The capsanthin-capsorubin synthase gene: a candidate gene for the y locus controlling the red fruit colour in pepper

The red colour of pepper fruits is determined by the y⁺ dominant allele and the yellow colour by the y recessive allele. The capsanthin-capsorubin synthase (CCS) gene is activated specifically during the final stages of pepper fruit ripening. RFLP and specific-PCR polymorphisms derived from the CCS gene were analysed in a F2 progeny of a red by yellow-fruited cross. They cosegregated completely with fruit colour. Our results support the hypothesis that the yellow phenotype might result from a deletion of the CCS gene. These specific markers were integrated into the genetic map and will be useful for marker assisted plant breeding.     

A candidate gene approach identified phytoene synthase as the locus for mature fruit color in red pepper (Capsicum spp.)

Abstrat  The color of mature pepper fruit is determined by the composition of carotenoids. The fruit color of red pepper is genetically determined by three loci, y, c1, and c2. We have been developing a genetic map of hot pepper using RFLP and AFLP markers in the F₂ population of an interspecific cross between Capsicum annuum cv TF68 and Capsicum chinense cv Habanero. The color of the ripe fruit of TF68 is red and Habanero is orange. The red color is dominant over orange in the F₁ and the locus controlling this character has been marked in our SNU Linkage Group 7. To identify the gene or markers tightly linked to the red/orange locus, several candidate genes involved in the carotenoid biosynthesis pathway, namely FPS, GGPS, PSY, PDS, LCY and CCS, were examined. One of the candidate genes, phytoene synthase, cosegregated completely with fruit color in the F₂ population. QTL analysis of the pigment content of F₂ individuals quantified by HPLC also indicated that phytoene synthase is the locus responsible for the development of fruit color. The color, pigment content and genetic behavior of Habanero also suggest that phytoene synthase may be responsible for the c2 gene discriminating between red and orange cultivars. Received: 15 March 2000 / Accepted: 16 August 2000     

A splicing mutation in the gene encoding phytoene synthase causes orange coloration in Habanero pepper fruits

Peppers (Capsicum spp.) display a variety of fruit colors that are reflected by the composition and amount of diverse carotenoid pigments accumulated in the pericarp. Three independent loci, c1, c2, and y, are known to determine the mature color of pepper fruits by their allelic combinations. We examined the inheritance of fruit color in recombinant inbred lines (RILs) derived from an interspecific cross between C. annuum cv. TF68 (red) and C. chinense cv. Habanero (orange). The c2 gene encodes phytoene synthase (PSY), a rate-limiting enzyme in the carotenoid biosynthesis pathway. TF68 has a dominant c2+ allele whereas Habanero is homozygous for the recessive c2 allele, which determined RIL fruit color. Here we report that the recessive c2 allele has a point mutation in the PSY gene that occurs at a splice acceptor site of the fifth intron leading to both a frame shift and premature translational termination, suggesting that impaired activity of PSY is responsible for orange fruit color. During ripening, PSY is expressed at a significantly high level in orange colored fruits compared to red ones. Interestingly, the PSY gene of red Habanero has a conserved splice acceptor dinucleotide AG. Further analysis suggests that red Habanero is a wild type revertant of the PSY mutant orange Habanero.     

Molecular identification of the yellow fruit color (c) locus in diploid strawberry: a candidate gene approach

A candidate gene approach was used to determine the likely molecular identity of the c locus (yellow fruit color) in Fragaria vesca, a diploid (2n=2x=14) strawberry. Using PCR with degenerate primer pairs, intron-containing segments of structural genes coding for chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS) and one Del-like regulatory gene in the anthocyanin biosynthetic pathway, were amplified, cloned and sequenced. Intron length polymorphisms for each of these genes were detected among three diploid varieties: F. vesca Alpine variety ’Yellow Wonder’ (YW) (Europe); DN1C, a F. vesca clone collected from Northern California; and Fragaria nubicola FRA520, a U.S.D.A. accession collected in Pakistan. Using F₂ generations of the crosses DN1C×YW and YW×FRA520 as mapping populations, the six candidate genes were mapped in relation to previously mapped randomly amplified polymorphic DNA (RAPD) markers and morphological markers. The F3H gene was linked without recombination to the c locus in linkage group I, while the other five candidate genes mapped to different linkage groups. These results suggest that the wild-type allele (C) of the c (yellow fruit color) locus encodes an F3H necessary for red fruit color in F. vesca. Received: 28 August 2000 / Accepted: 21 December 2000     

Orange, yellow and white-cream: inheritance of carotenoid-based colour in sunflower pollen

Inheritance of pollen colour was studied in sunflower (Helianthus annuus L.) using three distinct pollen colour morphs: orange, yellow and white‐cream. Orange is the most common colour of sunflower pollen, while the yellow morph is less frequent. These two types were observed in the inbred lines F11 and EF2L, respectively. White‐cream pollen is a rare phenotype in nature, and was identified in a mutant, named white‐cream pollen, recovered in the R₂ generation of an in vitro regenerated plant. The F11 inbred line was used as starting material for in vitro regeneration. The carotenoid content of these three pollen morphs differed, and was extremely reduced in white‐cream pollen. The phenotype of F₁ populations obtained by reciprocal crosses revealed that the orange trait was dominant over both white‐cream and yellow. Segregation of F₂ populations of both crosses, orange × yellow and orange × white‐cream, approached a 3:1 ratio, indicating the possibility of simple genetic control. By contrast, a complementation cross between the two lines with white‐cream and yellow pollen produced F₁ plants with orange pollen. The F₂ populations of this cross‐segregated as nine orange: four white‐cream: four yellow. A model conforming to the involvement of two unlinked genes, here designated Y and O, can explain these results. Accessions with yellow pollen would have the genotype YYoo, the white‐cream pollen mutant would have yyOO and the accession with orange pollen would have YYOO. Within F₂ populations of the cross white‐cream × yellow a new genotype, yyoo, with white‐cream pollen was scored. The results of the cross yyoo × YYoo produced only F₁ plants with yellow pollen, supporting a recessive epistatic model of inheritance between two loci. In this model, yy is epistatic on O and o. In F₂ populations, the distributions of phenotypic classes suggested that the genetic control of carotenoid content is governed by major genes, with large effects segregating in a background of polygenic variation. These three pollen morphs can provide insight into the sequence in which genes act, as well into the biochemical pathway controlling carotenoid biosynthesis in anthers and the transfer of these different pigments into pollenkitt.     

Two loss-of-function alleles of the glutathione S-transferase (GST) gene cause anthocyanin deficiency in flower and fruit skin of peach (Prunus persica)

Flower and fruit colors are important agronomic traits. To date, there is no forward genetic evidence that the glutathione S-transferase (GST) gene is responsible for the white flower color in peach (Prunus persica). In this study, genetic analysis indicated that the white-flower trait is monogenetic, is recessive to the non-white allele, and shows pleiotropic effects with non-white-flowered types. The genetic locus underpinning this trait was mapped onto chromosome 3 between 0.421951 and 3.227115 Mb by using bulked segregant analysis in conjunction with whole-genome sequencing, and was further mapped between 0 and 1.178149 Mb by using the backcross 1 (BC₁) population. Finally, the locus was fine-mapped within 535.974- and 552.027-kb intervals by using 151 F₂ individuals and 75 individuals from a BC₁ self-pollinated (BC₁S₁) population, respectively. Pp3G013600, encoding a GST that is known to transport anthocyanin, was identified within the mapping interval. The analysis of genome sequence data showed Pp3G013600 in white flowers has a 2-bp insertion or a 5-bp deletion in the third exon. These variants likely render the GST non-functional because of early stop codons that reduce the protein length from 215 amino acids to 167 and 175 amino acids, respectively. Genetic markers based on these variants validated a complete correlation between the GST loss-of-function alleles and white flower in 128 peach accessions. This correlation was further confirmed by silencing of Pp3G013600 using virus-induced gene silencing technology, which reduced anthocyanin accumulation in peach fruit. The new knowledge from this study is useful for designing peach breeding programs to generate cultivars with white flower and fruit skin.     

不同果色辣椒CCS基因克隆及表达分析

果实颜色是辣椒重要的商品性状之一。本研究以观赏椒GS6、Z1，甜椒SP01及黄色突变体SP02为材料，探究辣椒红素/辣椒玉红素合酶(CCS)基因在不同成熟果色辣椒中的序列差异和表达特性，初步解析辣椒不同成熟果色形成分子机理。研究结果显示：成熟色为红色的GS6、Z1和SP01中均能克隆到CCS全长基因，且序列无差异，其全长1497bp，编码498个氨基酸，只包含一个开放阅读框序列，没有内含子序列；而黄色突变体SP02中未能克隆出CCS基因；聚类和系统进化分析发现辣椒CCS基因与茄科作物的番茄、中华辣椒和灯笼辣椒等植物的亲缘关系较近；qRT-PCR分析结果显示：在GS6中，CCS基因在花中的表达量最高；在Z1和SP01中，CCS基因在果实中的表达量显著高于其他组织，在根中表达量最低；而在SP01的茎和叶以及SP02的所有组织中，CCS基因均未表达。在果实不同发育时期，CCS基因在SP01花后30 d（Ⅲ期）、GS6和Z1花后40d（Ⅳ期）表达量显著上升。研究结果表明，甜椒黄色突变体SP02果实颜色的形成可能和CCS基因的缺失或变异密切相关，而在成熟色为红色的辣椒中CCS基因的表达可能在果实颜色形成过程中发挥着重要的作用。     

CHROMOPLAST ULTRASTRUCTURE AS AFFECTED BY GENES CONTROLLING GRANA RETENTION AND CAROTENOIDS IN FRUITS OF CAPSICUM ANNUUM

Plastids in the fruits of isogenic lines of pepper (Capsicum annuum) were examined by electron microscopy with reference to four genotypes determining the carotenoid composition and the colors red, yellow, brown, and green of the ripe fruit. One gene pair (y⁺/y) influences carotenoid content and the other pair (cl⁺/cl) controls the chlorophyll. The retention of the grana and chlorophyll in the ripe fruits of the brown and green phenotypes is correlated with the cl cl genotype. The y⁺ gene increases the total carotenoids and promotes the formation of red pigments. Giant grana were found in the yellow and green phenotypes, but during ripening these disappeared in the yellow. Unusual dichotomous and concentric grana were observed in the green. Globule-associated carotenoids forming fibrillar crystalloids were present in all color types, although to a lesser degree in the yellow fruit. Membrane-associated carotenoids occurred only in the yellow and green phenotypes.     

QTL analysis and candidate gene mapping for skin and flesh color in sweet cherry fruit (<Emphasis Type="Italic">Prunus avium</Emphasis> L.)

Sweet cherry (Prunus avium L.) skin and fruit colors vary widely due to differences in red and yellow pigment profiles. The two major market classes of sweet cherry represent the two color extremes, i.e., yellow skin with red blush and yellow flesh and dark mahogany skin with mahogany flesh. Yet, within these extremes, there is a continuum of skin and flesh color types. The genetic control of skin and flesh color in sweet cherry was investigated using a quantitative trait locus (QTL) approach with progeny derived from a cross between cherry parents representing the two color extremes. Skin and flesh colors were measured using a qualitative color-card rating over three consecutive years and also evaluated quantitatively for darkness/lightness (L*), red/green (a*), and yellow/blue (b*). Segregations for the color measurements (card, L*, a*, and b*) did not fit normal distributions; instead, the distributions were skewed towards the color of the dark-fruited parent. A major QTL for skin and flesh color was identified on linkage group (LG) 3. Two QTLs for skin and flesh color were also identified on LG 6 and LG 8, respectively, indicating segregation for minor genes. The significance and magnitude of the QTL identified on LG 3 suggests the presence of a major regulatory gene within this QTL interval. A candidate gene PavMYB10, homologous to apple MdMYB10 and Arabidopsis AtPAP1, is within the interval of the major QTL on LG 3, suggesting that PavMYB10 could be the major determinant of fruit skin and flesh coloration in sweet cherry.     

Attraction of fruit flies of the genus Bactrocera to colored mimics of host fruit

In tests on feral populations of polyphagous Bactrocera tryoni (Froggatt) adults on host guava trees, both sexes were significantly more attracted to Tangletrap‐coated 50 mm diameter spheres colored blue or white than to similar spheres colored red, orange, yellow, green, or black or to Tangletrap‐coated 50 mm diameter yellow‐green guava fruit. In contrast, in tests on feral populations of oligophagous Bactrocera cacuminata (Hering) on host wild tobacco plants, both sexes were significantly more attracted to Tangletrap‐coated 15 mm diameter spheres colored orange or yellow than to other colors of spheres or to Tangletrap‐coated 15 mm diameter green wild tobacco fruit. Both sexes of both tephritid species were significantly more attracted to blue (in the case of B. tryoni) or orange (in the case of B. cacuminata) 50 mm spheres displayed singly than to blue or orange 15 mm spheres displayed in clusters, even though fruit of wild tobacco plants are borne in clusters. Finally, B. tryoni adults were significantly less attracted to non‐ultraviolet reflecting bluish fruit‐mimicking spheres than to bluish fruit‐mimicking spheres having a slightly enhanced level of ultraviolet reflectance, similar to the reflectance of possible native host fruit of B. tryoni, whose bluish skin color is overlayed with ultraviolet‐reflecting waxy bloom. Responses to fruit visual stimuli found here are discussed relative to responses found in other tephritid species.     

Linkage among the R, Y and BI loci in table beet

The primary pigments in table beet are the betalains, which are comprised of the red-violet betacyanins and the yellow betaxanthins. The presence of dominant alleles at two linked loci (R and Y) condition the qualitative production of betalain pigment in the beet plant. Red-pigmented roots are observed only in the presence of dominant alleles at both the R and Y loci, while white roots are conditioned by recessive alleles at the Y locus, and yellow roots by the genotype rrY-. A newly described gene ’blotchy’ (bl) conditions a blotchy or irregular pigment patterning in either red or yellow roots. The objective of the present investigation was to characterize the linkage relationships between the R and Y lociand the bl gene by evaluating segregating progenies developed from a series of matings of colored and white table beets. Due to epistatic interactions among the R, Y, and bl loci, algorithms for estimating linkage were developed using maximum-likelihood estimators for each cross. The two-point linkage estimate between the R and Y loci pooled over eight crosses was 7.4±1.7 cM. Segregation data indicated the bl gene is linked to the R and Y loci.The recombination fraction between R and bl was estimated from a pooled sample of four crosses at 16.7±10.8 cM. The most-likely gene order was R-Y-bl. These data demonstrate that the bl gene is a third locus conditioning betalain pigment production in table beet. The R-Y-bl genomic region is therefore important in the genetic control of betalain biosynthesis in table beet. Received: 18 May 1999 / Accepted: 29 August 1999     

The Mendelian inheritance of rare flesh and shell colour variants in the black‐lipped pearl oyster (Pinctada margaritifera)

Pinctada margaritifera is French Polynesia's most economically important aquaculture species. This pearl oyster has the specific ability to produce cultured pearls with a very wide range of colours, depending on the colour phenotypes of donor oysters used. Its aquaculture is still based on natural spat collection from wild stocks. We investigated three rare colour variants of P. margaritifera – orange flesh, and red and white shell colour phenotypes – in comparison with the wild‐type black flesh and shell commonly found in this species. The study aimed to assess the geographic distribution and genetic basis of these colour variants. Colour frequencies were evaluated during transfer and graft processes of pearl oyster seed captured at collector stations. Among the collection locations studied, Mangareva Island showed the highest rate of the orange flesh phenotype, whereas Takaroa and Takume atolls had relatively high rates of red and white shell phenotypes respectively. Broodstocks were made of these rare colour variants, and crosses were performed to produce first‐ and second‐generation progenies to investigate segregation. The results were consistent with Mendelian ratios and suggest a distinct model with no co‐dominance: (i) a two‐allele model for flesh trait, whereby the orange allele is recessive to the black fleshed type, and (ii) a three‐allele model for shell trait, whereby the black wild‐type allele is dominant to the red coloration, which is dominant to the white shell. Furthermore, the proposed model provides the basis for producing selected donor pearl oyster lines through hatchery propagation.     

Inheritance of coat colour in the Anatolian Shepherd dog

The predominant colour of the Anatolian Shepherd dog varies from a dark fawn to light red, with a variable black muzzle and face (mask). Evidence is presented that the colour is due to the dominant yellow allele (A ^y) of the agouti locus. Two other frequent colours are white spotting, due to the piebald allele (s ^p), and the chinchilla allele (ch). Two rarer colours are the agouti wolf-grey wild type (A ⁺) and a light fawn with a blue facial mask, due to the dilution allele (d).     

A mutation in Zeaxanthin epoxidase contributes to orange coloration and alters carotenoid contents in pepper fruit (Capsicum annuum)

Phytoene synthase (PSY1), capsanthin-capsorubin synthase (CCS), and pseudo-response regulator 2 (PRR2) are three major genes controlling fruit color in pepper (Capsicum spp.). However, the diversity of fruit color in pepper cannot be completely explained by these three genes. Here, we used an F₂ population derived from Capsicum annuum ‘SNU-mini Orange’ (SO) and C. annuum ‘SNU-mini Yellow’ (SY), both harboring functional PSY1 and mutated CCS, and observed that yellow color was dominant over orange color. We performed genotyping-by-sequencing and mapped the genetic locus to a 6.8-Mb region on chromosome 2, which we named CaOr. We discovered a splicing mutation in the zeaxanthin epoxidase (ZEP) gene within this region leading to a premature stop codon. HPLC analysis showed that SO contained higher amounts of zeaxanthin and total carotenoids in mature fruits than SY. A color complementation assay using Escherichia coli harboring carotenoid biosynthetic genes showed that the mutant ZEP protein had reduced enzymatic activity. Transmission electron microscopy of plastids revealed that the ZEP mutation affected plastid development with more rod-shaped inner membrane structures in chromoplasts of mature SO fruits. To validate the role of ZEP in fruit color formation, we performed virus-induced gene silencing of ZEP in the yellow-fruit cultivar C. annuum ‘Micropep Yellow’ (MY). The silencing of ZEP caused significant changes in the ratios of zeaxanthin to its downstream products and increased total carotenoid contents. Thus, we conclude that the ZEP genotype can determine orange or yellow mature fruit color in pepper.     

Recessive black is allelic to the yellow plumage locus in Japanese quail and associated with a frameshift deletion in the ASIP gene

The recessive black plumage mutation in the Japanese quail (Coturnix japonica) is controlled by an autosomal recessive gene (rb) and displays a blackish-brown phenotype in the recessive homozygous state (rb/rb). A similar black coat color phenotype in nonagouti mice is caused by an autosomal recessive mutation at the agouti locus. An allelism test showed that wild type and mutations for yellow, fawn-2, and recessive black in Japanese quail were multiple alleles (*N, *Y, *F2, and *RB) at the same locus Y and that the dominance relationship was Y*F2 > Y*Y > Y*N > Y*RB. A deletion of 8 bases was found in the ASIP gene in the Y*RB allele, causing a frameshift that changed the last six amino acids, including a cysteine residue, and removed the normal stop codon. Since the cysteine residues at the C terminus are important for disulphide bond formation and tertiary structure of the agouti signaling protein, the deletion is expected to cause a dysfunction of ASIP as an antagonist of alpha-MSH in the Y*RB allele. This is the first evidence that the ASIP gene, known to be involved in coat color variation in mammals, is functional and has a similar effect on plumage color in birds.