucleo-tide sequence comparisons with VvmybA1ITA and VvmybA1RUO allele sequences (Kobayashi et al. 2004)showed that VvmybA1ALF and VvmybA1ROD alleles were more related with the VvmybA1ITA allele, while the VvmybA1SUB sequence was quite divergent from all of other. Apart from the existence of a large num-ber of SNPs between VvmybA1SUB and the other
Fig.
2Molecular phenotypes of table grape cultivars at the VvmybA1 locus. a PCR assay for the functional (-ALF and SUB -above) and null (-ITA -below) alleles. Eight cultivars were chosen to illustrate all the di V erent molecular phenotypes found: MUH Muscat Hamburg (coloured ); SUR Sultanine Rosée (coloured );CAC Cape Currant (coloured ); DOM Dominga (white ); SUB Sul-tanine (white ); ROD Roditis (white ); ALF Alphonse Lavallée (coloured ); BKS Black Seedless (coloured ). M molecular weight marker
Table 1VvmybA1 genotypes and colour phenotypes observed in the analyzed table grape cultivars a
Alleles based on the PCR ampli W cation of null (ITA VvmybA1ITA ) and/or functional (ALF VvmybA1ALF , SUB VvmybA1SUB or RUO VvmybA1RUO ) alleles (see Materials and methods )b
Berry skin colour: W white (yellow or yellow-green); C coloured (pink to dark blue)
VvmybA1 PCR based genotypes Colour
phenotypes b Total
Genotype
Alleles a W
C
ITA
ALF SUB
RUO
1+915962++
152533++
410144+017175+
+
0226
+
+077Total
96
93189
Fig.3VvmybA1 expression in the berry skin of particular culti-vars. RT-PCR ampli W cation on total RNA from Muscat Ham-burg (MUH ), Roditis (ROD ), Sultanine (SUB ), Sultanine Rosée (SUR ) and Sultanine Rouge (SUG ). White cultivar Roditis and coloured cultivar Muscat Hamburg (both displaying the same PCR molecular phenotype) showed identical expression for VvmybA1. Despite presenting identical coding sequences as Sul-tanine Rosée and Sultanine Rouge, no VvmybA1 expression was detected in Sultanine. M molecular weight marker
VvmybA1 alleles, the Sultanine allele carries three short insertions, two in the 5?-UTR region and one in the second intron (Fig.4). Comparison of DNA sequences between VvmybA1ALF and VvmybA1ROD revealed six SNPs di V erences and the existence of a deletion of the last 135 coding nucleotides of the Rod-itis gene (Fig.4). The Roditis allele (VvMybA1ROD) encodes a truncated protein and might represent a loss of function allele responsible for the absence of colour in the skin of Roditis berries. In fact, this carboxyl ter-minus has been shown to function as activation domain in C1 (Go V et al. 1991) and is also proposed to have a relevant functional role in VvMYBA1 (Kobayashi et al. 2005). Still, without a functional proof, we cannot completely discard the possibility that this allele is par-tially active and Roditis’s lack of colour results from genetic variation at some other locus.
Comparative analysis of VvmybA1SUB sequences from coloured and white berry cultivars showed that they were identical, indicating that the colour di V er-ence could not be explained by sequence variation at the VvMybA1 coding sequence. These results might suggest either the existence of an uncovered tranposon insertion in the upstream promoter region of VvMybA1 or of a second regulatory locus controlling berry colour in grape. In any case, the transposon insertion or the second locus would regulate VvMybA1 expression (Fig.3).
In summary, with the exception of the Roditis’s truncated mRNA, all white berry cultivars analyzed show lack of VvmybA1 expression either due to the insertion of Gret1 in the VvmybA1 promoter region as in the VvmybA1ITA/VvmybA1ITA cultivars or to the down-regulation of functional alleles like in the white VvmybA1ITA/VvmybA1SUB cultivars.
Molecular basis of colour somatic variation
Colour somatic variation is a well studied phenomenon in plants and has been studied in many fruit crops including apple (Arakawa et al. 1999; Awad et al. 2000; Ben-Yehudah et al. 2005), bilberry (Jaakola et al. 2002), citrus (James and Howard 1989) and pear (van Dyk et al. 2005). The generation of somatic coloured variants from white cultivars is a relatively common phenomenon in table grape (Franks et al. 2002; Hocquigny et al. 2004; Thompson and Olmo 1963). In Ruby Okuyama, a red berry somatic variant of Italia, the coloured phenotype was associated with the recov-ery of VvmybA1 expression and related to the intra-LTR recombination of the Gret1 retrotransposon (Kobayashi et al. 2004).
Fig.4
Comparison of nucleotide sequences for VvmybA1 alleles.
a Sequence alignment of VvmybA1 alleles. All sequences are com-pared to the reference allele VvmybA1ITA (ITA) (Kobayashi et al. 2004). The position of the polymorphic sites and the VvmybA1SUB (SUB) and VvmybA1ROD(ROD) speci W c indels, and their locations in the 5?-UTR region, introns (I) and exons (E) are indi-cated on top. Start position corresponds to the location of primer
b (see panel b). TS duplicated target site for the Gret1 insertion. b Schemati
c representation of the alleles an
d approximat
e position o
f the primers used in the di V erent PCR assays
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We have investigated the molecular basis of somatic colour variation in seven additional white berry culti-vars homozygous for the VvmybA1ITA alleles (Beba, Chasselas Doré, Jaén Blanco, Moscatel de Grano Gordo, Moscatel de Grano Menudo, Sugraone and Xarel.lo) and their colour somatic variants. As shown in Fig.5a, in six somatic variants, colour recovery was associated with the ampli W cation of the solo-LTR PCR fragment (RUO) initially identi W ed in Ruby Okuyama (Kobayashi et al. 2004). Interestingly, in the remaining variants analyzed (Benitaka, Cape Currant, Chasselas Rouge, Moscatel de Grano Menudo Rojo and Jaén Rosado-Fig.5a) colour recovery was not related to the appearance of the solo-LTR PCR fragment. Both clas-ses of somatic variants were further analyzed to under-stand the molecular basis of colour recovery. For the W rst class, two independent spontaneous colour vari-ants of Sugraone, Ralli Seedless and Super Red were analyzed. Both cultivars displayed VvmybA1RUO alle-les (Figs.4b, 5a). Sequencing of the solo-LTRs from Ralli Seedless and Super Red and comparison with the 5? and 3?-LTR sequences in Sugraone showed that these two red somatic variants derived from recombi-nation events taking place at two di V erent positions within the LTRs. This result was obtained thanks to the identi W cation of 5 SNPs between the 5? and 3?LTRs of Sugraone that were enough to trace the recombination events giving rise to each colour cultivar (Fig.5b). It proofs that intra-LTR recombination is the colour recovery mechanism for these somatic variants and con W rms the independent nature of recombination events giving rise to them.
For the remaining colour somatic variants neither a solo-LTR nor any other functional allele could be PCR ampli W ed (Fig.5a), even when the six di V erent primer combinations detailed in Fig.5c (left) were used. A DNA blot hybridization analysis with Beni-taka, Chasselas Rouge, Italia and Ruby Okuyama genomic DNA allowed for the detection of the restriction fragment corresponding to the VvmybA1ITA allele as well as additional hybridizing bands with di V erent size in each genotype that could be attributable to the presence of VvmybA1 func-tional alleles (Fig.5c right). Retrotransposon inser-tions are known to frequently generate deletions, likely through mechanisms of illegitimate recombina-tion. In rice and Arabidopsis these deletions have been proposed as a possible cause for genome size reduction (Ma et al. 2004). We argue that illegitimate
recombination in the region of Gret1 insertion could produce deletion of the retrotransposon and addi-tional sequences in the 5? region of VvmybA1 pre-venting PCR primers hybridization.
Somatic variation is a very relevant phenomenon creating berry colour variation in grape. We have shown that this variation is mostly based on the genetic instability associated with the presence of Gret1at locus Fig.
5Molecular basis of somatic variation for grape skin colour.
a PCR assay for the functional (-RUO-above) and null (-ITA-be-low) alleles from eight white cultivars and their colour somatic variants: [SUO Sugraone/RAL Ralli Seedless and SPR Super Red], [BEB Beba/CLR Calop Rojo], [MGG Moscatel de Grano Gordo/MUR Moscatel Rosa], [XAL Xarel’lo/XAR Xarel’lo Rosa-do], [CHD Chasselas Doré/CHR Chasselas Rosé], [MGM Mosca-tel de Grano Menudo/MGR Moscatel de Grano Menudo Rojo and CAC Cape Currant], [JAB Jaén Blanco/JAR Jaén Rosado], [ITA Italia/RUO Ruby Okuyama and BEN Benitaka]. M molecular weight marker.
b Schemati
c representation of the intra-LTR recombination events that took place in the white cultivar Sugra-one (SUO) to generate somatic coloure
d variants Ralli (RAL) and Super Red (SPR) (top). Sequenc
e alignment o
f SUO 5?-LTR and 3?-LTR together with the solo recombinant LTR s of RAL and SUR. Arrows indicate the region where recombination events took place (bottom). TS duplicated target site (ACACA in blue). c Schematic representation of the putative VvmybA1 organization in the coloured cultivar Benitaka and primers used in the corre-spondin
g PCR assay. Vertical arrows indicate the approximate po-sition of the Apa I restriction sites used for the DNA blot hybridization assay. P VvmybA1 probe (left). DNA blot hybridiza-tion analysis of Benitaka (BEN) and Chasselas Rose (CHR). Italia (ITA) and Ruby Okuyama (RUO) were used as controls (right)
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VvmybA1 and can result from homologous recombina-tion between LTRs as well as putative illegitimate recombination events.
Conclusions
In summary, berry colour variation in grapevine is strongly associated with genetic variation at the VvmybA1 gene. We have shown that this gene under-lies the major colour locus mapped in segregating populations. Three major haplotypes, ITA, ALF and SUB, with allelic frequencies of 69, 23 and 4% respec-tively, have been identi W ed in the analyzed sample (Table S1). Among them ALF seems to represent a major functional allele while ITA and SUB are pres-ent in colourless cultivars. Interestingly, all cultivars carrying two functional VvmybA1 alleles such as Black Seedless, Almeria Nera, Dattier Noir or Negra Tardía are known by names that allude to their dark skin colour (see Table S1 and synonymies at the Vitis International Variety Catalogue: http://www.genres. de/idb/vitis/). Sequence variation at the VvmybA1 locus explains berry colour in over 95% of the ana-lyzed cultivars. While in the remaining cultivars, berry colour is always associated to VvmybA1 expres-sion. The null Gret1 insertion allele, ITA, behaves as an unstable allele generating additional derivative alleles from Gret1 related recombination events. Gret1 related instability can be associated to most cases of somatic colour variation with the exception of the Sultanine derived somatic variants that suggest instability at a second transposon insertion or a sec-ond locus. Additional loci will further contribute to berry colour variation, either through the regulation of VvmybA1 or through their role in the anthocyanin biosynthetic pathway, such as those encoding X avonoid hydroxylases (Bogs et al. 2006; Castellarin et al. 2006) which could be responsible for dark blue colours.
Acknowledgment We thank Juan Negueroles (AFREXPORT S.A.) and María del Carmen Martínez (Misión Biológica de Ga-licia-CSIC) for some of the plant materials used in DNA and RNA analyses. We also thank Cheo Machín for careful editing of the manuscript. This work was funded by a grant from the CDTI (Spanish Ministry of Science and Technology).
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武汉天喻信息产业股份有限公司 员工绩效考核制度 一．总则 1.公司实施考核直接目的是客观地分析和评价员工工作职责的履行和工作实际的效果， 适时提供真实可靠的人力资源管理和开发相关数据，并且根据考核结果正确实施奖惩，合理配置人力资源，全面提升员工绩效，保障公司的可持续高速发展。 2.本制度适用于武汉天喻信息产业股份有限公司行政管理系列（总部、综合管理部、物 财中心、产品研发中心的研发管理部、技术服务中心的生产部、营销中心的市场部）、产品研发系列、技术实施系列、市场营销系列员工。 二．考核原则 3.公开、公平、公正、客观原则：根据各职务职能特点制定考核标准和程序，采用关键 事件失误法制定考核量表及指标，以期激励各岗位员工各项工作按公司规定的要求和流程做的到位且高效。 4.绝对性考核原则：以岗位职责和具体事实为依据，按照职务职能标准对员工的工作行 为进行考核，而非人与人之间的对比考核。 5.分析性考核原则：按事先确定的职务职能考核要素及重点逐条进行考核，而不是对人 进行模糊的整体评价。 6.一个主体原则：采用直接上级考核直接下级的办法，并且考核结果应由直接上级与直 接下级沟通确认。 三．考核内容 7.公司行政管理系列员工主要对岗位职责进行考核。岗位职责应包括岗位职能的关键性 职责及具体工作规范。 8.公司产品研发、技术实施系列员工主要考核内容为： （1）月度项目计划与实际完成进度； （2）产品质量 ●产品功能和性能是否满足设计要求 ●文档质量 ●产品代码的易读性
●产品代码的可移植性 ●产品代码的可重用性 ●开发过程中的BUG的数量统计和分析 ●用户界面清晰 ●接口合理性 （3）各类开发文档是否齐全 （4）协作精神（项目组内、部门之间） （5）流程和规范的执行情况 （6）工作量是否饱满。 9.公司市场营销系列员工主要对工作业绩（合同额、回款额）、工作态度、团队合作等 进行考核。 四．考核程序 10.本制度实行前，由综合管理部负责组织有关考核制度的沟通、学习，务必使每名员工 在考核进行前了解掌握该制度。 11.月度考核工作由各部门自行组织。考核结果是由被考核者与其直接上级共同完成，即 每次考核时，被考核者的直接上级应与被考核者至少进行一次沟通。 12.总部人员的考核由综合管理部组织。考核者为执行总裁和总裁。 13.各部门于每月的5号前将上月每个员工的考核量表及部门的考核系数汇总表交至综 合管理部，由综合管理部完成对数据的统计。 14.综合管理部于每月的7号前将每个员工上月根据考核系数计算的应发工资额及相关 奖励额报至财务部，财务部将在10号前将工资发给员工。 五. 考核表 15.考核工作使用公司综合管理部统一印制的考核表。 16.考核表的考核标准由部门制定，关键事件失误扣分数值由部门自行确定，分管副总确 认，各部门制定出样表并与被考核者充分沟通后交综合管理部审核备案。 17.考核表的计分细则。各考核者每月对被考核人填写一份考核表，“考核结果”一栏为 “空”（合格）或对应考核标准的扣分值（不合格），并计算出考核系数（＝1－考核结果之和）填写在“月考核系数”栏。 18.考核人与被考核人沟通确认后将考核表交综合管理部备案。 六. 考核结果
一、教学设计说明： 本节课选自小学英语三年级上学期Lesson 9“What colour is it? ”.本课时主要学习用英语表达事物的颜色，其内容贴近小学生的生活和学习实际，有利于他们在真实的语言情境中培养运用英语进行交际的能力。教材强调学生在学习过程中的感悟、体验、实践、参与以及思维能力的发展，突出兴趣培养，重视学科内容的有效融合，注重培养学生自主学习的意识和能力。 二、学情分析： 本课的教学对象是刚刚涉足英语的三年级小学生，学生的好奇心强，大部分学生对于英语有着强烈的兴趣，敢于大胆开口说英语，愿意同他人合作，在交际中学习英语，感悟英语，在游戏中体验英语，认识英语。但三年级的新生刚刚入学，自制力较差，注意力不易集中。 三、设计理念： 《英语课程标准》中明确指出，基础教育阶段学习英语的目的旨在激发学生学习英语的兴趣，培养他们学习英语的积极态度，使他们建立初步的学习英语的自信心；培养学生一定的语感和良好的语音、语调基础，使他们形成初步用英语进行简单交流的能力，为进一步学习打下良好的基础。 要激发学生学习英语的兴趣，我们就要让学生变被动为主动地跟着教师参与教学活动，运用一些学生喜闻乐见的教学方法及措施。因此，教师在教学中发挥主导作用，以学生为主体，采用直观情境教学法、TPR(全身反应法)，活动式教学法。充分采用了任务型教学途径，为学生提供了较大的实践空间，能较好地发挥学生的主动性和创造性。任务型教学途径适合多种活动类型和组织形式（如，游戏、比赛、竞猜等），它不但可以独立操作，也可以利于合作学习，使学生在乐中学、学中乐。 四、教法学法设计： 在本课中，我采用任务教学法，直观演示法、情境教学法、游戏教学法相结合的方法。在教学设计中一开始就把任务交给学生，让学生在任务的驱动下学习语言知识，利用多媒体课件，实物等创设情境，采用学生易于接受的游戏等方式组织课堂教学，提高了课堂效率。 生活化的情景有利于培养学生的语言运用能力，“情景交际法”是本课主要的教学方法。通过颜色学习，由green（绿色）red(红色)yellow（黄色）引向对学生的交通意识教育，打破学科本位的传统模式，将实际生活渗透于英语教学之中。 TPR教学法主要针对儿童的身心特点设计的，运用该法设计红绿灯绕口令，能充分调动学生的各种感官参与学习，有助于对知识的理解和内化。在学习方法上，主要运用全程式自主学习方式，从学生的需求入手，将学习内容与学生的日常生活经验相结合，在协同学习的环境中体验、参与，从而建构个人的经验，创造个人的知识。 五、教学内容： 1、五个新单词: blue, green, yellow,orange, red. 2、句型: What colour is it? It’s… 六、教学目标： (一) 知识目标：1、能听、说、认读五个颜色单词blue, green, yellow,orange, red，并能在实际情景中进行运用。 2、能掌握句型：What colour is it? It’s…,并且能灵活运用。 （二）技能目标：让学生在游戏活动中熟悉运用所学颜色单词，并能对身边的事物进行简单的描述。让学生学会用What colour is it ?和It’s…来提问和回答周围的事物的颜色。培养学生在课堂上进行自主评价的能力。 （三）情感目标：关注学生的内在需要，在活动中培养学生的协作精神和竞争意识，培养学生热爱生活的积极情感态度。
做效果图之基本配色方案及色标大全（新手颜色搭配必学） 基本配色——奔放 ----------------------------------------------------------------------------------- 藉由使用象朱红色这种一般最令人熟知的色彩，或是它众多的明色和暗色中的一个，都能在一般设计和平面设计上展现活力与热忱。中央为红橙色的色彩组合最能轻易创造出有活力、充满温暖的感觉。 这种色彩组合让人有青春、朝气、活泼、顽皮的感觉，常常出现在广告中，展示精力充沛的个性与生活方式。把红橙和它的补色——蓝绿色——搭配组合起来，就具有亲近、随和、活泼、主动的效果，每当应用在织品、广告和包装上，都是非常有效。 基本配色——传统 ----------------------------------------------------------------------------------- 传统的色彩组合常常是从那些具有历史意义的色彩那里仿来的。 蓝、暗红、褐和绿等保守的颜色加上了灰色或是加深了色彩，都可表达传统的主题。 例如，绿，不管是纯色或是加上灰色的暗色，都象征财富。
狩猎绿（hunter green）配上浓金或是暗红或是黑色表示稳定与富有。这种色彩常出现在银行和律师事务所的装潢上，因为它们代表恒久与价值。 基本配色——低沉 ----------------------------------------------------------------------------------- 不同于其它色彩展现柔和，低沉之美的灰紫色没有对比色。灰紫色调合了红紫色、灰色和白色，是个少见的彩色。 任何颜色加上少许的灰色或白色，能表达出的柔和之美，有许多种包括灰蓝色、灰绿色等。但若灰紫色本身被赋其它彩度或亮度，则可能掩盖了原颜色的原有意境。使用补色，或比原色更生动的颜色，可使这些展现柔和之美的颜色顿时生意昂然，但要保持自然的柔美，亮度的变化应尽少使用。 基本配色——动感 ----------------------------------------------------------------------------------- 最鲜艳的色彩组合通常中央都有原色——黄色。黄色代表带给万物生机的太阳，活力和永恒的动感。当黄色加入了白色，它光亮的特质就会增加，产生出格外耀眼的全盘效果。 高度对比的配色设计，像黄色和它的补色紫色，就含有活力和行动的意味，尤其是出现在圆形的空间里面。身处在黄色或它的任何一
ChinaDRM介绍 中国广播影视数字版权管理论坛 中国广播影视数字版权管理论坛(ChinaDRM Forum，以下称ChinaDRM)成立于2004年11月24日。该论坛是在广电总局的大力支持下以中央电视台和清华大学为牵头单位，由江苏广播电视总台、重庆电视台、中国广播电影电视节目交易中心、中央数字电视传媒有限公司、辽宁电视台、上海文广新闻传媒集团、上海文广互动电视有限公司、中央人民广播电台、中国国际广播电台、北京电视台、国家广播电影电视总局电视规划院、中国传媒大学、北京永新同方数字电视技术有限公司、爱迪德技术（北京）有限公司、索尼（中国）有限公司、北京捷成世纪媒体科技有限公司、中视网络发展有限公司、THOMSON宽带研发（北京）有限公司、联合信源数字音视频技术(北京)有限公司、松下电器研究开发（中国）有限公司、北京算通科技发展有限公司共同发起成立，其宗旨是促进全社会对数字版权管理的重视,推动数字版权管理的发展，保障数字媒体内容发布链中所有参与者的权利，建立一个良好的内容发布和消费环境。论坛重在研究探讨中国数字媒体版权管理的应用需求、解决方案、技术标准、发展方向、发展战略及其与国家数字媒体发展密切相关的问题，关注最新的DRM技术动态，促进会员间的交流与合作，为构建一个良好的数字媒体版权管理发展环境创造条件。 论坛旨在呼吁业内外人士站在媒体事业发展的高度认识数字版权管理的紧迫性、必要性和重要性；同时，倡导各方抓住数字电视、网络电视发展的机遇，本着严谨务实的科学精神，研究媒体数字版权管理的先进技术，探讨怎样才能形成由内容、服务、设备提供者和内容消费者多方权益共享的、可持续发展的数字媒体产业链的方法，促进媒体产业的蓬勃发展，为媒体产业做强、做大创造条件，为广大的受众提供更加高质量的影视节目和更加便捷的获取途径。论坛也希望通过自身的工作，唤起全社会的版权管理意识，并最终促进数字媒体的数字版权管理体制的建立和法律法规的制订。 截至2010年7月16日，至此，ChinaDRM论坛已发展会员单位81家，其中：媒体单位和内容提供商15家；方案技术提供商41家；终端设备制造商19家；学校和研究机构5家；运营商1家。ChinaDRM标准起草委员会目前有会员单位67家+6家关联单位。 ChinaDRM已完成《中国广播影视数字版权管理需求白皮书》的撰写起草工作。目前，已起草完成《广播影视数字版权管理广播电视内容制作—数字内容版权标识》、《广播电视数字版权管理元数据标准》、《移动多媒体广播：业务与内容保护技术规范》、《广播影视数字版权管理广播电视内容分发—广播控制标记》、《IPTV内容分发数字版权管理技术标准(规范)草案》、《B2B内容分发数字版权管理技术标准(规范)草案(CD)》、《家庭网络技术标准(规范)草案(CD)》等标准草案。
《Colours》教学设计 万年县第二小学吴妤 一、设计理念 在本课的教学中，我主要按照新课标中的理念，以人为本、面向全体，采用任务型教学方式，让学生通过感知、体验、实践、参与、合作交流来实现任务目标。我在整个活动中只起一个活动者、组织者、指导者、参与者的作用，留足够的时间和空间给学生完成任务，努力做到教师用“爱”促进教育，用“趣”构建教育模式，用“玩”组织教学活动，使学生“为用而学、边用边学、学完就用”，充分发挥师生的互动作用，在“玩中学、玩中练、玩中见成效”。 二、教学内容：Colours 三、教学目标： （一）知识目标 1、能听、说、认读单词red,yellow,blue,green,orange. 2、能听懂、会说句型It’s red/yellow／．．．。Ｔhe…is red/yellow/…。 (二)能力目标 培养学生在游戏中熟练运用单词的能力、知识迁移能力、灵活运用交际用语的能力，能听懂指令，并能按照指令做出各种动作。 （三）情感、文化等有关目标 1、情感态度：激发学生学习英语的兴趣，培养学生学习英语的积极态度，使学生乐于合作参与，勇于进行交际实践。 2、学习策略：注重合作学习。 四、教学重点难点 （一）重点： 单词red,yellow,blue,green,orange的教学。 句型It’s red/yellow/…的教学。 （二）难点： Blue及green的正确发音。 五、教学准备 1、CAI课件。 2、学生自备有颜色的实物。 3、调查表。
4、录音机、磁带、五种颜色的花瓣单词卡片、彩虹图。 5、玻璃杯、水、颜料。 六、教学过程 Step1:Warming up. 师生共同表演唱彩虹歌《I can sing a rainbow》。 Step2:Revision. (1)课件出示一只鸟。T：What’s this? Ss:It’s a bird.又飞来了二只鸟，T:Do you like birds? Ss:Yes,I like birds./No,I don’t like birds. (2)课件出示一只兔子，T:Is this a bird? Ss:No,it’s a rabbit.又跑来了三只兔子，T：Do you like rabbits? Ss:Yes,I like rabbits./No,I don’t like rabbits. (3)课件出示一只猴。T：What can you see? Ss:I can see a monkey.又跑来了三只猴子，T:Do you like monkeys? S:Yes ,I like monkeys./No, I don’t like monkeys. Step3:Learn the new words. 1.出示彩虹图。T：Do you like rainbow? Ss:I like rainbow.T:I like rainbow,too.Because it’s colourful.Today we’ll learn colours.(出示课题，贴彩虹图)。I have divided you into five teams.(红队、黄队、蓝队、绿队、桔黄队，老师边说队名边贴代表队的卡在黑板)看看今天哪队表现得最好. 2.课件出示一面红旗。T：The flag is red .It’s red .课件显示句子It’s red.教师贴红色花瓣单词卡red ,教读red,指名读red,纠正读音，然后手拿一朵红花教It’s red.T:Pass the flower and say “It’s red .”生边传递花边说It’s red .再教读The flower is red .然后教师请学生拿着课前准备的实物，用句型The …is red .说说红色的实物。 3.课件出示一只黄色的鸭子。T：Look,it’s a duck.The duck is yellow.It’s yellow.课件显示句子It’s yellow.贴黄色花瓣单词卡yellow.用不同的语气教读yellow，（咦yellow，啊yellow，嘿yellow，哎yellow），再让学生表演读yellow,指名读，教读It’s yellow.The duck is yellow.生跟读之后，教师又让学生拿课前准备的实物说说黄色的物品。 4.课件出示大海，T：The sea is blue.It’s blue.课件显示句子It’s
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colours微评课 一、新旧结合，复习导入。 每节课的教与学都不是孤立的，它有承上启下的作用，是旧知识的拓展与深化。因此，在新课开始之前，对与本节课内容有关的知识与技能进行复习与巩固，既能弥补学生旧知识掌握中的缺漏，又能使学生很快地投入新知识的最佳学习状态。微课中教师主要教授的句型是What’s your favourite colo ur?及其回答My favourite colour is_______.教师在引出句型之前运用卡片复习了颜色单词，唤醒了学生的旧知，同时为本节课句型学习做好知识准备。 二、游戏操练，突破难点。 在英语单词教学中，如果总是采用教师领读、学生跟读的方法，学生会感到枯燥，所以不太愿意跟读。低年级学生活泼好动，乐于接受新奇、趣味性强的事物，在教学单词时融入游戏对帮助他们理解与母语迥异的另一种语言是必不可少的，是英语教学的重要手段。微课中favourite这个单词是今天所学的句型中的重点和难点，学生对其意思的理解和单词的发音掌握起来都具有一定的困难。教师在操练单词时设计了一个小游戏，出示笑脸学生就说favourite,不是笑脸就不能说，这个游戏可以防止学生注意力不集中，跟
读时偷懒，能让每个学生主动参与到游戏中，牢固掌握单词发音的同时也能对favourite的意思加深印象，为后面的句型教学扫除了障碍。 三、师生互动，实时评价。 教师从复习颜色，解决教学难点，到进入核心句型的学习，整个过程循序渐进，符合学生的认知和接受能力，过渡十分自然。颜色是学生平时生活中容易接触到并有所感知的话题，所以教师直接通过介绍自己最喜欢的颜色切入主题句型，并用What’s your favourite colour?询问，让学生一一进行操练。等学生较好的掌握答句之后，再通过全班问，个别答的方式巩固问句，关注发挥学生的主体作用，让学生真实的去感受知识，体验知识，积极参与，努力实践，在活动中学会用语言表达交流，较好的体现了从不懂到懂，从不会到会，从不熟练到熟练的过程。教学过程中，教师准备了各种不同颜色的小卡片，只要学生用正确的句型表达出自己最喜欢的颜色，就能得到相应的颜色卡片，这个小小的设计不仅能让学生体会到语言学习的功能性，营造出较真实自然的交际情景，而且学生所得到的卡片也可以视为教师对其敢于表达的及时评价和奖励，把学生对卡片的向往转化成课
平面设计常见的配色方案及色标 粉红代表浪漫。粉红色是把数量不一的白色加在红色里面，造成一种明亮的红。象红色一样，粉红色会引起人的兴趣与快感，但是比较柔和、宁静的方式进行。 浪漫色彩设计，藉由使用粉红、淡紫和桃红（略带黄色的粉红色），会令人觉得柔和、典雅。和其它明亮的粉彩配合起来，红色会让想起梦幻般的６月天和满满一束夏日炎炎下娇柔的花朵。
基本配色——流行
今天“流行”的，明天可能就“落伍”了。流行的配色设计看起来挺舒服的，但却有震撼他人目光的效果。 淡黄绿色（chartreuse）就是一个很好的例子，色彩醒目，适用于青春有活力且不寻常的事物上。 从棒球运动鞋到毛衣，这种鲜明的色彩在流行服饰里创造出无数成功的色彩组合。黄绿或淡黄绿色和它完美的补色——苯胺红（magenta）搭配起来，就是一种绝妙的对比色彩组合。
基本配色——平静 -----------------------------------------------------------------------------------
在任何充满压力的环境里，只要搭配出一些灰蓝或淡蓝的明色色彩组合，就会制造出令人平和、恬静的效果。 中间是淡蓝的配色设计，会给人安心的感觉，因为它看起来诚实、直接。 带着明色的寒色可保持安宁、平和的感觉。补色和这些强调平静的色彩在明暗度方面一定要类似，这点很重要，因为要是色彩太鲜明，会制造出不必要的紧张。
基本配色——强烈 -----------------------------------------------------------------------------------------
天喻信息2020年上半年财务分析详细报告 一、资产结构分析 1.资产构成基本情况 天喻信息2020年上半年资产总额为290,214.62万元，其中流动资产为249,529.6万元，主要以应收账款、存货、交易性金融资产为主，分别占流动资产的47.7%、16.83%和16.82%。非流动资产为40,685.02万元，主要以固定资产、长期股权投资、递延所得税资产为主，分别占非流动资产的41.21%、17.54%和15.5%。 资产构成表(万元) 项目名称 2018年上半年2019年上半年2020年上半年 数值百分比(%) 数值百分比(%) 数值百分比(%) 总资产 217,987.48 100.00 209,640.55 100.00 290,214.62 100.00 流动资产 166,798.47 76.52 169,495.69 80.85 249,529.6 85.98 应收账款 101,897.95 46.74 87,512.97 41.74 119,024.47 41.01 存货28,011.14 12.85 25,684.25 12.25 41,991.44 14.47 交易性金融资产0 - 19,989.38 9.54 41,975.34 14.46 非流动资产51,189.01 23.48 40,144.85 19.15 40,685.02 14.02
固定资产18,719.89 8.59 15,527.7 7.41 16,766.26 5.78 长期股权投资6,078.7 2.79 7,123.63 3.40 7,138.03 2.46 递延所得税资产3,883 1.78 4,627.95 2.21 6,306.93 2.17 2.流动资产构成特点 企业流动资产中被别人占用的、应当收回的资产数额较大，约占企业流动资产的48.93%，应当加强应收款项管理，关注应收款项的质量。企业持有的货币性资产数额较大，约占流动资产的32.81%，表明企业的支付能力和应变能力较强。但应当关注货币资金的投向。 流动资产构成表(万元) 项目名称 2018年上半年2019年上半年2020年上半年 数值百分比(%) 数值百分比(%) 数值百分比(%) 流动资产 166,798.47 100.00 169,495.69 100.00 249,529.6 100.00 应收账款 101,897.95 61.09 87,512.97 51.63 119,024.47 47.70 存货28,011.14 16.79 25,684.25 15.15 41,991.44 16.83 交易性金融资产0 - 19,989.38 11.79 41,975.34 16.82 货币资金26,724.52 16.02 22,352.38 13.19 39,889.14 15.99 其他应收款1,887.47 1.13 2,648.69 1.56 3,064.7 1.23 其他流动资产760.66 0.46 7,475.58 4.41 1,987.54 0.80 预付款项4,776.73 2.86 1,152.44 0.68 1,487.61 0.60
https://www.doczj.com/doc/16106924.html,/tresearch/a/83740115cid00001 课题名称 Colours 教学内容词汇 red、yellow、blue、green、pink、purple、orenge 句型 A：What colour is it? B：It’s… 教学目标 1、知识目标 学习怎样用英语表达事物的颜色，掌握相关的单词： red\yellow\blue\green\pink\purple\orenge和句型What colour is it?It’s…并能综合运用新、旧知识组织对话完成一定交际任务。 2、情感目标 激发学生学习英语的兴趣，调动学习积极性，引导学生积极与他人合作，在活动中培养学生的协作精神和竞争意识。 3、能力目标 培养学生的观察力、想象力、动手操作能力，提高学生感受美、欣赏美的水平，发展学生综合运用语言的能力。 4、德育目标 培养学生热爱大自然、保护环境的良好品德。 教学重点 创设语言情景，使学生正确理解和运用所学知识用英语表达事物颜色。 教学难点 激发学生的求知欲，创设各种真实或接近真实的语言环境，让学生体验参与，主动学习事物颜色的表达法。 课前准备
1、CAI课件； 2、“red、yellow、blue、green、pink、purple、orenge”单词卡片及“What colour is it?It’s…”句型卡片； 3、调色板、水彩颜料、铅画纸若干及一些带颜色的实物； 4、小动物头饰、色块卡； 5、歌曲Colour Song录音、投影器； 6、在教室里挂上一些五彩气球和彩带，营造课堂气氛，同时作为奖品，激发学生兴趣。 学生情况分析 学生的年龄在九、十岁左右，生性活泼好动，喜欢直观形象思维，对游戏、竞赛、画画特别感兴趣。学生学英语不久，有可能说的不好，有的还不敢说，课堂上要以表扬为主，注重培养学习英语的兴趣，鼓励他们大胆说、积极做、努力唱！ 教学步骤 Step1 Free talk 1、Sing a song:“Morning Song” 2、T：Good morning,boys and girls.Nice to see you. Ss：Nice to see you,too. 分别与单个学生对话：Hello!/What’s your name?/How are you?/Where’s your nose?/Touch your face,please./Show me your pencil./What’s this?(头、手、铅笔、橡皮、蜡笔……) Step2 Introduce T:What’s this? CAI演示：一阵雷雨过后，太阳露出了笑脸，天边出现了一道美丽的彩虹。 引出rainbow T:Wow,how beautiful!Today we’ll learn a lesson about the colours of the rainbow.
服装配色方案及色标大全(设计篇) 已有 100 次阅读2011-9-21 01:11|个人分类:理论|色彩, 设计, 色标, 颜色, 服装 基本配色——奔放 ----------------------------------------------------------------------------------- 藉由使用象朱红色这种一般最令人熟知的色彩，或是它众多的明色和暗色中的一个，都能在一般设计和平面设计上展现活力与热忱。中央为红橙色的色彩组合最能轻易创造出有活力、充满温暖的感觉。 这种色彩组合让人有青春、朝气、活泼、顽皮的感觉，常常出现在广告中，展示精力充沛的个性与生活方式。把红橙和它的补色——蓝绿色——搭配组合起来，就具有亲近、随和、活泼、主动的效果，每当应用在织品、广告和包装上，都是非常有效。 基本配色——传统 ----------------------------------------------------------------------------------- 传统的色彩组合常常是从那些具有历史意义的色彩那里仿来的。 蓝、暗红、褐和绿等保守的颜色加上了灰色或是加深了色彩，都可表达传统的主题。 例如，绿，不管是纯色或是加上灰色的暗色，都象征财富。 狩猎绿（hunter green）配上浓金或是暗红或是黑色表示稳定与富有。这种色彩常出现在银行和律师事务所的装潢上，因为它们代表恒久与价值。 基本配色——低沉
-------------- 不同于其它色彩展现柔和，低沉之美的灰紫色没有对比色。灰紫色调合了红紫色、灰色和白色，是个少见的彩色。 任何颜色加上少许的灰色或白色，能表达出的柔和之美，有许多种包括灰蓝色、灰绿色等。但若灰紫色本身被赋其它彩度或亮度，则可能掩盖了原颜色的原有意境。使用补色，或比原色更生动的颜色，可使这些展现柔和之美的颜色顿时生意昂然，但要保持自然的柔美，亮度的变化应尽少使用。 基本配色——动感 ----------------------------------------------------------------------------------- 最鲜艳的色彩组合通常中央都有原色——黄色。黄色代表带给万物生机的太阳，活力和永恒的动感。当黄色加入了白色，它光亮的特质就会增加，产生出格外耀眼的全盘效果。 高度对比的配色设计，像黄色和它的补色紫色，就含有活力和行动的意味，尤其是出现在圆形的空间里面。身处在黄色或它的任何一个明色的环境，几乎是不可能会感到沮丧的。 基本配色——丰富 ----------------------------------------------------------------------------------- 要表现色彩里的浓烈、富足感可藉由组合一个有力的色彩和它暗下来的补色。例如，深白兰地酒红色就是在红色中加了黑色，就像产自法国葡萄园里陈年纯美的葡萄酒，象征财富。白兰地酒红色和深森林绿如果和金色一起使用可表现富裕。- 这些深色、华丽的色彩用在各式各样的织料上，如皮革和波纹皱丝等等，可创造出戏剧性、难以忘怀的效果。这些色彩会给人一种财富和地位的感觉。 基本配色——高雅
平面设计常见的配色方案 及色标 粉红代表浪漫。粉红色是把数量不一的白色加在红色里面，造成一种明亮的红。象红色一样，粉红色会引起人的兴趣与快感，但是比较柔和、宁静的方式进行。 浪漫色彩设计，藉由使用粉红、淡紫和桃红（略带黄色的粉红色），会令人觉得柔和、典雅。和其它明亮的粉彩配合起来，红色会让想起梦幻般的６月天和满满一束夏日炎炎下娇 柔的花朵。
基本配色——流行
今天“流行”的，明天可能就“落伍”了。流行的配色设计看起来挺舒服的，但却有震撼他人目光的效果。 淡黄绿色（chartreuse）就是一个很好的例子，色彩醒目，适用于青春有活力且不寻常的事物上。 从棒球运动鞋到毛衣，这种鲜明的色彩在流行服饰里创造出无数成功的色彩组合。黄绿或淡黄绿色和它完美的补色——苯胺红（magenta）搭配起来，就是一种绝妙的对比色彩组合。
基本配色——平静 -----------------------------------------------------------------------------------
在任何充满压力的环境里，只要搭配出一些灰蓝或淡蓝的明色色彩组合，就会制造出令人平和、恬静的效果。 中间是淡蓝的配色设计，会给人安心的感觉，因为它看起来诚实、直接。 带着明色的寒色可保持安宁、平和的感觉。补色和这些强调平静的色彩在明暗度方面一定要类似，这点很重要，因为要是色彩太鲜明，会制造出不必要的紧张。
基本配色——强烈 -----------------------------------------------------------------------------------------
项目管理流程 编制：宋登琼 审核： 批准： 日期：2012年10月
目录 1.目的 (4) 2.适用范围 (4) 3.项目分类 (4) 3.1.技术研发型 (4) 3.2.产品研发型 (4) 3.3.项目研发型 (4) 4.职责 (4) 4.1.立项建议人 (4) 4.2.高层管理者 (5) 4.3.技术总监 (5) 4.4.项目经理 (5) 4.5.项目组成员 (5) 4.6.客户 (5) 4.7.销售与售前 (6) 4.8.CM工程师（配置管理工程师） (6) 4.9.QA工程师（质量管理工程师） (6) 4.10.EPG (6) 4.11.技术支持人员 (6) 5.具体内容 (7) 5.1.项目立项流程 (7) 5.1.1.流程图 (7) 5.1.2.工作流程 (7) 5.2.项目策划流程 (9) 5.2.1.流程图 (9) 5.2.2.工作流程 (9) 5.3.项目监控流程 (11) 5.3.1.流程图 (11) 5.3.2.工作流程 (11) 5.4.项目结项流程 (13) 5.4.1.流程图 (13) 5.4.2.工作流程 (13) 5.4.3.项目的异常暂停或终止 (14) 5.5.过程模板 (15) 6.参考文件 (15) 7.说明 (15)
1.目的 本流程定义了公司研发项目的立项、策划、监控、结项和风险管理的全标准化过程和活动，达到为项目和人员在项目实施和操作过程中提供规范化指导的作用。 2.适用范围 本规定适用于研发中心所有立项实施的项目。 3.项目分类 3.1.技术研发型 指以实现技术为目标，形成产品的时机还不成熟的项目； 3.2.产品研发型 指以形成产品为目标，实现自定义的应用的项目； 3.3.项目研发型 指按照市场的输入定制的常规项目； 4.职责 4.1.立项建议人 ?负责制定产品概念阶段进度计划； ?协调相关人员开展立项调研，参与编制《产品可行性分析报告》； ?负责编写《项目立项建议书》，提出立项建议，申请立项评审。
Pep小学英语三年级上册教学设计 Unit2 Colour 指导思想： 以学生为主体，激发和培养学生学习英语的兴趣，帮助他们建立学习的成就感和自信心，使他们在学习过程中发展综合语言运用能力，培养创新精神。 知识目标： 1. 能听说、认读颜色单词：blue, green, yellow, red, 2. 通过用“Show me...”的指令来练习和运用有关颜色的单词。 能力目标： 辨色彩，感受生活。 情感目标： 1. 通过游戏活动和歌曲培养学生说英语、学英语的兴趣。 2. 爱色彩，热爱自然，保护环境。 3. 通过用哑语说颜色，培养学生们关心他人的优秀品质。 教学重难点： 1. 听、说、认、读五个颜色单词。 2. 会说唱 Let's do，并会听音做出相应的反应。 教具准备： 1. 教师准备 blue, green, yellow, red, 的单词卡片和颜色卡片。 2. 教师和学生都准备红、黄、蓝、绿、紫色的蜡笔。 3. 教师准备颜色的教学课件。 4. 教师准备学生学过的文具并放在书包中。 教学教程： Warm-up: （1）教师生之间相互问候： T: Good morning/ Good afternoon/ Hello, boys and girls. S: Good morning/ Good afternoon/ Hello, teacher./... T: How are you? S: Fine, thank you./I'm fine, thank you./...
（2）游戏 T: Let's play a game, OK? S: OK. “猜一猜”的游戏。将学生学过的文具放入书包中，让学生摸一摸，猜一猜。从而复习学过的文具词。 （说明：“猜一猜”的游戏能调动学生大脑中所有相关记忆，使学生回忆起所有知道的有关文具的单词。） （3）课件展示学过的文具用品：橡皮（红色），钢笔（绿色），铅笔盒（蓝色），书（黄），书包（绿色），蜡笔。 Presentation: 1. Teach “red” (1) 教师从复习中引入新课，问：“What colour is the eraser?”教师自答：“It's red.” (2) 教师手拿红色的橡皮反复说“red”，让学生猜意思，并使他们明白“red”表示红色。 (3）教师领读“red”。 (4) 教师拿起其他红色的物体，指示说：“It's red.” (5) 让学生找出教室中红色的物体练习说：“It's red.”在练习过程中教师应注意纠正发音。 (6）教师板书“red”，让学生认读。 （说明：先练听说，后练认读，先易后难，循序渐进。此方式符合三年级学生的认知规律，难度低于出示单词并领读的传统教法。） 2. 用这种方法逐一介绍 blue，green，yellow，purple 并出示单词卡片让生认读。 注意：“green”一词的发音较难，教教师要多带读，引导学生感悟发音，从而了解英语的语音语调的知识。 3. 猜词游戏： a. 教师从颜色卡片中抽出一张，请每小组派一名学生猜，猜对的小组可加1分。
中国《电脑商报》杂志「中国电脑商500强」之「行业软件十佳方案商」 行业方案提供商top10 政府 1、长城计算机软件与系统有限公司 2、沈阳东软软件股份有限公司 3、神州数码（中国）有限公司 4、中国软件与技术服务股份有限公司 5、长江计算机（集团）公司 6、北京北大千方科技有限公司 7、太极计算机股份有限公司 8、中电科长江数据股份有限公司 9、航天信息股份有限公司 10、江苏苏源高科技有限公司 教育 1、清华同方股份有限公司 2、清华紫光股份有限公司 3、浙江浙大图灵软件技术有限公司 4、江苏天技科技实业有限公司 5、上海复旦光华信息科技股份有限公司 6、太极计算机股份有限公司 7、江西思创数码科技股份有限公司
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小学三年级英语课文《Colour》的教 学评课稿 一、活动的设计有趣味性 1、陈老师能根据小学生的年龄特点，准备了很多五彩缤纷的实物，有黄的、红的、蓝的、紫的、绿的等，都是学生喜闻乐见的玩具，并设计生动活泼的课堂活动，如：通过图片猜颜色、听音涂色等游戏，特别在教授单词时，运用了多种形式，如：“大小声”、“火车接龙”等，避免了没趣、机械重复的操练，既培养了学生的语言运用能力，又培养了学生的注意力、观察力、思维力和想象力。激发学生兴趣！ 2、关注教学方法，体现了一个活字.教师的教学方法灵敏，新单词呈现形式多样.应该说整堂课中，教师在引入新词时，都是比较新奇而又自然，而且具有生活化的.教师还注意利用实物，图片，卡片，身体语言，表情动作等作为教学资源，创设讲解，操练和运用英语的情景.季老师能贯彻以学生为中心的原则，关注教学过程，尽可能发挥学生的主体作用，让学生真实的去感受知识，体验知识，积极参与，努力实践，在活动中学会用语言表达交流，较好的体现了从不懂到懂，从不会到会，从不烂熟到烂熟的过程. 二、活动的设计有层次性 本课中，陈老师在教学中先让学生学说表示颜色的单词，如：red让学生掌握单词的音、型、意。再让学生找一找身边有什么东西是红色的，用简单的句型进行表达，如:Thisis red.My pencil is red.What colour is it? What colour do you like?最后，让学生运用所学语言进行拓展性练习，挖掘学生创造使用语言的能力，从教师的语言输入转为学生的语言输出，符合语言的习得规律。 三、活动设计有实用性 陈老师在培养学生在实际情景中运用英语方面花了很多功夫，积极践行“学中用，用中学”。新授完颜色单词之后，敖老师因势引导，过渡到句型“Ilike…What colour is it?What colour do you