a comparison of the carotenoid accumulation in capsicum varieties that show different ripening...

Journal of Experimental Botany, Vol. 58, No. 12, pp. 3135–3144, 2007

doi:10.1093/jxb/erm132 Advance Access publication 28 August, 2007

RESEARCH PAPER

A comparison of the carotenoid accumulation in Capsicumvarieties that show different ripening colours: deletionof the capsanthin-capsorubin synthase gene is nota prerequisite for the formation of a yellow pepper

Sun-Hwa Ha1,*, Jung-Bong Kim1, Jong-Sug Park1, Shin-Woo Lee2 and Kang-Jin Cho1

1 National Institute of Agricultural Biotechnology, RDA, Suwon 441-707, Republic of Korea2 Department of Crops Biotechnology, Jinju National University, Jinju 660-758, Republic of Korea

Received 16 April 2007; Revised 21 May 2007; Accepted 23 May 2007

Abstract

Ripe pepper (Capsicum sp.) fruits can display

a range of colours from white to deep red. To un-

derstand better the regulatory mechanisms of the

carotenoid biosynthetic pathways that underlie these

ripening colours, Capsicum varieties that show seven

different fully ripe colour types were analysed. The

levels and composition of the carotenoid accumulation

in these samples at different stages of ripening were

measured, and the resulting data were analysed in

conjunction with the expression patterns of the carot-

enoid biosynthetic genes. It was found that red

peppers accumulate increasing levels of total carote-

noids during ripening, whereas non-red peppers accu-

mulate lower levels of total carotenoids of varying

composition. The expression levels of the phytoene

synthase, phytoene desaturase, and capsanthin-

capsorubin synthase (Ccs) genes are high in peppers

with high levels of total carotenoid, whereas one or

two of these genes are not expressed in peppers with

lower levels of total carotenoid. Surprisingly, it was

found that the Ccs gene is present in two Capsicum

varieties whose ripe colour is yellow. This gene has

never previously been shown to be present in yellow

peppers. Sequence analyses of the Ccs gene further

revealed two structural mutations in yellow peppers

that may result in either a premature stop-codon or

a frame-shift. Taken together with the fact that the Ccs

transcript is not detectable in yellow peppers, our

current results suggest that nonsense-mediated tran-

scriptional gene silencing of Ccs and not the deletion

of this gene is responsible for yellow ripening in

Capsicum.

Key words: Capsanthin-capsorubin synthase, capsanthin,

carotenoid, pepper (Capsicum sp.), promoter.

Introduction

Among the different species of crops, the pepper(Capsicum sp.) harbours the most evolved carotenoidbiosynthetic pathway (Fig. 1). Although capsanthin andcapsorubin are the key components that cause the typicalred colour during the ripening process in the pepper, thereare many other types of carotenoids that can accumulatein pepper fruits (Deli et al., 2001; Maoka et al., 2001). Inaddition to fruit colour changes during ripening, which aredue to alternations in carotenoid composition, diverse ripecolours from white to deep red can also be found acrossthe different pepper varieties. Pepper fruits are thereforea good model system to study the regulatory mechanismsunderlying carotenoid biosynthesis.In plants, carotenoids play indispensable roles in light

harvesting, photo-protection, and as precursors for abscisicacid (ABA) synthesis. Plants attract insects and otheranimals to act as pollinators and vehicles of seed dis-persion by using stimuli generated by carotenoid pig-ments, including red, orange, and yellow, which arepresent in fruits and flowers (Bartley and Scolnik, 1995).Carotenoids have also been used for many years as naturalcolorants in both animal and human foodstuffs, and someof the b-carotenoids are essential components of the

* To whom correspondence should be addressed. E-mail: [email protected]

ª The Author [2007]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.For Permissions, please e-mail: [email protected]

human diet as pro-vitamin A. Moreover, some carotenoidcomponents have been shown to have important biologi-cal functions as antioxidants and free-radical scavengers,which can reduce the risk of cancer (Hornero-Mendezet al., 2000; Hirschberg, 2001; Maoka et al., 2001).Many of the genes that are involved in carotenoid

biosynthesis have now been cloned from several plantsincluding Arabidopsis, tomato, pepper, daffodil, andmarigold (DellaPenna and Pogson, 2006). These caroteno-genic genes are developmentally regulated and alsodirectly connected with the colour phenotype of theseplants, as shown in tomato fruits and merigold petals(Ronen et al., 1999, 2000; Moehs et al., 2001). Thesestudies have demonstrated that carotenoid accumulation ismainly controlled by the transcriptional regulation ofcarotenoid biosynthetic genes.Pepper fruit is one of the oldest and most widely used

natural food additives. The typical red colour found inCapsicum is derived from the capsanthin and capsorubincomponents that are exclusively synthesized and accumu-lated during fruit ripening in this genus (Mınguez-Mosquera and Hornero-Mendez, 1994). Hence, red cultivarsof Capsicum have been the focus of a number of studiesover many years on the composition of carotenoidpigments (Cholnoky et al., 1955; Deli et al., 2001).The quantitative and qualitative changes in the caroten-

oid profile in red paprika have previously been comparedwith both yellow and black paprika varieties (Matus et al.,1991; Deli et al., 1992). In addition, early efforts to

identify the factors that determine the colour of ripeninghave also been made in Capsicum using genetic ap-proaches (Kormos and Kormos, 1960; Hurtado-Hernandezand Smith, 1985). Since diverse colour phenotypes in theF2 segregation from a cross between white and red pepperfruits were observed, three independent loci, y, c1, andc2, were proposed as putative genes that determine fruitpigmentation. Among these candidates in the pepper,a single dominant gene corresponding to the y locus wassubsequently determined to be the capsanthin-capsorubinsynthase (Ccs) gene, the deletion of which results ina yellow ripening phenotype (Lefebvre et al., 1998). Morerecently, the orange fruit colour in Capsicum was alsofound to result from the absence of the Ccs gene(Popovsky and Paran, 2000; Lang et al., 2004). Popovskyand Paran further demonstrated that this orange colourmight originate from two possible genotypes that are eitherdominant or recessive for Ccs (Popovsky and Paran, 2000).To delineate the relationship between carotenoid accu-

mulation and the expression of carotenoid biosyntheticgenes in the pepper further, Capsicum varieties wereselected that show different ripening fruit colours to analysethese properties. These results show that the number ofgenes that are active during ripening appears to correlatewith the total carotenoid levels. It is also reported here forthe first time that the Ccs gene is present in two Capsicumvarieties whose ripening colour is yellow, which differsfrom previous reports about other yellow peppers where theCcs gene was found to be deleted.

Fig. 1. Carotenoid biosynthetic pathway in the red Capsicum. AGGPS, geranylgeranyl pyrophosphate synthase; PSY, phytoene synthase; PDS,phytoene desaturase; ZDS, f-carotene desaturase; b-LCY, lycopene-b-cyclase; e-LCY, lycopene-e-cyclase; b-CH, b-carotene hydroxylase; e-CH,e-carotene hydroxylase; ZE, zeaxanthin epoxydase; VDE, violaxanthin de-epoxidase; NXS, neoxanthin synthase; CCS, capsanthin-capsorubin synthase.

3136 Ha et al.

Materials and methods

Plant materials

Capsicum varieties were selected from the Genebank of theNational Institute of Agricultural Biotechnology (Rural Develop-ment Administration, Suwon, Korea) on the basis of their differentcolour types in the fully ripe fruit. Their introduction numbers (IT)are as follows: 158770 (C. baccatum var. pendulum, LR2), 158773(C. chinense, R2), 158782 (C. annuum, W), 158806 (C. baccatum,LR1), 163499 (C. annuum, DR), 164918 (C. chinense, Y3), 191655(C. chinense, PYO), 203499 (C. chinense, O), 709424(C. pubescens, R1), 800065 (C. chinense, Y2), and 800070(C. baccatum, Y1) (see Supplementary Fig. 1 at JXB online forphenotypic features and general Capsicum information). A com-mercial cultivar (C. annuum cv. Nockwang, R3) was also includedin these analyses, which was purchased from HungNong SeedsCompany (Seoul, Korea). All 12 pepper varieties chosen for thisstudy were grown in the field and the fruit samples were harvestedat fully expanded stages where differences in their respectivecolours during ripening were evident. At this time, the fruit colourswere also visually recorded.

Extraction of carotenoid pigments and quantification

by HPLC

Pepper fruits that had been harvested at different ripening stages foreach pepper variety were homogenized under liquid N2, weighedafter freeze-drying, and subjected to carotenoid extraction asdescribed previously (Mınguez-Mosquera and Hornero-Mendez,1993). After saponification, the organic layer of the extracts wasvacuum-dried and then analysed by HPLC. The HPLC systemconsisted of a HP 1050 auto-sampler, a gradient pump, a columnthermo-regulator (Mistral, Spark, The Netherlands), and a C-30

carotenoid column (5 lm, 2534.6 mm, YMC Europe, Schermbeck,Germany) equipped with a pre-column (Nucleosil 5 lm-C18,1034.6 mm, Bischoff, Leonberg, Germany). The data wereacquired using HP3D-chemstation software (Hewlett-Packard).As authentic standards for the qualitative and quantitative analysesof carotenoids, a-carotene (b, e-carotene) and b-carotene(b, b-carotene) were purchased from Sigma-Aldrich Company(St Louis, Missouri, USA). Lutein [(3R, 3#R, 6#R)-b, e-carotene-3,3#-diol], zeaxanthin [(3R, 3#R)-b, b-carotene-3, 3#-diol], b-cryptoxanthin [(3R)-b, b-caroten-3-ol], and capsanthin [(3R, 3#S,5#R)-3, 3#-dihydroxy-b, j-carotene-6#-one] were purchased fromExtrasynthese SA (Genay, Cedex, France). Total carotenoid levelswere calculated as the sum of the six carotenoids a-carotene, lutein,b-carotene, b-cryptoxanthin, zeaxanthin, and capsanthin.

Northern blotting

Total RNAs were extracted from the same fruit samples used in theHPLC analysis. The cDNAs for phytoene synthase (Psy), phytoenedesaturase (Pds), b-carotene hydroxylase (Bch), and the Ccs geneswere amplified from Korean red pepper (C. annuum cv. Nockwang)for use as probes in northern blot analysis (Ha et al., 1999).Hybridization was then carried out overnight at 65 �C in 0.5 MNa2PO4 (pH 7.2) buffer, 1% bovine serum albumin fraction V and7% SDS with the 32P-labelled probes. After membranes were rinsedin 23 SSC solution, and then washed once in 13 SSC solution at65 �C for 15 min, and finally in 13 SSC and 0.1% (w/v) SDS at65 �C, they were exposed on the X-ray film to develop the signals.

PCR and sequencing of genomic DNA

Genomic DNA extracts were prepared from the ripe fruits of eachof the pepper varieties under study using a modified cetyltriammo-nium bromide (CTAB) method. PCR was then performed using

Fig. 2. (A) Photographs showing the colours of different varieties of red pepper fruits at different stages of ripening. (B) Measurements of the totalcarotenoid contents in red Capsicum varieties during ripening. Fruits at each stage of ripening (S1–4) were used for the extraction of carotenoids andtotal RNA. Fruits at ripening stage S3 of the LR1 pepper, S4 of the R1 pepper, and S2 and S3 of both the LR2 and R2 peppers are not shown here.

Carotenoid accumulation in Capsicum varieties that show different ripening colours 3137

Super taq PLUS (SuperBio, Suwon, Korea) with 100 ng of eachDNA template and 10 pmol of gene-specific primers for geranylger-anyl pyrophosphate synthase (Ggps), Psy, and Ccs (Ha et al., 1999),and also the promoter region of the Ccs genes (forward primer/reverse primer; 5#-TTGAACCTCCTTGATAAAA-3#/3#-AGGTA-GAGGAAATGAAAGG-5#). The PCR conditions were: 95 �C for10 min, and then 30 cycles of 95 �C for 15 s, 58 �C for 15 s, and72 �C for 1 min. PCR fragments were then subcloned into a readingframe A cassette of the Gateway vector conversion system throughpDONA201 by BP and LR recombination (Invitrogen, Carlsbad,CA) and further sequenced with a Big Dye terminator v3.1 cyclesequencing kit (Applied Biosystems, Foster City, CA). Analyses ofDNA and protein sequences were performed using the Lasergene(DNASTAR, Madison, WI) and BioEdit programs (v7.0.5,Carlsbad, CA).

Results

Assessment of the accumulation of carotenoids at thefully ripe stage in Capsicum fruits

The Capsicum fruit colour phenotype is determined byboth the amount and composition of the carotenoids thathave accumulated at the chromoplasts. Fruits of differentCapsicum varieties showing a diversity of mature coloursthat includes deep red (DR), red (R), light-red (LR), orange(O), pale-yellow orange (PYO), yellow (Y), and white (W),were analysed by HPLC to compare the total levels andcomposition of their carotenoids at the fully ripe stage.As shown in Fig. 2, in pepper varieties whose ripening

colour is red, the DR pepper accumulates the highest levelof total carotenoids (1043 mg kg�1 dry weight). ThreeR peppers (R1–3) accumulate total carotenoids at lower

levels compared with the DR pepper, but at higher levelsthan the two LR peppers of LR1 and LR2. Interestingly,in Fig. 3, it was found that for pepper varieties whose ripecolour is non-red, both O and PYO peppers accumulatetotal carotenoids at lower levels than the three Y peppers(Y1–3). No carotenoids are detectable in ripe W pepper.All of the ripe pepper fruits, with the exception of the

W and Y (Y1-3) peppers, were found to accumulatecapsanthin at various levels. These capsanthin levels aredecreased in ripened fruits with a lighter red pigmentationin the order DR, R, LR, O, and PYO, suggesting that theydirectly correlate with the red colour-saturation in thesepepper varieties. Capsanthin was found to be the majorcomponent (80%) of the total carotenoids in the ripe fruitsof red peppers. The levels of other b-carotenoids in-cluding b-carotene, b-cryptoxanthin, and zeaxanthin var-ied from 1.5% to 16%, and that of lutein was neverdetected above 0.3% of the total carotenoid content inmost of the red peppers. In terms of the main carotenoidcontent, the O pepper was a surprising exception amongpepper varieties as it displayed capsanthin and luteinlevels of 39% and 48%, respectively. Similarly high luteinlevels were detected in the Y1, Y2, and Y3 varietieswith levels of 40.5%, 49.8%, and 67%, respectively,whereas capsanthin in undetectable in yellow peppers.Alpha-carotene was detectable only in the Y1–3 samplesshowing a mature colour and was found to be present atlevels ranging from 10.6% to 13.3% of the totalcarotenoids. These results thus indicate that the non-redpeppers, O and Y, accumulate a-carotenoids such as

Fig. 3. (A) Photographs showing the colours of different varieties of non-red pepper fruits at different stages of ripening. (B) Measurements ofthe total carotenoid contents in non-red Capsicum varieties during ripening. Fruits at each stage of ripening (S1–4) were again used for the extractionof carotenoids and total RNA.

3138 Ha et al.

lutein and a-carotene at higher levels and maintain totalcarotenoids at lower levels than red peppers (see Supple-mentary Fig. 1 at JXB online for the detailed carotenoidcomposition of each pepper variety).

Changes to the carotenoid composition during theripening of Capsicum fruits

To examine whether changes in fruit colour in the peppercan be correlated with changes in the carotenoid compo-sition of this fruit, the samples were harvested at differentstages of ripening and their carotenoid contents wereanalysed by HPLC. In the fully expanded non-ripe pepperfruits (commonly S1 stage in all varieties), the totalcarotenoid profile mainly comprises the chlorophyll-dependent carotenoids such as lutein and b-carotene. Thesecompounds were detectable at their highest levels of(11.8 mg kg�1 and 7.3 mg kg�1 dry weight, respectively)in the Y2 pepper that showed the deepest green colour atmaturation. By contrast, the W and LR1 varieties that donot show any green colour resulting from chlorophyllcontain much lower levels of lutein (1.28 mg kg�1and0.35 mg kg�1 dry weight, respectively).As shown in Fig. 2, the total carotenoid levels increase

with ripening in the red Capsicum varieties and werefound to increase by 11–92-fold in the order LR1, LR2,

R1, R2, R3, and DR during this process. These increasesin the total carotenoid levels are principally due to theincreased capsanthin levels during ripening. In the fourred pepper samples LR1, LR2, R2, and R3, the levels ofall of the b-carotenoids tested, including capsanthin,steadily increase until the final stages of ripening. Theaccumulation of capsanthin in R1 and DR is also similarto these other four red varieties, reaching maximum levelsof 180.7 mg kg�1 and 1013.2 mg kg�1 dry weight,respectively, at their fully ripe stages. The accumulationof other b-carotenoids in R1 and DR differs, however, asit reaches a maximum level just before the fully ripe stageand then decreases from 12.7 mg kg�1 to 5.0 mg kg�1 dryweight for R1, and from 49.4 mg kg�1 to 27.9 mg kg�1

dry weight for DR.By contrast with the red peppers, the total carotenoid

levels in the W, Y, PYO, and O peppers do not increasesignificantly during ripening (Fig. 3). In the W pepper, thelutein and b-carotene levels that are present at the earlystages of ripening were found to have completelydisappeared at the fully ripe stage. In the three Y peppers,the levels of lutein that are present at relatively high levelsbefore ripening are maintained during ripening. Inaddition, a-carotene was found to be produced de novoand b-carotene was observed to be depleted in order to

Fig. 4. Expression of carotenoid biosynthetic genes during ripening in both the red and non-red Capsicum varieties indicated. The fruit ripeningstages shown are identical to those depicted in Figs 2 and 3.

Fig. 5. PCR amplification of carotenoid biosynthetic genes from the genomes of the indicated Capsicum varieties. The Ggps, Psy, and Ccs geneswere amplified in the genomic region corresponding to their cDNAs (GenBank P80042, X68017, and X76165) and the Ccs promoters were amplifiedfrom a region corresponding to 920 bp upstream of the translational start site (GenBank Y14165).


synthesize other b-carotenoids, without affecting the totalcarotenoid levels during ripening in the Y samples.Of the two non-red varieties that accumulate capsanthin

(O and PYO) the PYO pepper showed a small increase inits total carotenoid content (1.4-fold) with evidence ofcapsanthin accumulation de novo. In the O pepper, thetotal carotenoid levels had slightly decreased in parallelwith the reduction of b-carotene during ripening. Beta-carotene appears to be mainly converted into capsanthinand small amounts of zeaxanthin at the fully ripe stage inthe O pepper (see Supplementary Fig. 1 at JXB online fora detailed description of the carotenoid composition ofeach pepper variety).

The carotenogenic gene expression profile in ripeningpeppers of different colours

To investigate whether the observed differences incarotenoid accumulation in our pepper sample’s cohortcorrelated with the expression profile of the carotenoidbiosynthetic genes, northern blotting of the same samplesused in the HPLC analysis was performed (Fig. 4).Transcripts for Psy, Pds, Bch, and Ccs were barelydetectable at the fully expanded non-ripe stage (S1) inany of these varieties. The expression of these genesbegins to be induced at the onset of ripening at differentlevels in each variety, except for the W pepper that showsno expression of any of these genes at the ripening stageas expected.

At least two, and sometimes three of these four genesare expressed in the Y, PYO, and LR1 peppers that accu-mulate low levels of total carotenoids (below 28 mg kg�1

dry weight). By contrast, all four genes are expressed inthe five R pepper samples (LR2, R1, R2, R3, and DR) thataccumulate high total carotenoid levels (greater than163 mg kg�1 dry weight). Interestingly, the expressionpatterns of these four genes are similar in LR2, R1, andR2, which accumulate similar levels of total carotenoid(163, 186, and 226 mg kg�1 dry weight), with a stronginduction of Psy evident during ripening. The expressionlevels of these genes are higher in R3 and DR, which isconsistent with their higher levels of total carotenoids(400 mg kg�1 and 1043 mg kg�1 dry weight), and a moredramatic induction of Ccs expression is observed in thesevarieties compared with the other genes. Hence, theseresults reveal that the activity of each of the fourcarotenoid biosynthetic genes tested is required to achievehigh levels of carotenoid accumulation. Moreover, thestrong induction of the Psy and Ccs genes appears to berequired during ripening in red peppers.

Structural variations in the carotenogenic genes amongpeppers of different ripening colours

Any alteration in either the coding and/or promoter regionof a gene can result in a reduction or even a failure inexpression. Since the expression levels of the carotenoidbiosynthetic genes were found to correlate closely with

Fig. 6. Comparisons between the Ccs gene structure in the 5#-upstream region for the indicated Capsicum varieties. (A) Structural variations areevident by alignment with the nucleotide sequence of the bell pepper (C. annuum). Positions at which an insertion/deletion has occurred are indicatedby the open and closed arrowheads (closed symbol indicates deletion). (B) Phylogenetic tree of the Ccs gene generated by multiple alignments of thenucleotide sequences in the 5#-upstream region.

3140 Ha et al.

the total carotenoid accumulation, the genomic sequencesof several of these genes were examined to determinewhether defects in the gene structure could account fordifferent ripe-colours in Capsicum. As shown in Fig. 5,genomic PCR analysis of the different pepper samplesreveals that the Ggps gene is equivalently sized in all ofthe varieties tested in this study. This is also the case forthe Psy gene with the exception of the W pepper. Both thecoding and promoter regions of Ccs are absent fromthe genomes of the W and Y1 peppers, but are present inthe remaining varieties.The coding region of the Ccs gene has previously been

shown to have the same 1.5 kb size in the bell pepper(Bouvier et al., 1994) that was observed in this study’ssamples, but the promoter regions of this gene differbetween Capsicum varieties and can be grouped into threepatterns; 920/922 bp, 998/999 bp, and 1175 bp.Interestingly, both the coding and promoter regions ofthe Ccs gene are present in the genomes of two of theY varieties in the sample cohort, Y2 and Y3 (Fig. 5), bothof which share a yellow ripe-colour and a similarcarotenoid composition with the Y1 pepper, that is lackingin capsanthin. To our knowledge, this is the first evidenceof a yellow ripe-colour pepper that harbours the Ccscoding region.As shown in Fig. 6A, multiple alignments of the

nucleotide sequences of the Ccs promoter regions amongthe different pepper varieties in this study’s samples haveidentified three kinds of insertions/deletions, in addition toseveral minor variations, when compared with the pre-viously reported sequence for the bell pepper (Bouvieret al., 1998). The 11 bp-deletion and 88 bp-insertion thatwere identified in these analyses occur in seven of thevarieties tested, but not in three Capsicum annuum

species. A 176 bp-insertion is observed in all fourC. chinense species except for the O pepper, which in factshows a higher Ccs promoter sequence homology with theC. baccatum species and is quite closely related to thisgenus in the phylogenic tree (Fig. 6B). It is concludedfrom these data, therefore, that differences in the sequenceof the Ccs gene promoter region reflect species-specificvariations in Capsicum (see Supplementary Fig. 2 at JXBonline for the detailed sequence comparison data).Multiple alignments of the nucleotide sequences from

the Ccs coding regions were also performed and thepresence of two structural mutations in the Y2 and Y3peppers was found (Fig. 7; see Supplementary Fig. 3 atJXB online for the detailed sequence alignment data). Oneof these mutations results in a frame-shift, thus causingearly translation-termination, via an 8 bp-insertion atposition 1431 of the gene in Y2. The Ccs mutation in Y3causes a premature stop-codon via a single base change atposition 599. It is interesting that both of these mutationsoccur in peppers where the Ccs gene is present but whichhave a yellow ripe-colour.

Discussion

In general, carotenoid accumulation does not appear toincrease until a particular fruit is fully expanded. In thefruits of the red Capsicum, the concentration of lutein,a major chloroplast pigment, decreases during the ripeningprocess from its highest level during the non-ripe maturestage, whereas the levels of b-carotene gradually increaseand other b-carotenoids including b-cryptoxanthin, zeax-anthin, and capsanthin begin to be synthesized de novo.As a result of this, the total carotenoid levels in red pepper

Fig. 7. Schematic representation of the structural mutations in the Ccs gene among different Capsicum varieties. The nucleotide sequences werealigned on the basis of the bell pepper sequence (C. annuum). Mutations in the nucleotide sequences are highlighted in bold and the resultingmissense mutations in the amino acid coding are underlined. ATG and TGA indicate the start and stop codons, respectively. The numbers depict theamino acid positions and the asterisk indicates the stop codon.


varieties increase as they ripen. In the present study, it wasobserved that the increases in the total carotenoid contentduring ripening in red pepper varieties are highly variable(from 11–92-fold) and are dependent on the ripe colours(Fig. 1). Such a large variation is not unexpected becausethe red varieties sampled for this study were selected onthe basis of seven different ripe colours.In non-red pepper fruits, in contrast, the total carotenoid

levels do not increase and remain low during ripening.Moreover, the composition of the total carotenoids in non-red pepper fruits significantly differs from red pepperfruits where more than 80% of this total is capsanthin.In particular, the lutein content of the Y peppers is farhigher than any of the R peppers at the non-ripe stagesand remains high, at 41–67% of the total carotenoids, infully ripe Y peppers. Based on these results, this study’spepper varieties could be categorized into two groups: oneshowing an accumulation of increasing levels of totalcarotenoids, and one that maintains low levels ofcarotenoids that vary in composition. The fact that all redpeppers seem to accumulate increasing amounts of totalcarotenoids with a large proportion of capsanthin suggeststhat capsanthin production is mainly responsible for thisincrease. Accordingly, the structure and expression levelsof the Ccs gene could be considered to be the mostimportant determinant of whether high levels of totalcarotenoids are produced in the pepper.All non-red peppers were found to accumulate limited

amounts of total carotenoids regardless of whethercapsanthin accumulation occurred (Fig. 3). In the case ofthe O pepper, which shows low amounts of totalcarotenoid, it is possible that an alteration in theexpression of the Psy gene causes the orange-ripeningphenotype in spite of the capsanthin accumulation, asdescribed previously (Huh et al., 2001). Huh et al. (2001)reported here that the orange pepper (C. chinense cv.Habanero) produces aberrant Psy transcripts by abnormalsplicing, resulting in a reduced activity of PSY.The present results are consistent with those of Bouvier

et al. (1994) in showing that all red peppers accumulatehigh levels of capsanthin and express the Ccs gene at highlevels, which accounts for the high levels of totalcarotenoid in these fruits. In addition, it was also observedthat high levels of total carotenoid in Capsicum requiredthe induction of the Psy, Pds, and Bch genes in addition tothe Ccs gene during ripening, even though these patternsvary between the different red pepper varieties (Fig. 4).Minimal levels of expression of other genes such asb-lycopene cyclase (b-Lcy) and violaxanthin de-epoxidase(Vde) also appear to be required for the accumulation ofhigh levels of carotenoid during ripening in red peppers(data not shown). Our current data indicating that theexpression levels of the carotenoid biosynthetic genes arethe key regulators of the high levels of total carotenoidaccumulation in peppers are in good agreement with

previous results obtained in tomato fruits (Pecker et al.,1996; Ronen et al., 1999, 2000), marigold petals (Moehset al., 2001), and red peppers (Romer et al., 1993;Hugueney et al., 1996; Ha et al., 1999).This relationship between the presence of structural

genes for carotenoid biosynthesis and the phenotypicvariability in ripe colours has been studied for severaldecades using genetic approaches in diverse Capsicumspecies (Kormos and Kormos, 1960; Hurtado-Hernandezand Smith, 1985; Lefebvre et al., 1998; Popovsky andParan, 2000; Thorup et al., 2000; Lang et al., 2004). Inparticular, several studies have reported that the Ccs geneis either deleted or absent in yellow and orange colourpepper fruits (Bouvier et al., 1994; Lefebvre et al., 1998;Popovsky and Paran, 2000; Lang et al., 2004). Bouvieret al. (1994) have also demonstrated that the Ccs gene isnot present in two yellow pepper cultivars, Jaune dePignerolle and Golden Summer. Interestingly, Lang et al.(2004) and Popovsky and Paran (2000) reported similarresults, showing that the Ccs gene is deleted in yellowfruits at 211 bp and 220 bp from the 3#-end, respectively.Nucleotide sequences of Ccs genes were analysed in thepepper varieties in the present study and it was revealedthat the coding sequence of this gene and its promoterregion are present in two of the pepper varieties whoseripe-colour is yellow (Fig. 5). The Ccs gene, however,is not expressed in the corresponding yellow peppers(Fig. 4), probably due to the presence of mutations in thecoding region that generate a premature stop-codon (Y2)and a frame-shift (Y3) (Fig. 7).Neither unusual cis-acting regulatory sequences nor

critical mutations are evident that would impair thepromoter activity of the Ccs genes in the two yellowpepper samples, Y2 and Y3. The Y2 pepper can producea truncated CCS protein of 495 amino acids that containsa novel 18 amino acid stretch at its C-terminus caused byan 8 bp insertion in the coding region of the gene. Thepremature translation-termination codon (PTC) caused bythe single base change in the Ccs gene of the Y3 peppercan generate a truncated CCS protein of 199 amino acids.Such PTCs or nonsense mutations can result in two kindsof yellow ripe colours. Such a phenomenon can beexplained by a mechanism called ‘nonsense-mediatedmRNA decay’, in which aberrant mRNAs harbouringpremature translation-termination codons decay rapidlybecause the resulting C-terminally truncated proteins canfunction as dominant negative inhibitors of the full-lengthprotein (Buhler et al., 2005). Recently, a 1.9-fold higheractivity of PSY has been reported in the high pigment-1(hp-1) tomato mutant without any increase of geneexpression compared with the wild-type tomato, indicatingtranslational or post-transcriptional control of caroten-oid gene expression (Cookson et al., 2003). A post-transcriptional regulation also appears to play a role incarotenoid metabolism. Our elucidation of structural

3142 Ha et al.

mutations in the Ccs gene in yellow peppers is likely to beanother such example of this regulation and may be usefulin increasing our understanding of carotenoid metabolismin the future.

Supplementary data

Supplementary data in the form of three figures can befound at JXB online. Figure 1: Phenotypic features,carotenoid compositions and general information for theCapsicum varieties used in this study. Figure 2: Sequencecomparisons in the region of the Ccs promoter among theindicated Capsicum varieties. Figure 3: Multiple align-ments of the nucleotide and predicted amino acidsequences of the Ccs coding genes among the indicatedCapsicum varieties.

Acknowledgements

This work was supported by research funds of the National Instituteof Agricultural Biotechnology (NIAB 06-2-11-10-2 and NIAB06-2-12-6-1 to S-H Ha) and the Ministry of Science andTechnology through the Crop Functional Genomics Center (grantto S-H Ha) in Korea.

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3144 Ha et al.

1

Supplementary figure legends 1

2

Supplementary Fig. 1. Phenotypic features, carotenoid compositions and general 3

information for the Capsicum varieties used in this study. The introduction numbers 4

(IT), as given by the Genebank of National Institute of Agricultural Biotechnology 5

(Rural Development Administration, Suwon, Korea) are: 158770 (C. baccatum var. 6

pendulum, LR2), 158773 (C. chinense, R2), 158782 (C. annuum, W), 158806 (C. 7

baccatum, LR1), 163499 (C. annuum, DR), 164918 (C. chinense, Y3), 191655 (C. 8

chinense, PYO), 203499 (C. chinense, O), 709424 (C. pubescens, R1), 800065 (C. 9

chinense, Y2) and 800070 (C. baccatum, Y1). 10

11

Supplementary Fig. 2. Sequence comparisons in the region of the Ccs promoter 12

among the Capsicum varieties under study. The blue boxes indicate remarkably 13

variable sequences: Box 1, 11bp-deletion; Box 2, 88bp-insertion; Box 3, 176bp-14

insertion. 15

16

Supplementary Fig. 3. Multiple alignments of the nucleotide (A and B) and 17

predicted amino acid (C and D) sequences of the Ccs coding genes among the 18

indicated Capsicum varieties. Two decisive structural mutations in the two Y-pepper 19

varieties 164918 (Y3) and 800065 (Y2) are enclosed in blue boxes. 20

IT 158770 (LR2)

Composition of carotenoids

Fruit color during ripening process

S1 S4

Korean Introduction No. IT158770Capsicum Species Capsicum baccatum var. pendulum

English name ajiCultivar name VAR.PENDULUM(WILLD) ESHBAUGH

Origin RussiaDonor contury Taiwan

Donor & Donor No. AVRDC C00774

Capsicum Information

Variety Stage α-Carotene Lutein β-Carotene β-Cryptoxanthin Zeaxanthin Capsanthin Totalcarotenoids

158770 S1 1.51 1.60 3.11(48.60) (51.40)

S4 0.43 1.61 0.79 160.57 163.39(0.26) (0.98) (0.48) (98.27)

Numbers in parenthesis refer to percentage in total amounts of carotenoid.

(μg/g)

IT 158773 (R2)

Fruits of stage (S) 2 and S3 are not shown here.

S1

S4



Capsicum Information Korean Introduction No. IT158773

Capsicum Species Capsicum chinenseEnglish name Tabasco pepperCultivar name OSE UTORO

Origin Nigeria Donor contury Taiwan

Donor & Donor No. AVRDC C00776-1


158773 S1 2.98 2.01 4.98(59.75) (40.25)

S2 0.31 2.67 1.54 3.58 20.23 28.33(1.11) (9.41) (5.42) (12.65) (71.40)

S3 0.35 9.46 5.17 5.57 132.30 152.85(0.23) (6.19) (3.38) (3.64) (86.56)

S4 0.43 11.71 10.19 13.71 189.65 225.68(0.19) (5.19) (4.51) (6.07) (84.04)


(μg/g)

S1 S2

IT 158782 (W)




Capsicum Species Capsicum annuum var. annuumEnglish name pepperCultivar name

Origin EnglandDonor contury Taiwan




158782 S1 1.28 0.73 2.06(63.55) (36.45)

S2 0.00

(μg/g)

S2 S4

S1

S1

IT 158806 (LR1)




Fruit of stage 3 (S3) is not shown here.

Korean Introduction No. IT158806Capsicum Species Capsicum baccatum var. pendulum

English name ajiCultivar name

Origin EnglandDonor contury Taiwan



158806 S1 0.35 0.19 0.13 1.90 2.57(13.53) (7.53) (5.05) (73.88)

S2 0.13 0.04 0.46 4.59 5.22(2.44) (0.85) (8.84) (87.87)

S3 0.30 0.22 1.52 10.85 12.89(2.35) (1.73) (11.75) (84.17)

S4 0.05 0.45 0.36 2.65 24.85 28.36(0.19) (1.58) (1.26) (9.34) (87.64)


(μg/g)

S4

S1

S2

S3

IT 163499 (DR)




Capsicum Species Capsicum annuum var. annuumEnglish name pepperCultivar name

Origin BrazilDonor contury USA

Donor & Donor No. USDA/ARS PI21312


163499 S1 7.56 3.70 11.28(67.10) (32.90)

S2 0.91 5.03 2.33 6.60 179.43 194.36(0.47) (2.59) (1.22) (3.40) (92.32)

S3 1.72 20.90 11.52 16.94 809.00 860.09(0.20) (2.43) (1.34) (1.97) (94.06)

S4 2.08 15.90 5.00 6.98 1013.15 1043.20(0.20) (1.53) (0.48) (0.67) (97.12)


(μg/g)

S1

S2

IT 164918 (Y3)




Capsicum Species Capsicum chinenseEnglish nameCultivar name Yellow

OriginDonor contury India

Donor & Donor No.


164918 S1 5.28 2.46 7.74(68.22) (31.78)

S2 1.56 5.42 3.29 1.05 2.07 13.40(11.66) (40.48) (24.55) (7.87) (15.44)


(μg/g)

Fruits of break point (Br) stage are not used for carotenoid analysis.

S2

S1

S2

S1

S1 Br S2

1 2 3 3

IT 191655 (PYO)





191655-1 S1 1.54 0.95 2.48(61.91) (38.09)

S2 0.16 2.88 3.04(5.26) (94.74)

191655-2 S1 1.72 1.22 2.94(58.51) (41.49)

S2 0.16 5.63 5.79(2.76) (97.24)

191655-3 S1 3.49 1.16 4.65(61.91) (38.09)

S2 0.06 5.47 5.53(1.08) (98.92)

Korean Introduction No. IT191655Capsicum Species Capsicum chinense

English nameCultivar name

Origin MexicoDonor contury Taiwan



(μg/g)

S1

S2

S3

Fruits of stage 2 are not used for carotenoid analysis.

IT 203499 (O)




Capsicum Species Capsicum chinenseEnglish nameCultivar name Habanero Chilli

Origin AustraliaDonor contury Australia

Donor & Donor No.


203499 S1 9.16 4.78 0.42 14.35(63.79) (33.31) (2.90)

S3 4.90 0.29 1.00 3.98 10.17(48.18) (2.85) (9.83) (39.13)


(μg/g)

Fruits of mature red stage (S4) are not shown here. Fruits of break point (Br) are not used for carotenoid analysis.

S1S2

S3

Br

Purple flower

IT 709424 (R1)



Capsicum Information Korean Introduction No. Temporary IT709424

Capsicum Species Capsicum pubescensEnglish name rocotoCultivar name Locoto

Origin Bolivia Donor contury Bolivia

Donor & Donor No.


709424 S1 3.91 1.18 5.08(76.87) (23.13)

S2 2.13 0.90 3.04(70.25) (29.75)

S3 1.33 2.73 4.54 2.45 5.71 16.76(7.93) (16.29) (27.09) (14.62) (34.07)

S4 0.67 1.73 1.43 1.81 180.67 186.31(0.36) (0.93) (0.77) (0.97) (96.97)


(μg/g)

800070 800065 800065

S1

S2

S1

S2

IT 800070 (Y1) & IT 800065 (Y2)



Capsicum Information Korean Introduction No. Temporary IT800065 Temporary IT800070

Capsicum Species Capsicum chinense Capsicum baccatumEnglish nameCultivar name

OriginDonor contury Bolivia Bolivia

Donor & Donor No.


800070 S1 9.87 6.19 0.91 16.97(58.14) (36.49) (5.37)

S2 1.67 10.58 1.91 1.63 15.79(10.57) (67.00) (12.13) (10.30)

800065 S1 11.78 7.31 0.92 20.02(58.85) (36.54) (4.61)

S2 2.64 9.89 4.81 0.45 2.08 19.86(13.30) (49.78) (24.23) (2.24) (10.45)


(μg/g)

S1 S2 S3 S4

Nockwang (R3)



Capsicum Information Korean Introduction No.

Capsicum Species Capsicum annuum var. annuumEnglish nameCultivar name Nockwang

Origin Republic of KoreaDonor contury Republic of Korea

Donor & Donor No. HungNong Seeds Company


Nockwang S1 4.32 2.90 3.36 10.57(40.85) (27.40) (31.75)

S2 1.25 3.84 0.62 6.54 43.35 55.61(2.26) (6.90) (1.12) (11.77) (77.95)

S3 0.90 3.67 1.86 8.38 104.33 119.14(0.76) (3.08) (1.56) (7.03) (87.58)

S4 0.79 8.97 7.51 16.65 372.15 406.08(0.20) (2.21) (1.85) (4.10) (91.65)


(μg/g)

Supplementary Fig. 1. Phenotypic features, carotenoid compositions and general

information for the Capsicum varieties used in this study. The introduction numbers

(IT), as given by the Genebank of National Institute of Agricultural Biotechnology

(Rural Development Administration, Suwon, Korea) are: 158770 (C. baccatum var.

pendulum, LR2), 158773 (C. chinense, R2), 158782 (C. annuum, W), 158806 (C.

baccatum, LR1), 163499 (C. annuum, DR), 164918 (C. chinense, Y3), 191655 (C.

chinense, PYO), 203499 (C. chinense, O), 709424 (C. pubescens, R1), 800065 (C.

chinense, Y2) and 800070 (C. baccatum, Y1). Color abbreviations mean deep red

(DR), red (R), light-red (LR), orange (O), pale-yellow orange (PYO), yellow (Y)

and white (W).

800065 TTGAACCTCCTTGATAAAAAATTTTGACTCCGCCATTACTACAAGGTAGAACCTCCAACAATCACCAATACCTAAATTGCAAAAATTTCAGTTCGTTTTTTAATTTCTATTTTGGGAAGAGGAATACTACAAGGCCTCCAACAATCACCAATACCTAAATTGCAAAAATTTCAGTTTGTT 180




203499 TTGAACCTCCTTGATAAAAAATTTTGACTCCGCCACTGCTACAAGGTAGAACCTCCAACAATCACCAATACCTAAATTGCAAAAATTTCAATTCGTTTTTTAATTTCTGTTTTGGGAAGAGGAATACTACAAGGCCTCCAACAATCACCAATACCTAAATTGCAAAAATTTTAGTTTGTT 180

158806 TTGAACCTCCTTGATAAAAAATTTTGACTCCGCCACTGCTACAAGGTAGAACCTTCAACAATCACCAATACCTAAATTGCAAAAATTTCAGTTCGTTTTTTAGTTTCTGTTTTGGGAAGAG-AATACTACAAGGCCTCCAACAATCTCCAATATCTAAATTGCAAAAATTTCAGTTTGTT 179

158770 TTGAACCTCCTTGATAAAAAATTTTGACTTCGCCACTGCTACAAGGTAGAACCTTCAACAATCACCAATACCTAAATTGCAAAAATTTCAGTTCGTTTTTTAGTTTCTGTTTTGGGAAGAG-AATACTACAAGGCCTCCAACAATCACCAATATCTAAATTGCAAAAATTTCAGTTTGTT 179

Nockwang TTGAACCTCCTTGATAAAAAATTTTGACTCCGCCATTGCTACAAGGTAGAACCTCCAACAATCACCAATACCTAAATTGCAAAAATTTCAGTTCGTTTTTTAATTTCTGTTTTGGGAAGAGGAATACTACAAGGCCTCCAACAATCACCAATACCTAAATTGCAAAAATTTCAGTTTGTT 180

Subicho TTGAACCTCCTTGATAAAAAATTTTGACTCCGCCATTGCTACAAGGTAGAACCTCCAACAATCACCAATACCTAAATTGCAAAAATTTCAGTTCGTTTTTTAATTTCTGTTTTGGGAAGAGGAATACTACAAGGCCTCCAACAATCACCAATACCTAAATTGCAAAAATTTCAGTTTGTT 180

163499 TTGAACCTCCTTGATAAAAAATTTTGACTCCGCCATTGCTACAAGGTAGAACCTCCAACAATCACCAATACCTAAATTGCAAAAATTTCAGTTCGTTTTTTAATTTCTGTTTTGGGAAGAGGAATACTACAAGGCCTCCAACAATCACCAATACCTAAATTGCAAAAATTTCAGTTTGTT 180

800065 TTTTAGTTTCTGTTTTGGGAAGAGGAATACTACAAGGTAGGGCCTCCAACAATCACCAATACCTAAATTGCAA-----------CGTTTTTTAGTTTCTGTTTTGGGAAGAGGAACACTACAAGGAAGGGCCTCCAACAATCACCAATACCTAAATTGCAAAAATTTCAGTTCGTATTTT 349




203499 TTTTAGTTTCTGTTTTGGGAAGAGGAATACTACAAGGTAGGGCCTCCAACAATCACCAATACCTAAATTGCAA-----------CGGTTTTTAGTTTCTATTTTGGGAAGAGGAATACTACAAGGTAGGGCCTCCAACAATCACCAATACCTAAATTGCAAAAATTTCAGTTCGTATTTT 349

158806 TTTTAGTTTCTGTTTTGGGAAGAG-AATACTACAAGGTAGGGCCTCCAACAATCACCAATACCTAAATTACAA-----------CGTGTTTTAGTTTCTGATTTGGGAAGAGGAATACTACAAGGTAGGGCCTCCAACAATCACCAATACCTAAATTGCAAAAATTTCAGTTCGTATTTT 347

158770 TTTTAGTTTCTGTTTTGGGAAGAG-AATACTACAAGGTAGGGCCTCCAACAATCACCAATACCTAAATTACAA-----------CGTTTTTTAGTTTCTGATTTGGGAAGAGGAATACTACAAAGTAGGGCCTCCAACAATCACCAATACCTAAATTGCAAAAATTTCAGTTTGTATTTT 347

Nockwang TTTTAGTTTCTGTTTTGGGAAGAGGAATACTACAAGGTAAGGCCTCCAACAATCACCAATACCTAAATTGCAAAAATTTCAGTTCGTATTTTCGTTTCTATTTTGGGAAG---------------------------------------------------------------------- 290

Subicho TTTTAGTTTCTGTTTTGGGAAGAGGAATACTACAAGGTAAGGCCTCCAACAATCACCAATACCTAAATTGCAAAAATTTCAGTTCGTATTTTCGTTTCTATTTTGGGAAG---------------------------------------------------------------------- 290

163499 TTTTAGTTTCTGTTTTGGGAAGAGGAATACTACAAGGTAAGGCCTCCAACAATCACCAATACCTAAATTGCAAAAATTTCAGTTCGTATTTTCGTTTCTATTTTGGGAAG---------------------------------------------------------------------- 290

800065 CGTTTCTATTTTGGGAAGTGGAATAGTATAAGGTAGGACCTCCAACAATCACCAATACCTAAATTGCAAAAGTTCCGATTCATTTTTTAGTTTCTGTTTTGGAAAGAGAAATACTACAAGGTAGGGCCTCCAACAATCACCAGTACCTAAATTGTAAAAATTTCAGTTCGTTTTTTAGTT 529




203499 CGTTTCTATTTTGGGAAGTGGAATAGTATAAGGTAGGACCTCCAACAATCACCAATACCTAAATTGCAAAAGTTCCGATTCATTTTTTAGTTTCTGTTTTAGAAAGAGAAATACTACAAGGTAGGGCCTCCAACAATCACCAGTACCTAAATTGTAAAAATTTCAGTTCGTTTTTTAGTT 529

158806 CATTTTTATTTTGGGAAGTGGAATAGTATAAGGTAGGACCTCCAACAATCACCAATACCTAAATTGCAAAAGTTCCGATTCATTTTTTAGTTTCTGTTTTGGGAAGAGGAATACTATAAGGTAGGGCCTCCAACAATCACGAATACCTAAATTGCAAAAATTTCAGTTCGTATTTTCGTT 527

158770 CATTTCTATTTTGGGAAGTGGAATAGTATAAGGTAGGACCTCCAACAATCACCAATACCTAAATTGCAAAAGTTCCGATTCATTTTTTAGTTTCTGTTTTGGGGAGAGGAATACTATAAGGTAGGGCCTCCAACAATCACGAATACCTAAATTGCAAAAATTTCAGTTAGTATTTTCGTT 527

Nockwang ------------------TGGAATAGTATAAGGTAGGACCTCCAACAATCACCAATACCTAAATTGCAAAAGTTCCGATTCATTTTTTAGTTTCTGTTTTGGAAAGAGAAATACTACAAGGTAGGGTCTCCAACAATCACCAGTACCTAAATTGTAAAAATTTCAGTTCGTTTTTTAGTT 452

Subicho ------------------TGGAATAGTATAAGGTAGGACCTCCAACAATCACCAATACCTAAATTGCAAAAGTTCCGATTCATTTTTTAGTTTCTGTTTTGGAAAGAGAAATACTACAAGGTAGGGTCTCCAACAATCACCAGTACCTAAATTGTAAAAATTTCAGTTCGTTTTTTAGTT 452

163499 ------------------TGGAATAGTATAAGGTAGGACCTCCAACAATCACCAATACCTAAATTGCAAAAGTTCCGATTCATTTTTTAGTTTCTGTTTTGGAAAGAGAAATACTACAAGGTAGGGTCTCCAACAATCACCAGTACCTAAATTGTAAAAATTTCAGTTCGTTTTTTAGTT 452

800065 TCTATTTTGGGAAGTGGAATAGTATAAGGTAGGACCTCCAACAATCACCAATATCTAAATTGCAAAAGTTCCGATTCTTTTTTTAGTTTCTGTTTTGGAAAGAGAAATACTACAAGGTAGGGCTTTCAACAATCACCAGTACCTAAATTGTAAAAATTTCAGTTCATTTTTTAGTTTCTA 709

164918 TCTATTTTGGGAAGTGGAATAGCATAAGGTAGGACCTCCAACAATCACCAATATGTAAATTGCAAAAGTTCCGATTCTTTTTTTAGTTTCTGTTTTGGAAAGAGAAATACTACAAGGTAGGGCTTTCAACAATCACCAGTACCTAAATTGTAAAAATTTCAGTTCATTTTTTAGTTTCTA 709



203499 TCTATTTTGGGAAGTGGAATAGTATAAGGTAGGACCTCCAACAATCACCAATACATAAATTGCAAAAGTTCCGATTCATTTTTTAGTTTCTGTTTTGGAAAGAGAAATACTACAAGGAAGGACCTTCAACGATCACCAATACCTAAATTGCAAAAATTTCAGTTCCTTTTTTAGTTTCTG 709

158806 TCTATTTTGGGAAGTGGAATAGTATAAGGTAGGACCTCCAACAATCACCAATACCTAAATTGCAAAAGTTCCGATTCATTTTTTAGTTTCTGTTTTGGAAAGAGAAATACTACAAGGTAGGACATTCAACAATCACCAATACCTAAATTGCAAAAATTTCAGTTCATTTTTTAGTTTCTG 707

158770 TCTATTTTGGGAAGTGGAATAGTATAAGGTAGGACCTCCAACAATCACCAATACCTAAATTGCAAAAGTTCCGATTCATTTTTTAGTTTCTGTTTTGGAAAGAGAAATACTACAAGGTAGGACATTCAACAATCACCAATACCTAAATTGCAAAAATTTCAGTTCATTTTTTAGTTTCTG 707

Nockwang TCTATTTTGGGAAGTGGAATAGTATAAGGTAGGACCTCCAACAATCACCAATACCTAAATTGCAAAAGTTCCGATTCTTTTTTTAGTTTCTGTTTTGGAAAGAGAAATACTACAAGGTAGGACCTTCAACAATCACCAATACCTAAATTGCAAAAACTTCAGTTCATTTTTTAGTTTCTG 632

Subicho TCTATTTTGGGAAGTGGAATAGTATAAGGTAGGACCTCCAACAATCACCAATACCTAAATTGCAAAAGTTCCGATTCTTTTTTTAGTTTCTGTTTTGGAAAGAGAAGTACTACAAGGTAGGACCTTCAACAATCACCAATACCTAAATTGCAAAAACTTCAGTTCATTTTTTAGTTTCTG 632

163499 TCTATTTTGGGAAGTGGAATAGTATAAGGTAGGACCTCCAACAATCACCAATACCTAAATTGCAAAAGTTCCGATTCTTTTTTTAGTTTCTGTTTTGGAAAGAGAAATACTACAAGGTAGGACCTTCAACAATCACCAATACCTAAATTGCAAAAACTTCAGTTCATTTTTTAGTTTCTG 632

800065 TTTTGGGAAGTGGAATAGTATAAGGTAGGACCTCCAACAATCACCAATACCTAAATTGCAAAAGTTCCGATTCTTTTTTTAGTTTCTGTTTCGGAAAGAGAAATACTACGAGGTAGGACCTTCAACAATCACCAATACCTAAATTGCAAAAATTTCAGTTCATTTTTTAGTTTCTGTTTT 889

164918 TTTTGGGAAGTGGAATAGTATAAGGTAGGACCTCCAACAATCACCAATACCTAAATTGCAAAAGTTCCGATTCTTTTTTTAGTTTCTGTTTCGGAAAGAGAAATACTACAAGGTAGGACCTTCAACAATCACCAATACCTAAATTGCAAAAATTTCAGTTCATTTTTTAGTTTCTGTTTT 889



203499 TTTTGGGAAGAAGAATA------------------------------------------------------------------------------------------------------------------------------------------------------------------- 726

158806 TTTTGGAAAGAAGAATA------------------------------------------------------------------------------------------------------------------------------------------------------------------- 724


Nockwang TTTTGGGAAGAAGAATA------------------------------------------------------------------------------------------------------------------------------------------------------------------- 649

Subicho TTTTGGGAAGAAGAATA------------------------------------------------------------------------------------------------------------------------------------------------------------------- 649


800065 GGGATGAAGAATACTAGAAGATAACAATCACCAATACCTAAATTAAAAATTTCAGTTAGTTTTTTAGTTTCTGTTTTTGGGAAGAGGAATACTTTCTTTTGCTATATAAAGCCAA-GTAGGTACCTATAAGCATCAATATTTTGTATTGCTTAGTGATTCCCCTAGTTCAGTATTTCAAT 1068

164918 GGGATGAAGAATACTAGAAGATAACAATCACCAATACCTAAATTAAAAATTTCAGTTAGTTTTTTAGTTTCTGTGTTTGGGAAGAGGAATACTTTCTTTTGCTATATAAAGCCAA-GTAGGTACCTATAAGCATCAATATTTTGTATTGCTTAGTGATTCCCCTAGTTCAGTATTTCAAT 1068

191655 GGGATGAAGAATACTAGAAGATAACAATCACCAATACCTAAATTAAAAATTTCAGTTAGTTTTTTAGTTTCTGTTTTTGGGAAGAGGAATACTTTCTTTTGCTATATAAAGCCAA-GTAGGTACATATAAGCATCAATATTTTGTATTGCTTAGTGATTCCCCTAGTTCAGTATTTCAAT 1068

158773 GGGATGAAGAATACTAGAAGATAACAATCACCAATACCTAAATTAAAAATTTCAGTTAGTTTTTTAGTTTCTGTTTTTGGGAAGAGGAATACTTTCTTTTGCTATATAAAGCCAA-GTAGGTACCTATAAGCATCAATATTTTGTATTGCTTAGTGATTCCCCTAGTTCAGTATTTCAAT 1068

203499 -------------CTACAAGGTAACAATCACCAATATCTAAATTAAAAATTTCAGTTAGTTTTTTAGTTTCTGTTTTTGGGAAGAGGAATACCTTCTTTTGCTATATAAAGCCAAAGTAGGTACCTATAAGCATCAATATTTTGTATTGCTTAGTGATTCCACTAGTTCAGTATTTCAAA 893

158806 -------------CTACAAGGTAACAATCACCAATACCTAAATTAAAAATTTCAGTTAGTTTTTTAGTTTCTGTTTTTGGGAAGAGGAATACTTTCTTTTGCTATATAAAGCCAAAGTAGGTACCCATAAGCATCAATATTTTGTATTGCTTAGTGATTCCCCTAGTTCAGTATTTCAAT 891

158770 -------------CTACCAGGTAACAATCACCAATACCTAAATTAAAAATTTCAGTTAGTTTTTTAGTTTCTCTTTTTGGGAAGAGGAATACTTTCTTTTGCTATATAAAGCCAAAGTAGGTGCCTATAAGCATCAATATTTTGTATTGCTTAGTGATTCCCCTAGTTCAGTATTTCAAT 891

Nockwang -------------CTTCAAGGTAACAATCACCAATACCTAAATTAAAAATTTCAGTTAGTTTTTTAGTTTCTGTTTTTGGGAAGAGGAATACTTTCTTTTGCTATATAAAGCCAAAGTAGGTACCTATAAGCATCAATATTTTGTATTGCTTAGTGATTCCCCTAGTTCGGTATTTCA-T 815

Subicho -------------CTTCAAGGTAACAATCACCAATACCTAAATTAAAAATTTCAGTTAGTTTTTTAGTTTCTGTTTTTGGGAAGAGGAATACTTTCTTTTGCTATATAAAGCCAAAGTAGGTACCTATAAGCATCAATATTTTGTATTGCTTAGTGATTCCCCTAGTTCGGTATTTCA-T 815

163499 -------------CTTCAAGGTAACAATCACCAATACCTAAATTAAAAATTTCAGTTAGTTTTTTAGTTTCTGTTTTTGGGAAGAGGAATACTTTCTTTTGCTATATAAAGCCAAAGTAGGTACCTATAAGCATCAATATTTTGTATTGCTTAGTGATTCCCCTAGTTCGGTATTTCA-T 815

800065 TTTTTTTCACTATACTACATCACCTCCTCCCATAAACAGCCATTATCAATCTTGCATTTTCTCTAATGGAAACCCTTCTAAAGCCTTTTCCATCTCCTTTACTTTCC 1175

164918 TTTTTTTCACTATACTATATCACCTCCTCCCATAAACAGCCATTATCAATCTTGCATTTTCTCTAATGGAAACCCTTCTAAAGCCTTTTCCATCTCCTTTACTTTCC 1175



203499 TTTTTTTCACTATACTATATCACCTCCTCCCATAAATAGCCATTATCAATCTT-CATTTTCTCTAATGGAAACCCTTCTAAAGCCTTTTCCATCTCCTTTACTTTCC 999

158806 TTTTTTTCACTATACTATATCACCTCCTCCCATAAATAGCCATTATCAATCTTGCATTTTCTCTAATGGAAACCCTTCTAAAGCCTTTTCCATCTCCTTTACTTTCC 998

158770 TTTTTTTCACTATACTATATCACCTCCTCCCATAAATAGCCATTATCAATCTTGCATTTTCTCTAATGGAAACCCTTCTAAAGCCTTTTCCATCTCCTTTACTTTCC 998

Nockwang TTTTTTTCACTATACTATATCACCTCCTCTCATAAATAGCCATTATAAATCTTGCATTTTCTCTAATGGAAACCCTTCTAAAGCCTTTTCCATCTCCTTTACTTTCC 922

Subicho TTTTTTTCACTATACTATATCACCTCCTCTCATAAATAGCCATTATAAATCTTGCATTTTCTCTAATGGAAACCCTTCTAAAGCCTTTTCCATCTCCTTTACTTTCC 922

163499 TTTTTTTCACTATACTATATCACCTCCTCTCATAAATAGCCATTATAAATCTTGCATTTTCTCTAATGGAAACCCTTCTAAAGCCTTTTCCATCTCCTTTACTTTCC 922

Box 1

Supplementary Fig. 2

Box 2

Box 3

Supplementary Fig. 2. Sequence comparisons in the region of the Ccs promoter among the Capsicum varieties

under study. The blue boxes indicate remarkably variable sequences: Box 1, 11bp-deletion; Box 2, 88bp-insertion;

Box 3, 176bp-insertion.

163499 ATGGAAACCCTTCTAAAGCCTTTTCCATCTCCTTTACTTTCCATTCCTACTCCTAACATGTATAGTTTCAAACACAACTCCACTTTTCCAAATCCAACCAAACAAAAAGATTCAAGAAAGTTCCATTATAGAAACAAAAGCAGTACACATTTTTGTAGCTTTCTTGATTTAGCACCCACA 180

Subicho ATGGAAACCCTTCTAAAGCCTTTTCCATCTCCTTTACTTTCCATTCCTACTCCTAACATGTATAGTTTCAAACACAACTCCACTTTTCCAAATCCAACCAAACAAAAAGATTCAAGAAAGTTCCATTATAGAAACAAAAGCAGTACACATTTTTGTAGCTTTCTTGATTTAGCACCCACA 180

Nockwang ATGGAAACCCTTTTAAAGCCTTTTCCATCTCCTTTACTTTCCATTCCTACTCCTAACATGTATAGTTTCAAACACAACTCCACTTTTCCAAATCCAACCAAACAAAAAGATTCAAGAAAGTTCCATTATAGAAACAAAAGCAGTACACATTTTTGTAGCTTTCTTGATTTAGCACCCACA 180

158770 ATGGAAACCCTTCTAAAGCCTTTTCCATCTCCTTTACTTTCCATTCCTACTCCTAACATGTATAGTTTCAAACACAACTCCACTTTTCCAAATCCAACCAAACAAAAAGATTCAAGAAAATTCCATTATAGAAACAAAAGCAGTACACATTTTTGTAGCTTTCTTGATTTAGCACCCACA 180



158773 ATGGAAACCCTTCTAAAGCCTTTTCCATCTCCTTTACTTTCCATTCCTACTCCTAACATGTATAGTTTCAAACACAACTCCACCTTTCCAAATCCAACCAAACAAAGAGATTCAAGAAAGTTCCATTCTAGAAACAAAAGCAGTACACATTTTTGTAGCTTTCTTGATTTAGCACCCACA 180

191655 ATGGAAACCCTTCTAAAGCCTTTTCCATCTCCTTTACTTTCCATTCCTACTCCTAACATGTATAGTTTCAAACACAACTCCACTTTTCCAAATCCAACCAAACAAAGAGATTCAAGAAAGTTCCATTCTAGAAACAAAAGCAGTACACATTTTTGTAGCTTTCTTGATTTAGCACCCACA 180



163499 TCAAAGCCAGAGTCTTTAGATGTTAACATCTCATGGGTTGATACTGATCTGGACCGGGCTGAATTCGACGTGATCATCATTGGAACTGGCCCTGCCGGGCTTCGGCTAGCTGAACAAGTTTCTAAGTATGGTATCAAGGTATGTTGCGTTGACCCTTCACCACTTTCCATGTGGCCAAAT 360

Subicho TCGAAGCCAGAGTCTTTAGATGTTAACATCTCATGGGTTGATACTGATCTGGACCGGGCTGAATTCGACGTGATCATCATTGGAACTGGCCCTGCCGGGCTTCGGCTAGCTGAACAAGTTTCTAAATATGGTATTAAGGTATGTTGCGTTGACCCTTCACCACTTTCCATGTGGCCAAAT 360

Nockwang TCAAAGCCAGAGTCTTTAGATGTTAACATCTCATGGGTTGATACTGATCTGGACCGGGCTGAATTCGACGTGATCATCATTGGAACTGGCCCTGCCGGGCTTCGGCTAGCTGAACAAGTTTCTAAATATGGTATTAAGGTATGTTGCGTTGACCCTTCACCACTTTCCATGTGGCCAAAT 360

158770 TCAAAGCCAGAGTCTTTAGATGTTAACATCTCATGGGTTGATACTGATCTGGACCGGGCTGAATTCGACGTGATCATCATTGGAACTGGCCCTGCCGGGCTTCGGCTAGCTGAACAAGTTTCTAAATATGGTATTAAGGTATGTTGTGTTGACCCTTCACCACTTTCCATGTGGCCAAAT 360


203499 TCAAAGCCAGAGTCTTTAGATGTTAACATCTCATGGGTTGATACTGATCTGGACCGGGCTGAATTCGATGTGATCATCATTGGAACTGGCCCTGCCGGGCTTCGGCTAGCTGAACAAGTTTCTAAATATGGAATTAAGGTATGTTGCGTTGACCCTTCACCACTTTCCATGTGGCCAAAT 360

158773 TCAAAGCCAGAGTCTTTAGATGTTAACATCTCATGGGTTGATACTGATCTGGACCGGGCTGAATTCGACGTGATCATCATTGGAACTGGCCCTGCCGGGCTTCGGCTAGCTGAACAAGTTTCTAAATATGGAATTAAGGTATGTTGCGTTGACCCTTCACCACTTTCCATGTGGCCAAAT 360




163499 AATTATGGTGTTTGGGTTGATGAGTTTGAAAAGTTGGGATTAGAAGATTGTCTAGATCATAAGTGGCCTGTGAGTTGTGTTCATATAAGTGATCACAAGACTAAGTATTTGGACAGACCATATGGTAGAGTAAGTAGAAAGAAGTTGAAGTTGAAATTGTTGAATAGTTGTGTTGAAAAT 540

Subicho AATTATGGTGTTTGGGTTGATGAGTTTGAAAAGTTGGGATTAGAAGATTGTCTGGATCATAAGTGGCCTGTGAGTTGTGTTCATATAAGTGATCACAAGACTAAGTATTTGGACAGACCATATGGTAGAGTAAGTAGAAAGAAGTTGAAGTTGAAATTGTTGAATAGTTGTGTTGAAAAT 540

Nockwang AATTATGGTGTTTGGGTTGATGAGTTTGAAAAGTTGGGATTAGAAGATTGTCTAGATCATAAGTGGCCTGTGAGTTGTGTTCATATAAGTGATCACAAGACTAAGTATTTGGACAGACCATATGGTAGAGTAAGTAGAAAGAAGTTGAAGTTGAAATTGTTGAATAGTTGTGTTGAAAAT 540


158806 AATTATGGTGTTTGGGTTGATGAGTTTGAAAAGTTGGGATTAGAAGATTGTCTAGATCATAAGTGGCCTGTGAGTTGTGTTCATATAAGTGATCACAAGACTAAGTATTTGGACAGACCATATGGTAGAGTAAGTAGAAAGAAGTTGAAGTTGAAATTGTTGAATAGTTGTGTTGGAAAT 540






163499 AGAGTGGAGTTTTATAAAGCCAAGGTTTTGAAAGTGAAGCATGAAGAATTTGAGTCTTCGATTGTTTGTGATGATGGTAGGAAGATAAGTGGTAGCTTGATTGTTGATGCAAGTGGCTATGCTAGTGATTTTATAGAGTATGACAAGCCAAGAAACCATGGTTATCAAGTTGCTCATGGG 720

Subicho AGAGTGAAGTTTTATAAAGCCAAGGTTTTGAAAGTGAAGCATGAAGAATTTGAGTCTTCGATTGTTTGTGATGATGGTAGGAAGATAAGTGGTAGCTTGATTGTTGATGCAAGTGGCTATGCTAGTGATTTTATAGAGTATGACAAGCCAAGAAACCATGGTTATCAAGTTGCTCATGGG 720

Nockwang AGAGTGAAGTTTTATAAAGCCAAGGTTTTGAAAGTGAAGCATGAAGAATTTGAGTCTTCGATTGTTTGTGATGATGGTAGGAAGATAAGTGGTAGCTTGATTGTTGATGCAAGTGGCTATGCTAGTGATTTTATAGAGTATGACAAGCCAAGAAACCATGGTTATCAAGTTGCTCATGGG 720

158770 AGAGTGAAGTTTTATAAAGCCAAGGTTTTGAAAGTGAAGCATGAAGAATTTGAGTCTTCGATTGTTTGTGATGATGGTAGGAAGATAAGTGGTAGCTTGATTGTTGATGCAAGTGGCTATGCTAGTGATTTTATAGAGTATGACAAGCCAAGAAACCATGGTTATCAAGTTGCTCATGGG 720





164918 AGAGTGAAGTTTTATAAAGCCAAGGTTTTGAAAGTGAAGCATGAAGAATTTGAGTCTTAGATTGTTTGTGATGATGGTAGGAAGATAAGTGGTAGCTTGATTGTTGATGCAAGTGGCTATGCTAGTGATTTTATAGAGTATGACAAGCCAAGAAACCATGGTTATCAAGTTGCTCATGGG 720


163499 ATTTTAGCAGAAGTTGATAATCATCCATTTGATTTGGATAAAATGATGCTTATGGATTGGAGGGATTCTCATTTAGGTAATGAGCCATATCTGAGGGTGAAGAATACTAAAGAACCAACATTCTTGTATGCAATGCCATTTGATAGGAATTTGGTATTCTTGGAAGAGACTTCTTTAGTG 900

Subicho ATTTTAGCAGAAGTTGATAATCATCCATTTGATTTGGATAAAATGATGCTTATGGATTGGAGGGATTCTCATTTAGGTAATGAGCCATATCTGAGGGTGAAGAATACTAAAGAACCAACATTCTTGTATGCAATGCCATTTGATAGGAATTTGGTATTCTTGGAAGAGACTTCTTTAGTG 900

Nockwang ATTTTAGCAGAAGTTGATAATCATCCATTTGATTTGGATAAAATGATGCTTATGGATTGGAGGGATTCTCATTTAGGTAATGAGCCATATCTGAGGGTGAAGAATACTAAAGAACCAACATTCTTGTATGCAATGCCATTTGATAGGAATTTGGTATTCTTGGAAGAGACTTCTTTAGTG 900

158770 ATTTTAGCAGAAGTTGATAATCATCCATTTGATTTGGATAAAATGATGCTTATGGATTGGAGGGATTCTCATTTAGGTAATGAGCCATATCTGAGGGTGAAGAATACTAAAGAACCAACATTCTTGTGTGCAATGCCATTTGATAGGAATTTGGTATTCTTGGAAGAGACTTCTTTAGTG 900

158806 ATTTTAGCAGAAGTTGATAATCATCCATTTGATTTGGATAAAATGATGCTTATGGATTGGAGGGATTCTCATTTAGGTAATGAGCCATATCTAAGGGTGAAGAATACTAAAGAACCAACATTCTTGTATGCAATGCCATTTGATAGGAATTTGGTATTCTTGGAAGAGACTTCTTTAGTG 900

203499 ATTTTAGCAGAAGTTGATAATCATCCATTTGATTTGGATAAAATGATGCTTATGGATTGGAGGGATTCTCATTTAGGTAATGAGCCATATCTGAGGGTGAAGAATACTAAAGAACCAACATTCTTGTATGCAATGCCATTTGATAAGAATTTGGTATTCTTGGAAGAGACTTCTTTAGTG 900





163499 AGTCGGCCTATGTTATCGTATATGGAAGTGAAAAGAAGGATGGTAGCAAGATTAAGACATTTGGGGATCAAAGTGAGAAGTGTCCTTGAGGAAGAGAAGTGTGTGATCACTATGGGAGGACCACTTCCGCGGATTCCTCAAAATGTTATGGCTATTGGTGGGACTTCAGGGATAGTTCAT 1080

Subicho AGTCGGCCTATGTTATCGTATATGGAAGTGAAAAGAAGGATGGTAGCAAGATTAAGACATTTGGGGATCAAAGTGAGAAGTGTCCTTGAGGAAGAGAAGTGTGTGATCACTATGGGAGGACCACTTCCGCGGATTCCTCAAAATGTTATGGCTATTGGTGGGACTTCAGGGATAGTTCAT 1080

Nockwang AGTCGGCCTATGTTATCGTATATGGAAGTGAAAAGAAGGATGGTAGCAAGATTAAGACATTTGGGGATCAAAGTGAGAAGTGTCCTTGAGGAAGAGAAGTGTGTGATCACTATGGGAGGACCACTTCCGCGGATTCCTCAAAATGTTATGGCTATTGGTGGGACTTCAGGGATAGTTCAT 1080

158770 AGTCGGCCTATGTTATCGTATATGGAAGTGAAAAGAAGGATGGTAGCAAGATTAAGACATTTGGGGATCAAAGTGAGAAGTGTCCTTGAGGAAGAGAAGTGTGTGATCCCTATGGGAGGACCACTTCCGCGGATTCCTCAAAATGTTATGGCTATTGGTGGGACTTCAGGGATAGTTCAT 1080





164918 AGTCGGCCTACGTTATCGTATATGGAAGTGAAAAGAAGGATGGTAGCAAGATTAAGACATTTGGGGATCAAAGTGAGAAGTGTCCCTGAGGAAGAGAAGTGTGTGATCACTATGGGAGGACCACTTCCGCGGATTCCTCAAAATGTTATGGCTATTGGTGGGACTTCAGGGATAGTTCAT 1080

800065 AGTCGGCCTATGTTATCGTATATGGAAGTGAAAAGAAGGATGGTAGCAAGATTAAGACATTTGGGGATCAAAGTGAGAAGTGTCCTTGAGGAAGAGGAGTGTGTGATCACTATGGGAGGACCACTTCCGCGGATTCCTCAAAATGTTATGGCTATTGGTGGGACTTCAGGGATAGTTCAT 1080

163499 CCATCGTCTGGGTACATGGTGGCTCGTAGCATGGCATTGGCACCAGTACTGGCTGAGGCCATCGTCGAAAGCCTTGGCTCAACAAGAATGATAAGAGGGTCTCAACTTTACCATAGAGTTTGGAATGGTTTGTGGCCTTCGGATAGAAGACGTGTTAGAGAATGTTATTGTTTCGGAATG 1260

Subicho CCATCGTCTGGGTACATGGTGGCTCGTAGCATGGCATTGGCACCAGTACTGGCTGAGGCCATCGTCGAAAGCCTTGGCTCAACAAGAATGATAAGAGGGTCTCAACTTTACCATAGAGTTTGGAATGGTTTGTGGCCTTCGGATAGAAGACGTGTTAGAGAATGTTATTGTTTCGGAATG 1260

Nockwang CCATCGTCTGGGTACATGGTGGCTCGTAGCATGGCATTGGCACCAGTACTGGCTGAGGCCATCGTCGAAAGCCTTGGCTCAACAAGAATGATAAGAGGGTCTCAACTTTACCATAGAGTTTGGAATGGTTTGTGGCCTTCGGATAGAAGACGTGTTAGAGAATGTTATTGTTTCGGAATG 1260

158770 CCATCGTCTGGGTACATGGTGGCTCGTAGCATGGCATTGGCACCAGTACTGGCTGAGGCCATCGTCGAAAGCCTTGGCTCAACAAGAATGATAAGAGGGTCTCAACTTTACCATAGAGTTTGGAGTGGTTTGTGGCCTTCGGATAGAAGACGTGTTAGAGAATGTTATTGTTTCGGAATG 1260



158773 CCATCGTCCGGGTACATGGTGGCTCGTAGCATGGCATTGGCACCAGTACTGGCTGAGGCCATCGTCGAAAGCCTTGGCTCAACAAGAATGATAAGAGGGTCTCAACTTTACCATAGAGTTTGGAATGGTTTGTGGCCTTCGGATAGAAGACGTGTTAGAGAATGTTATTGTTTCGGAATG 1260




163499 GAGACTTTGTTGAAGCTTGATTTGGAAGGTACTAGGAGATTGTTTGATGCTTTCTTTGATGTTGATCCCAAGTACTGGCACGGGTTCCTTTCTTCAAGATTGTCTGCCAAAGAACTTGCTGTACTCAGTTTGTACCTTTTTGGACATGCCTCTAATTTGGCTAGGTTGGA--------TA 1432

Subicho GAGACTTTGTTGAAGCTTGATTTGGAAGGTACTAGGAGATTGTTTGATGCTTTCTTTGATGTTGATCCCAAGTACTGGCACGGGTTCCTTTCTTCAAGATTGTCTGTCAAAGAACTTGCTGTACTCAGTTTGTACCTTTTTGGACATGCCTCTAATTTGGCTAGGTTGGA--------TA 1432

Nockwang GAGACTTTGTTGAAGCTTGATTTGGAAGGTACTAGGAGATTGTTTGATGCTTTCTTTGATGTTGATCCCAAGTACTGGCACGGGTTCCTTTCTTCAAGATTGTCTGTCAAAGAACTTGCTGTACTCAGTTTGTACCTTTTTGGACATGCCTCTAATTTGGCTAGGTTGGA--------TA 1432

158770 GAGACTTTGTTGAAGCTTGATTTGGAAGGTAGTAGGAGATTGTTTGATGCTTTCTTTGATGTTGATCCCAAGTACTGGCACGGGTTCCTTTCTTCAAGATTGTCTGTTAAAGAACTTGCTGTACTCAGTTTGTACCTTTTTGGACGTGCCTCTAATTTGGCTAGGTTGGA--------TA 1432

158806 GAGACTTTGTTGAAGCTTGATTTGGAAGGTAGTAGGAGATTGTTTGATGCTTTCTTTGATGTTGATCCCAAGTACTGGCACGGGTTCCTTTCTTCAAGATTGTCTGTCAAAGAACTTGCTGTACTCAGTTTGTACCTTTTTGGACGTGCCTCTAATTTGGCTAGGTTGGA--------TA 1432

203499 GAGACTTTGTTGAAGCTTGATTTGGAAGGTACTAGGAGATTGTTTGATGCTTTCTTTGATGTTGATCCCAAGTACTGGCACGGGTTCCTTTCTTCAAGATTGTCTGTCAAAGAACTTGCTGTACTCAGTTTGTACCTTTTTGGACATGCCTCTAATTTGGCTAGGTTGGA--------TA 1432

158773 GAGACTTTGTTGAAGCTTGATTTGGAAGGTACCAGGAGATTGTTTGATGCTTTCTTTGATGTTGATCCCAAGTACTGGCACGGGTTCCTTTCTTCAAGATTGTCTGTCAAAGAACTTGCTGTACTCAGTTTGTACCTTTTTGGACATGCCTCTAATTTGGCTAGGTTGGA--------TA 1432



800065 GAGACTTTGTTGAAGCTTGATTTGGAAGGTACCAGGAGATTGTTTGATGCTTTCTTTGATGTTGATCCCAAGTACTGGCACGGGTTCCTTTCTTCAAGATTGTCTGTCAAAGAACTTGCTGTACTCAGTTTGTACCTTTTTGGACATGCCTCTAATTTGGCTAGGTTGGAATGTTGGATA 1440

163499 TTGTTACAAAGTGCACTGTCCCCTTGGTTAAACTGCTGGGCAATCTAGCAATAGAGAGCCTTTGA 1497

Subicho TTGTTACAAAGTGCACTGTCCCCTTGGTTAAACTGCTGGGCAATCTAGCAATAGAGAGCCTTTGA 1497

Nockwang TTGTTACAAAGTGCACTGTCCCCTTGGTTAAACTGCTGGGCAATCTAGCAATAGAGAGCCTTTGA 1497

158770 TTGTTACAAAGAGCACTGTCCCCTTGGTTAAACTGCTGGGCAATCTAGCAATAGAGAGCCTTTGA 1497








Subicho ATGGAAACCCTTCTAAAGCCTTTTCCATCTCCTTTACTTTCCATTCCTACTCCTAACATGTATAGTTTCAAACACAACTCCACTTTTCCAAATCCAACCAAACAAAAAGATTCAAGAAAGTTCCATTATAGAAACAAAAGCAGTACACATTTTTGTAGCTTTCTTGATTTAGCACCCACA 180

Nockwang ATGGAAACCCTTTTAAAGCCTTTTCCATCTCCTTTACTTTCCATTCCTACTCCTAACATGTATAGTTTCAAACACAACTCCACTTTTCCAAATCCAACCAAACAAAAAGATTCAAGAAAGTTCCATTATAGAAACAAAAGCAGTACACATTTTTGTAGCTTTCTTGATTTAGCACCCACA 180




158773 ATGGAAACCCTTCTAAAGCCTTTTCCATCTCCTTTACTTTCCATTCCTACTCCTAACATGTATAGTTTCAAACACAACTCCACCTTTCCAAATCCAACCAAACAAAGAGATTCAAGAAAGTTCCATTCTAGAAACAAAAGCAGTACACATTTTTGTAGCTTTCTTGATTTAGCACCCACA 180




163499 TCAAAGCCAGAGTCTTTAGATGTTAACATCTCATGGGTTGATACTGATCTGGACCGGGCTGAATTCGACGTGATCATCATTGGAACTGGCCCTGCCGGGCTTCGGCTAGCTGAACAAGTTTCTAAGTATGGTATCAAGGTATGTTGCGTTGACCCTTCACCACTTTCCATGTGGCCAAAT 360

Subicho TCGAAGCCAGAGTCTTTAGATGTTAACATCTCATGGGTTGATACTGATCTGGACCGGGCTGAATTCGACGTGATCATCATTGGAACTGGCCCTGCCGGGCTTCGGCTAGCTGAACAAGTTTCTAAATATGGTATTAAGGTATGTTGCGTTGACCCTTCACCACTTTCCATGTGGCCAAAT 360

Nockwang TCAAAGCCAGAGTCTTTAGATGTTAACATCTCATGGGTTGATACTGATCTGGACCGGGCTGAATTCGACGTGATCATCATTGGAACTGGCCCTGCCGGGCTTCGGCTAGCTGAACAAGTTTCTAAATATGGTATTAAGGTATGTTGCGTTGACCCTTCACCACTTTCCATGTGGCCAAAT 360



203499 TCAAAGCCAGAGTCTTTAGATGTTAACATCTCATGGGTTGATACTGATCTGGACCGGGCTGAATTCGATGTGATCATCATTGGAACTGGCCCTGCCGGGCTTCGGCTAGCTGAACAAGTTTCTAAATATGGAATTAAGGTATGTTGCGTTGACCCTTCACCACTTTCCATGTGGCCAAAT 360






Subicho AATTATGGTGTTTGGGTTGATGAGTTTGAAAAGTTGGGATTAGAAGATTGTCTGGATCATAAGTGGCCTGTGAGTTGTGTTCATATAAGTGATCACAAGACTAAGTATTTGGACAGACCATATGGTAGAGTAAGTAGAAAGAAGTTGAAGTTGAAATTGTTGAATAGTTGTGTTGAAAAT 540

Nockwang AATTATGGTGTTTGGGTTGATGAGTTTGAAAAGTTGGGATTAGAAGATTGTCTAGATCATAAGTGGCCTGTGAGTTGTGTTCATATAAGTGATCACAAGACTAAGTATTTGGACAGACCATATGGTAGAGTAAGTAGAAAGAAGTTGAAGTTGAAATTGTTGAATAGTTGTGTTGAAAAT 540


158806 AATTATGGTGTTTGGGTTGATGAGTTTGAAAAGTTGGGATTAGAAGATTGTCTAGATCATAAGTGGCCTGTGAGTTGTGTTCATATAAGTGATCACAAGACTAAGTATTTGGACAGACCATATGGTAGAGTAAGTAGAAAGAAGTTGAAGTTGAAATTGTTGAATAGTTGTGTTGGAAAT 540






163499 AGAGTGGAGTTTTATAAAGCCAAGGTTTTGAAAGTGAAGCATGAAGAATTTGAGTCTTCGATTGTTTGTGATGATGGTAGGAAGATAAGTGGTAGCTTGATTGTTGATGCAAGTGGCTATGCTAGTGATTTTATAGAGTATGACAAGCCAAGAAACCATGGTTATCAAGTTGCTCATGGG 720

Subicho AGAGTGAAGTTTTATAAAGCCAAGGTTTTGAAAGTGAAGCATGAAGAATTTGAGTCTTCGATTGTTTGTGATGATGGTAGGAAGATAAGTGGTAGCTTGATTGTTGATGCAAGTGGCTATGCTAGTGATTTTATAGAGTATGACAAGCCAAGAAACCATGGTTATCAAGTTGCTCATGGG 720

Nockwang AGAGTGAAGTTTTATAAAGCCAAGGTTTTGAAAGTGAAGCATGAAGAATTTGAGTCTTCGATTGTTTGTGATGATGGTAGGAAGATAAGTGGTAGCTTGATTGTTGATGCAAGTGGCTATGCTAGTGATTTTATAGAGTATGACAAGCCAAGAAACCATGGTTATCAAGTTGCTCATGGG 720






164918 AGAGTGAAGTTTTATAAAGCCAAGGTTTTGAAAGTGAAGCATGAAGAATTTGAGTCTTAGATTGTTTGTGATGATGGTAGGAAGATAAGTGGTAGCTTGATTGTTGATGCAAGTGGCTATGCTAGTGATTTTATAGAGTATGACAAGCCAAGAAACCATGGTTATCAAGTTGCTCATGGG 720



Subicho ATTTTAGCAGAAGTTGATAATCATCCATTTGATTTGGATAAAATGATGCTTATGGATTGGAGGGATTCTCATTTAGGTAATGAGCCATATCTGAGGGTGAAGAATACTAAAGAACCAACATTCTTGTATGCAATGCCATTTGATAGGAATTTGGTATTCTTGGAAGAGACTTCTTTAGTG 900

Nockwang ATTTTAGCAGAAGTTGATAATCATCCATTTGATTTGGATAAAATGATGCTTATGGATTGGAGGGATTCTCATTTAGGTAATGAGCCATATCTGAGGGTGAAGAATACTAAAGAACCAACATTCTTGTATGCAATGCCATTTGATAGGAATTTGGTATTCTTGGAAGAGACTTCTTTAGTG 900

158770 ATTTTAGCAGAAGTTGATAATCATCCATTTGATTTGGATAAAATGATGCTTATGGATTGGAGGGATTCTCATTTAGGTAATGAGCCATATCTGAGGGTGAAGAATACTAAAGAACCAACATTCTTGTGTGCAATGCCATTTGATAGGAATTTGGTATTCTTGGAAGAGACTTCTTTAGTG 900

158806 ATTTTAGCAGAAGTTGATAATCATCCATTTGATTTGGATAAAATGATGCTTATGGATTGGAGGGATTCTCATTTAGGTAATGAGCCATATCTAAGGGTGAAGAATACTAAAGAACCAACATTCTTGTATGCAATGCCATTTGATAGGAATTTGGTATTCTTGGAAGAGACTTCTTTAGTG 900

203499 ATTTTAGCAGAAGTTGATAATCATCCATTTGATTTGGATAAAATGATGCTTATGGATTGGAGGGATTCTCATTTAGGTAATGAGCCATATCTGAGGGTGAAGAATACTAAAGAACCAACATTCTTGTATGCAATGCCATTTGATAAGAATTTGGTATTCTTGGAAGAGACTTCTTTAGTG 900






Subicho AGTCGGCCTATGTTATCGTATATGGAAGTGAAAAGAAGGATGGTAGCAAGATTAAGACATTTGGGGATCAAAGTGAGAAGTGTCCTTGAGGAAGAGAAGTGTGTGATCACTATGGGAGGACCACTTCCGCGGATTCCTCAAAATGTTATGGCTATTGGTGGGACTTCAGGGATAGTTCAT 1080

Nockwang AGTCGGCCTATGTTATCGTATATGGAAGTGAAAAGAAGGATGGTAGCAAGATTAAGACATTTGGGGATCAAAGTGAGAAGTGTCCTTGAGGAAGAGAAGTGTGTGATCACTATGGGAGGACCACTTCCGCGGATTCCTCAAAATGTTATGGCTATTGGTGGGACTTCAGGGATAGTTCAT 1080






164918 AGTCGGCCTACGTTATCGTATATGGAAGTGAAAAGAAGGATGGTAGCAAGATTAAGACATTTGGGGATCAAAGTGAGAAGTGTCCCTGAGGAAGAGAAGTGTGTGATCACTATGGGAGGACCACTTCCGCGGATTCCTCAAAATGTTATGGCTATTGGTGGGACTTCAGGGATAGTTCAT 1080

800065 AGTCGGCCTATGTTATCGTATATGGAAGTGAAAAGAAGGATGGTAGCAAGATTAAGACATTTGGGGATCAAAGTGAGAAGTGTCCTTGAGGAAGAGGAGTGTGTGATCACTATGGGAGGACCACTTCCGCGGATTCCTCAAAATGTTATGGCTATTGGTGGGACTTCAGGGATAGTTCAT 1080


Subicho CCATCGTCTGGGTACATGGTGGCTCGTAGCATGGCATTGGCACCAGTACTGGCTGAGGCCATCGTCGAAAGCCTTGGCTCAACAAGAATGATAAGAGGGTCTCAACTTTACCATAGAGTTTGGAATGGTTTGTGGCCTTCGGATAGAAGACGTGTTAGAGAATGTTATTGTTTCGGAATG 1260

Nockwang CCATCGTCTGGGTACATGGTGGCTCGTAGCATGGCATTGGCACCAGTACTGGCTGAGGCCATCGTCGAAAGCCTTGGCTCAACAAGAATGATAAGAGGGTCTCAACTTTACCATAGAGTTTGGAATGGTTTGTGGCCTTCGGATAGAAGACGTGTTAGAGAATGTTATTGTTTCGGAATG 1260

158770 CCATCGTCTGGGTACATGGTGGCTCGTAGCATGGCATTGGCACCAGTACTGGCTGAGGCCATCGTCGAAAGCCTTGGCTCAACAAGAATGATAAGAGGGTCTCAACTTTACCATAGAGTTTGGAGTGGTTTGTGGCCTTCGGATAGAAGACGTGTTAGAGAATGTTATTGTTTCGGAATG 1260







163499 GAGACTTTGTTGAAGCTTGATTTGGAAGGTACTAGGAGATTGTTTGATGCTTTCTTTGATGTTGATCCCAAGTACTGGCACGGGTTCCTTTCTTCAAGATTGTCTGCCAAAGAACTTGCTGTACTCAGTTTGTACCTTTTTGGACATGCCTCTAATTTGGCTAGGTTGGA--------TA 1432

Subicho GAGACTTTGTTGAAGCTTGATTTGGAAGGTACTAGGAGATTGTTTGATGCTTTCTTTGATGTTGATCCCAAGTACTGGCACGGGTTCCTTTCTTCAAGATTGTCTGTCAAAGAACTTGCTGTACTCAGTTTGTACCTTTTTGGACATGCCTCTAATTTGGCTAGGTTGGA--------TA 1432

Nockwang GAGACTTTGTTGAAGCTTGATTTGGAAGGTACTAGGAGATTGTTTGATGCTTTCTTTGATGTTGATCCCAAGTACTGGCACGGGTTCCTTTCTTCAAGATTGTCTGTCAAAGAACTTGCTGTACTCAGTTTGTACCTTTTTGGACATGCCTCTAATTTGGCTAGGTTGGA--------TA 1432

158770 GAGACTTTGTTGAAGCTTGATTTGGAAGGTAGTAGGAGATTGTTTGATGCTTTCTTTGATGTTGATCCCAAGTACTGGCACGGGTTCCTTTCTTCAAGATTGTCTGTTAAAGAACTTGCTGTACTCAGTTTGTACCTTTTTGGACGTGCCTCTAATTTGGCTAGGTTGGA--------TA 1432

158806 GAGACTTTGTTGAAGCTTGATTTGGAAGGTAGTAGGAGATTGTTTGATGCTTTCTTTGATGTTGATCCCAAGTACTGGCACGGGTTCCTTTCTTCAAGATTGTCTGTCAAAGAACTTGCTGTACTCAGTTTGTACCTTTTTGGACGTGCCTCTAATTTGGCTAGGTTGGA--------TA 1432

203499 GAGACTTTGTTGAAGCTTGATTTGGAAGGTACTAGGAGATTGTTTGATGCTTTCTTTGATGTTGATCCCAAGTACTGGCACGGGTTCCTTTCTTCAAGATTGTCTGTCAAAGAACTTGCTGTACTCAGTTTGTACCTTTTTGGACATGCCTCTAATTTGGCTAGGTTGGA--------TA 1432




800065 GAGACTTTGTTGAAGCTTGATTTGGAAGGTACCAGGAGATTGTTTGATGCTTTCTTTGATGTTGATCCCAAGTACTGGCACGGGTTCCTTTCTTCAAGATTGTCTGTCAAAGAACTTGCTGTACTCAGTTTGTACCTTTTTGGACATGCCTCTAATTTGGCTAGGTTGGAATGTTGGATA 1440


Subicho TTGTTACAAAGTGCACTGTCCCCTTGGTTAAACTGCTGGGCAATCTAGCAATAGAGAGCCTTTGA 1497

Nockwang TTGTTACAAAGTGCACTGTCCCCTTGGTTAAACTGCTGGGCAATCTAGCAATAGAGAGCCTTTGA 1497








163499 METLLKPFPSPLLSIPTPNMYSFKHNSTFPNPTKQKDSRKFHYRNKSSTHFCSFLDLAPT 60

Subicho METLLKPFPSPLLSIPTPNMYSFKHNSTFPNPTKQKDSRKFHYRNKSSTHFCSFLDLAPT 60

Nockwang METLLKPFPSPLLSIPTPNMYSFKHNSTFPNPTKQKDSRKFHYRNKSSTHFCSFLDLAPT 60




158773 METLLKPFPSPLLSIPTPNMYSFKHNSTFPNPTKQRDSRKFHSRNKSSTHFCSFLDLAPT 60




163499 SKPESLDVNISWVDTDLDRAEFDVIIIGTGPAGLRLAEQVSKYGIKVCCVDPSPLSMWPN 120

Subicho SKPESLDVNISWVDTDLDRAEFDVIIIGTGPAGLRLAEQVSKYGIKVCCVDPSPLSMWPN 120

Nockwang SKPESLDVNISWVDTDLDRAEFDVIIIGTGPAGLRLAEQVSKYGIKVCCVDPSPLSMWPN 120








163499 NYGVWVDEFEKLGLEDCLDHKWPVSCVHISDHKTKYLDRPYGRVSRKKLKLKLLNSCVEN 180

Subicho NYGVWVDEFEKLGLEDCLDHKWPVSCVHISDHKTKYLDRPYGRVSRKKLKLKLLNSCVEN 180

Nockwang NYGVWVDEFEKLGLEDCLDHKWPVSCVHISDHKTKYLDRPYGRVSRKKLKLKLLNSCVEN 180


158806 NYGVWVDEFEKLGLEDCLDHKWPVSCVHISDHKTKYLDRPYGRVSRKKLKLKLLNSCVGN 180






163499 RVEFYKAKVLKVKHEEFESSIVCDDGRKISGSLIVDASGYASDFIEYDKPRNHGYQVAHG 240

Subicho RVKFYKAKVLKVKHEEFESSIVCDDGRKISGSLIVDASGYASDFIEYDKPRNHGYQVAHG 240

Nockwang RVKFYKAKVLKVKHEEFESSIVCDDGRKISGSLIVDASGYASDFIEYDKPRNHGYQVAHG 240

158770 RVKFYKAKVLKVKHEEFESSIVCDDGRKISGSLIVDASGYASDFIEYDKPRNHGYQVAHG 240





164918 RVKFYKAKVLKVKHEEFES 199


163499 ILAEVDNHPFDLDKMMLMDWRDSHLGNEPYLRVKNTKEPTFLYAMPFDRNLVFLEETSLV 300

Subicho ILAEVDNHPFDLDKMMLMDWRDSHLGNEPYLRVKNTKEPTFLYAMPFDRNLVFLEETSLV 300

Nockwang ILAEVDNHPFDLDKMMLMDWRDSHLGNEPYLRVKNTKEPTFLYAMPFDRNLVFLEETSLV 300

158770 ILAEVDNHPFDLDKMMLMDWRDSHLGNEPYLRVKNTKEPTFLCAMPFDRNLVFLEETSLV 300


203499 ILAEVDNHPFDLDKMMLMDWRDSHLGNEPYLRVKNTKEPTFLYAMPFDKNLVFLEETSLV 300



164918


163499 SRPMLSYMEVKRRMVARLRHLGIKVRSVLEEEKCVITMGGPLPRIPQNVMAIGGTSGIVH 360

Subicho SRPMLSYMEVKRRMVARLRHLGIKVRSVLEEEKCVITMGGPLPRIPQNVMAIGGTSGIVH 360

Nockwang SRPMLSYMEVKRRMVARLRHLGIKVRSVLEEEKCVITMGGPLPRIPQNVMAIGGTSGIVH 360

158770 SRPMLSYMEVKRRMVARLRHLGIKVRSVLEEEKCVIPMGGPLPRIPQNVMAIGGTSGIVH 360





164918

800065 SRPMLSYMEVKRRMVARLRHLGIKVRSVLEEEECVITMGGPLPRIPQNVMAIGGTSGIVH 360

163499 PSSGYMVARSMALAPVLAEAIVESLGSTRMIRGSQLYHRVWNGLWPSDRRRVRECYCFGM 420

Subicho PSSGYMVARSMALAPVLAEAIVESLGSTRMIRGSQLYHRVWNGLWPSDRRRVRECYCFGM 420

Nockwang PSSGYMVARSMALAPVLAEAIVESLGSTRMIRGSQLYHRVWNGLWPSDRRRVRECYCFGM 420

158770 PSSGYMVARSMALAPVLAEAIVESLGSTRMIRGSQLYHRVWSGLWPSDRRRVRECYCFGM 420





164918


163499 ETLLKLDLEGTRRLFDAFFDVDPKYWHGFLSSRLSAKELAVLSLYLFGHASNLARLDIVT 480

Subicho ETLLKLDLEGTRRLFDAFFDVDPKYWHGFLSSRLSVKELAVLSLYLFGHASNLARLDIVT 480

Nockwang ETLLKLDLEGTRRLFDAFFDVDPKYWHGFLSSRLSVKELAVLSLYLFGHASNLARLDIVT 480

158770 ETLLKLDLEGSRRLFDAFFDVDPKYWHGFLSSRLSVKELAVLSLYLFGRASNLARLDIVT 480


203499 ETLLKLDLEGTRRLFDAFFDVDPKYWHGFLSSRLSVKELAVLSLYLFGHASNLARLDIVT 480



164918

800065 ETLLKLDLEGTRRLFDAFFDVDPKYWHGFLSSRLSVKELAVLSLYLFGHASNLARLECWI 480

163499 KCTVPLVKLLGNLAIESL 498

Subicho KCTVPLVKLLGNLAIESL 498

Nockwang KCTVPLVKLLGNLAIESL 498

158770 KSTVPLVKLLGNLAIESL 498





164918

800065 LLQSALSPWLNCWAI 495


Subicho METLLKPFPSPLLSIPTPNMYSFKHNSTFPNPTKQKDSRKFHYRNKSSTHFCSFLDLAPT 60

Nockwang METLLKPFPSPLLSIPTPNMYSFKHNSTFPNPTKQKDSRKFHYRNKSSTHFCSFLDLAPT 60









Subicho SKPESLDVNISWVDTDLDRAEFDVIIIGTGPAGLRLAEQVSKYGIKVCCVDPSPLSMWPN 120

Nockwang SKPESLDVNISWVDTDLDRAEFDVIIIGTGPAGLRLAEQVSKYGIKVCCVDPSPLSMWPN 120









Subicho NYGVWVDEFEKLGLEDCLDHKWPVSCVHISDHKTKYLDRPYGRVSRKKLKLKLLNSCVEN 180

Nockwang NYGVWVDEFEKLGLEDCLDHKWPVSCVHISDHKTKYLDRPYGRVSRKKLKLKLLNSCVEN 180


158806 NYGVWVDEFEKLGLEDCLDHKWPVSCVHISDHKTKYLDRPYGRVSRKKLKLKLLNSCVGN 180






163499 RVEFYKAKVLKVKHEEFESSIVCDDGRKISGSLIVDASGYASDFIEYDKPRNHGYQVAHG 240

Subicho RVKFYKAKVLKVKHEEFESSIVCDDGRKISGSLIVDASGYASDFIEYDKPRNHGYQVAHG 240

Nockwang RVKFYKAKVLKVKHEEFESSIVCDDGRKISGSLIVDASGYASDFIEYDKPRNHGYQVAHG 240






164918 RVKFYKAKVLKVKHEEFES 199



Subicho ILAEVDNHPFDLDKMMLMDWRDSHLGNEPYLRVKNTKEPTFLYAMPFDRNLVFLEETSLV 300

Nockwang ILAEVDNHPFDLDKMMLMDWRDSHLGNEPYLRVKNTKEPTFLYAMPFDRNLVFLEETSLV 300

158770 ILAEVDNHPFDLDKMMLMDWRDSHLGNEPYLRVKNTKEPTFLCAMPFDRNLVFLEETSLV 300


203499 ILAEVDNHPFDLDKMMLMDWRDSHLGNEPYLRVKNTKEPTFLYAMPFDKNLVFLEETSLV 300



164918



Subicho SRPMLSYMEVKRRMVARLRHLGIKVRSVLEEEKCVITMGGPLPRIPQNVMAIGGTSGIVH 360

Nockwang SRPMLSYMEVKRRMVARLRHLGIKVRSVLEEEKCVITMGGPLPRIPQNVMAIGGTSGIVH 360






164918

800065 SRPMLSYMEVKRRMVARLRHLGIKVRSVLEEEECVITMGGPLPRIPQNVMAIGGTSGIVH 360


Subicho PSSGYMVARSMALAPVLAEAIVESLGSTRMIRGSQLYHRVWNGLWPSDRRRVRECYCFGM 420

Nockwang PSSGYMVARSMALAPVLAEAIVESLGSTRMIRGSQLYHRVWNGLWPSDRRRVRECYCFGM 420

158770 PSSGYMVARSMALAPVLAEAIVESLGSTRMIRGSQLYHRVWSGLWPSDRRRVRECYCFGM 420





164918


163499 ETLLKLDLEGTRRLFDAFFDVDPKYWHGFLSSRLSAKELAVLSLYLFGHASNLARLDIVT 480

Subicho ETLLKLDLEGTRRLFDAFFDVDPKYWHGFLSSRLSVKELAVLSLYLFGHASNLARLDIVT 480

Nockwang ETLLKLDLEGTRRLFDAFFDVDPKYWHGFLSSRLSVKELAVLSLYLFGHASNLARLDIVT 480






164918

800065 ETLLKLDLEGTRRLFDAFFDVDPKYWHGFLSSRLSVKELAVLSLYLFGHASNLARLECWI 480


Subicho KCTVPLVKLLGNLAIESL 498

Nockwang KCTVPLVKLLGNLAIESL 498






164918

800065 LLQSALSPWLNCWAI 495

B.

A.

C. D.

Premature stop codon frame shift

1 bp change

8 bp insertion

early translation-terminationSupplementary Fig. 3

Supplementary Fig. 3. Multiple alignments of the nucleotide (A and B) and predicted amino acid (C and D)

sequences of the Ccs coding genes among the indicated Capsicum varieties. Two decisive structural mutations in

the two Y-pepper varieties 164918 (Y3) and 800065 (Y2) are enclosed in blue boxes.

a comparison of the carotenoid accumulation in capsicum varieties that show different ripening...

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