Molecular genetics of the y locus in pepper: its relation to capsanthin-capsorubin synthase and to fruit color

Classical genetic studies have determined that the yellow fruit color in pepper is recessive to red in the locus y. We studied the relation of the y locus with the gene coding for capsanthin-capsorubin synthase (CCS) that synthesizes the red carotenoid pigments in the mature fruit. Cosegregation of y and CCS in populations derived from crosses between plants bearing red×white and red×yellow fruits indicated the correspondence of the two genes. We obtained indications for the occurrence of a deletion in the CCS gene in plants containing the recessive y allele. This deletion did not contain the distal 220 bp of the 3′ end of the gene. We used the CCS gene to determine the genotype of peppers with different fruit colors at the y locus. In BC₁ segregants from a red×white cross, the red and peach-fruited progenies had the wild-type allele at the CCS locus, while the orange, yellow and white-fruited progenies had the mutant allele. Screening orange-fruited cultivars with CCS as well as segregation analysis of CCS in an additional red×white cross indicated two possible genotypes of the orange fruit color in this locus. Received: 25 January 1999 / Accepted: 16 August 1999     

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。     

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     

Induced point mutations in the phytoene synthase 1 gene cause differences in carotenoid content during tomato fruit ripening

In tomato, carotenoids are important with regard to major breeding traits such as fruit colour and human health. The enzyme phytoene synthase (PSY1) directs metabolic flux towards carotenoid synthesis. Through TILLING (Targeting Induced Local Lesions IN Genomes), we have identified two point mutations in the Psy1 gene. The first mutation is a knockout allele (W180*) and the second mutation leads to an amino acid substitution (P192L). Plants carrying the Psy1 knockout allele show fruit with a yellow flesh colour similar to the r, r mutant, with no further change in colour during ripening. In the line with P192L substitution, fruit remain yellow until 3 days post-breaker and eventually turn red. Metabolite profiling verified the absence of carotenoids in the W180* line and thereby confirms that PSY1 is the only enzyme introducing substrate into the carotenoid pathway in ripening fruit. More subtle effects on carotenoid accumulation were observed in the P192L line with a delay in lycopene and β-carotene accumulation clearly linked to a very slow synthesis of phytoene. The observation of lutein degradation with ripening in both lines showed that lutein and its precursors are still synthesised in ripening fruit. Gene expression analysis of key genes involved in carotenoid biosynthesis revealed that expression levels of genes in the pathway are not feedback-regulated by low levels or absence of carotenoid compounds. Furthermore, protein secondary structure modelling indicated that the P192L mutation affects PSY1 activity through misfolding, leading to the low phytoene accumulation.     

Induced mutation in β-CAROTENE HYDROXYLASE results in accumulation of β-carotene and conversion of red to orange color in pepper fruit

Pepper fruit is typically red, but green, orange and yellow cultivars are gaining consumer acceptance. This color variation is mainly due to variations in carotenoid composition. Orange color in pepper can result from a number of carotenoid profiles, but its genetic basis is only partly known. We identified an EMS-induced orange-fruited mutant using the wild-type blocky red-fruited cultivar ‘Maor’ as progenitor. This mutant accumulates mainly β-carotene in its fruit, instead of the complex pattern of red and yellow carotenoids in ‘Maor’. We identified an A⁷⁰⁹ to G transition in the cDNA of β-CAROTENE HYDROXYLASE2 in the orange pepper and complete co-segregation of this single-nucleotide polymorphism with the mutated phenotype. We therefore hypothesized that β-CAROTENE HYDROXYLASE2 controls the orange mutation in pepper. Interestingly, the expression of β-CAROTENE HYDROXYLASE2 and additional carotenogenesis genes was elevated in the orange fruit compared with the red fruit, indicating possible feedback regulation of genes in the pathway. Because carotenoids serve as precursors for volatile compounds, we compared the volatile profiles of the two parents. The orange pepper contained more volatile compounds than ‘Maor’, with predominant elevation of norisoprenoids derived from β-carotene degradation, while sesquiterpenes predominated in the red fruit. Because of the importance of β-carotene as a provitamin A precursor in the human diet, the orange-fruited mutant might serve as a natural source for pepper fruit biofortification. Moreover, the change in volatile profile may result in a fruit flavor that differs from other pepper cultivars.     

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.     

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.     

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).     

Chlorophyll breakdown during pepper fruit ripening in the <Emphasis Type="Italic">chlorophyll retainer</Emphasis> mutation is impaired at the homolog of the senescence-inducible stay-green gene

The pepper chlorophyll retainer (cl) mutation is characterized by inhibition of chlorophyll degradation during fruit ripening. Ripe fruit of cl pepper containing chlorophyll and red carotenoids is brown, while ripe fruit containing chlorophyll and yellow carotenoids is green. In addition to the inhibitory effect during fruit ripening caused by cl, we show that chlorophyll degradation is inhibited during natural and dark-induced leaf senescence. Therefore, the cl mutation has the characteristics of the stay-green (sgr) mutants described in many other species. Upon the recent discovery of the SGR gene in various plant species, we isolated pepper SGR (CaSGR) and found that it genetically cosegregates with cl in a BC1 mapping population. Furthermore, sequencing the wild-type and mutant alleles revealed an amino-acid substitution of tryptophan (aromatic amino acid) to arginine (basic amino acid) at position 114 in the protein sequence. The single-nucleotide polymorphism (SNP) that differentiates the wild-type and mutant alleles was exploited to develop a PCR marker useful for marker-assisted selection. Expression of CaSGR as measured by semiquantitative RT-PCR was mostly induced upon fruit ripening and to a lesser extent upon leaf senescence. Taking together, our genetic, sequence and expression data all indicate that CaSGR is a candidate for controlling the cl mutation in pepper.     

Inheritance of seed colour and identification of RAPD and AFLP markers linked to the seed colour gene in rapeseed (Brassica napus L.)

In China Polima cytoplasmic male sterility (cms) is currently the most important hybrid system used for the breeding of hybrids. In an effort to develop yellow-seeded Polima cms restorer lines, we used yellow-seeded, doubled haploid (DH) line No.2127-17 as the gene source in crosses with two elite black-seeded Polima cms R lines, Hui5148-2 and 99Yu42, which originated from our breeding programme. The inheritance of seed colour was investigated in the F₂, BC₁ and F₁-derived DH progenies of the two crosses. Seed colour was found to be under the control of the maternal genotype and the yellow seed trait to be partially dominant over the black seed trait. Segregation analysis revealed a single gene locus for the partial dominance of yellow seed colour. Of 810 randomly amplified polymorphic DNA (RAPD) primers, 240 (29.6%) revealed polymorphisms between the parents. Of the 240 RAPD primers and 512 amplified fragment length polymorphism (AFLP) primer pairs, four RAPDs and 16 AFLP pairs showed polymorphisms between the bulks, with two RAPD and eight AFLP markers being identified in the vicinity of the seed-coat colour gene locus using a DH progeny population—derived from the cross Hui5148-2×No.2127-17—of 127 individuals in combination with the bulked segregant analysis strategy. Seven of these latter ten markers were linked to the allele for yellow seed, whereas the other three were linked to the allele for black seed. The seed-coat colour gene locus was bracketed by two tightly linked markers, EA02MG08 (2.4 cM) and S1129 (3.9 cM). The partial dominance and single gene control of the yellow seed-coat colour trait together with the available molecular markers will greatly facilitate the future breeding of yellow-seeded hybrid varieties.     

Towards the saturation of the pepper linkage map by alignment of three intraspecific maps including known-function genes.

Three populations composed of a total of 215 doubled haploid lines and 151 F2 individuals were used to design an intraspecific consensus map of pepper (Capsicum annuum L.). The individual maps varied from 685 to 1668 cM with 16 to 20 linkage groups (LGs). The alignment of the three individual maps permitted the arrangement of 12 consensus major linkage groups corresponding to the basic chromosome number of pepper and displaying a complex correspondence with the tomato map. The consensus map contained 100 known-function gene markers and 5 loci of agronomic interest (the disease-resistance loci L, pvr2, and Pvr4; the C locus, which determines capsaicin content; and the up locus, controlling the erect habit of the fruits). The locations of three other disease-resistance loci (Tsw, Me3, and Bs3) and the y locus, which determines the yellow fruit colour, were also found on this consensus map thanks to linked markers. Here we report on the first functional detailed map in pepper. The use of candidate gene sequences as genetic markers allowed us to localize four clusters of disease-resistance gene analogues and to establish syntenic relationships with other species.     

The effect of chimeric transgene architecture on co-ordinated gene silencing

Two gene constructs were made consisting of a 244-bp sense fragment from the 5′ end of a polygalacturonase cDNA, the 3′ end of which was ligated to a 414-bp fragment from the 5′ end of a phytoene synthase cDNA. In the first construct, the phytoene synthase fragment was in a sense orientation (sense/sense chimeric gene) and in the second construct the phytoene synthase fragment was in an antisense orientation (sense/antisense chimeric gene). Both chimeric genes were inserted between a cauliflower mosaic virus promoter and terminator. Tomato (Lycopersicon esculentum Mill. cv. Ailsa Craig) plants transformed with each construct gave rise to transformants with three distinct phenotypes: plants with red fruit, plants with pure yellow fruit and plants with red and yellow sectored fruit. For both chimeric constructs, expression of the endogenous polygalacturonase and phytoene synthase genes were found to be co-ordinately suppressed in yellow tissue, but showed normal expression in red tissue. Data from microscopic analyses of fruit chromoplasts, from the three phenotypes, implied that phytoene synthase suppression from each construct predominantly had two states within a cell: on or off. Received: 31 July 1997 / Accepted: 28 August 1997     

Presence of the Dominant Extension Allele ED in Red and Mosaic Cattle

The melanocyte-stimulating hormone receptor (MC1-R) is a central regulator of mammalian coat colour, encoded by the extension locus. In cattle, the dominant extension allele E^D is associated with the production of black pigment in coloured areas. Genotyping of the MC1-R gene in a bull with mosaic expression of red vs. black pigment verified the existence of the E^D allele, in spite of the fact that the majority of the animal is red coloured. No further mutations were found within the E^D variant of the MC1-R gene, which was inherited from a completely red mother (genotype E^D/e).     

Mutations in the melanocortin 1 receptor (MC1R) gene are associated with coat colours in the domestic rabbit (Oryctolagus cuniculus)

We sequenced almost the complete coding region of the MC1R gene in several domestic rabbits (Oryctolagus cuniculus) and identified four alleles: two wild-type alleles differing by two synonymous single nucleotide polymorphisms (c.333A>G;c.555T>C), one allele with a 30-nucleotide in-frame deletion (c.304_333del30) and one allele with a 6-nucleotide in-frame deletion (c.280_285del6). A polymerase chain reaction-based protocol was used to distinguish the wild-type alleles from the other two alleles in 263 rabbits belonging to 37 breeds or strains. All red/fawn/yellow rabbits were homozygous for the c.304_333del30 allele. This allele represents the recessive e allele at the extension locus identified through pioneering genetic studies in this species. All Californian, Checkered, Giant White and New Zealand White rabbits were homozygous for allele c.280_285del6, which was also observed in the heterozygous condition in a few other breeds. Black coat colour is part of the standard colour in Californian and Checkered breeds, in contrast to the two albino breeds, Giant White and New Zealand White. Following the nomenclature established for the rabbit extension locus, the c.280_285del6 allele, which is dominant over c.304_333del30, may be allele E(D) or allele E(S).     

Pigment Bodies in Fruits of Crimson and High Pigment Lines of Tomatoes

Pigment bodies in fruits of crimson (og^c), high pigment (hp),and crimson-high pigment (og^c hp) lines of tomatoes were observedby electron and light microscopy and compared with those ofnormal red lines and a yellow cultivar. During chloroplast-chromoplasttransformation, two main structurally distinct bodies are produced,their total and relative amounts apparently accounting for theentire range of colours (from very deep red to yellow) characterizingthe mature fruits of these different colour lines. The longnarrow crystalloids, believed to be lycopene, form in associationwith an extended thylakoid system; in senescing (over-ripe)fruit many of these are reduced to shorter irregular forms.The rounded globules are believed to be beta-carotene dissolvedin lipid material derived from membrane lysis. Analytical resultscorroborate microscopic observations that the effect of theog^c gene, as compared with the r⁺ gene for normal red colour,is to increase the lycopene content and lower the beta-carotenecontent. The effect of the hp gene is to increase the levelsof both pigments. The results support the view that the genescontrol the development of fruit pigments which affect chromoplastultrastructure. Lycopersicon esculentum Mill, tomato, fruit, pigment bodies, beta-carotene, lycopene     

Inheritance of coat colour in the German Pinscher dog

The two primary colour varieties of the German Pinscher breed are the red and the black and tan. Respectively, these are produced by the dominant yellow allele A ^y and the black and tan allele a ^t of the agouti locus. The dilute gene d is present in the breed at a low frequency and produces the rarer isabella and blue and tan colours, with the genotypes A ^y -dd and a ^t dd, respectively.