A commercial cultivar C. annuum cv. “Sonia Gold” was used as non-red fruit color pepper cultivar. “Sonia Gold” is a bell (blocky) type sweet pepper with yellow fruit color that is commonly cultivated as vegetable (Figure 1(a)). As a comparison, a corn type red fruit hot pepper C. annuum cv. “Takanotsume” that is a traditional cultivar used as spice in Japan was used (Figure 1(b)). As for genotyping, each of the 16 cultivars of C. annuum and C. chinense were used (Table 1). The total DNA was extracted from leaves of each of the accessions with CTAB method [23].

Based on the genome sequence of C. annuum, six forward primers (Ccs5up_F-762, F-1671, F-2393, F-2745, F-3080, F-3784) were designed at 300 - 900 bp intervals in the 5' untranslated region of the Ccs gene. The numbers following F- in the primer names indicates the upstream position when the first nucleotide of the exon is defined as 1. Using the six forward primers with a fixed reverse primer Ccs_R+1494, diagnostic PCR was carried out using Tks Gflex DNA polymerase (Takara Bio) in 20 μL reaction volume with 40 ng genomic DNA as template. The condition of PCR amplification was 1 cycle at 94˚C for 1 min, 30 cycles at

^aThe materials segregated from commercial variety and maintained in our laboratory.

94˚C for 30 sec, 60˚C for 30 sec and 72˚C for 5 min, followed by a final extension at 72˚C for 3 min. Amplification was checked by electrophoresis in 1% agarose gel with 1 × TAE buffer. The primers used were listed in Table 2.

Using the normal allele specific and the large deletion specific forward primers (Ccs5del F1 and Ccs5del F2, respectively) with a fixed reverse primer (Ccs5del R) developed in this study, multiplex PCR was conducted for each of the 16 cultivars of C. annuum and C. chinense. PCR condition was the same as diagnostic PCR described above, except that the extension time was changed to 30 sec.

^aNames in the parenthesis are used in Lang et al. (2004) [13]. ^bThe artificial mismatch site to create restriction site is shown in bold and underlined.

To distinguish ccs-3 that is another mutated allele of Ccs rediscovered in this study, a dCAPS marker was developed. At first the region including single base indel polymorphic site at the 3' end was amplified by PCR using primers ccs3dCAPS F and R developed in this study. Reverse primer contains an artificial SNP changing A to G at the second nucleotide position from 3' end to create a recognition site of restriction enzyme NdeII (GATC). PCR was conducted in 20 μL volume with 40 ng template DNA. The condition of PCR amplification was 1 cycle at 94˚C for 1 min, 30 cycles at 94˚C for 30 sec, 60˚C for 30 sec and 72˚C for 20 sec, followed by a final extension at 72˚C for 3 min. Takara Taq (Takara Bio) that does not possess 3' to 5' exonuclease activity was used as DNA polymerase. Restriction enzyme reaction was carried out using 3 μL PCR product and 30 units of NdeII in 20 μL volume. The mixture was incubated at 37 ˚C overnight. The expected band size of the PCR product is 180 bp for wild type and 179 bp for ccs-3. It is expected that wild type is not digested with NdeII but ccs-3 is digested to produce a 40 bp shorter band. Electrophoresis was performed using 3% agarose gel with 1 × TBE buffer.

To investigate the expression of ccs-3, RT-PCR was performed for “Kiiro Togarashi”, a yellow fruit cultivar possessing ccs-3 (Figure 1(c)) and “Takanotsume”, a red fruit cultivar, as positive control. Total RNA was extracted from pericarp of fruits at color turning stage, using RNeasy Plant Mini Kit (QIAGEN). Reverse transcription reaction was carried out using PrimeScript II Reverse Transcriptase (Takara Bio). Two primer sets were applied, that is, Ccs F+174 and Ccs R+352 for upstream part of exon and Ccs F+1284 and Ccs R+1494 for downstream part of exon. Expression of ubiquitin was also examined as positive control. For all the primer sets, RT-PCR was carried out in 20 μL of reaction volume and the condition was 1 cycle at 94˚C for 1 min, 30 cycles at 94˚C for 30 sec, 60˚C for 30 sec and 72˚C for 30 sec, followed by a final extension at 72˚C for 30 sec.

To identify the large deletion in upstream region of the Ccs gene, diagnostic PCR was carried out using six forward primers designed at 300 - 900 bp intervals with a fixed reverse primer (Figure 2(a)). At first, to verify the amplification accuracy, PCR was performed for the red pepper variety “Takanotsume” having the intact Ccs gene. As expected, a stepwise amplification of intensive bands with different sizes was observed for the six primers, although some minor non- specific bands were shown (Figure 2(b)). Then diagnostic PCR using these six primers was conducted for the yellow pepper cultivar “Sonia Gold” (Figure 2(c)). Five downstream primers did not show clear single band amplification

and only the most upstream primer Ccs5up_F-3784 amplified an intensive single band. Since the second upstream primer was located at −3080 bp position from the first nucleotide of exon, it was deduced that at least 3000 bp was lack in the Ccs gene of “Sonia Gold”.

The approximately 800 bp fragment obtained by using the most upstream primer Ccs5up_F-3784 was sequenced and aligned with the published genome sequence of red pepper. As a result, it was revealed that 4430 bp from −3234 bp position in upstream untranslated region to 1196 bp position in exon was deleted in the Ccs gene of “Sonia Gold” (Figure 3). Since the accurate size and position of the large deletion could be successfully identified, hereafter we called this allele ccs-del. The size of the deletion of ccs-del was different from but near to ccs-p1 having 4879 bp deletion recently reported by Jeong et al. (2019) [14]. Especially the end of the deletion of ccs-del and ccs-p1 can be regarded to be identical depending on alignment.

Based on the results described above, we tried to develop PCR marker to distinguish ccs-del (Figure 4). Using the sequence information obtained, the 4430 bp large deletion specific primer Ccs5del F2 was designated astride the deletion

position (Figure 4(a)). In addition to this primer, normal allele specific primer Ccs5del F1 was designed inside the deleted region. Using these two primers at a time as forward with a single reverse primer Ccs5del R, multiplex PCR was carried out. According to the sequence information, it was expected that a 491 bp band was amplified for the normal allele and a 282 bp band was amplified for ccs-del. As shown in Figure 4(b), an approximately 500 bp single band was amplified for “Takanotsume” and an approximately 300 bp single band was amplified for “Sonia Gold” as expected. Thus, we successfully developed the PCR marker to distinguish the 4430 bp large deletion.

Using the developed PCR marker for ccs-del, the Ccs genes in 16 non-red fruit color cultivars of C. annuum were genotyped (Figure 5). As a result, among 16 cultivars, 14 had ccs-del. Out of them, msGTY-1 (lane 10) is the orange fruit cultivar that has been used and revealed to have a large deletion in Lang et al. (2002) [13]. This result indicates that the 4430 bp deletion is a common cause of non-red fruit color in C. annuum.

Two cultivars “Cayenne Pepper Golden” (lane 5) and “Kiiro Togarashi” (lane 16) showed normal allele bands. “Cayenne Pepper Golden” and “Kiiro Togarashi” are European and Japanese traditional cultivars, respectively, and both are hot pepper having yellow and long type fruit. The sequences of the Ccs gene of the two cultivars were determined. Then, it was clarified that the sequences of both the cultivars were identical, having a single base deletion at 1265 bp position from the start codon that results in a frameshift and generates a stop codon (Figure 6). This was the allele ccs-3 that have been previously reported in other C. annuum cultivars [14] [24]. Expression of ccs-3 in “Kiiro Togarashi” was investigated by RT-PCR with two primer sets, that is, upstream and downstream parts of exon (Figure 7(a). Both the upstream and downstream primer sets showed positive amplification in RT-PCR (Figure 7(b)). This result suggested that almost entire region of ccs-3, including even the region downstream of stop codon generated by frameshift due to the 1 bp deletion, was transcribed in the yellow cultivars. Therefore, the fruit color change might have been caused not by expressional change but by loss or weakening of function of ccs-3 due to the frameshift. A dCAPS marker was developed to distinguish ccs-3 (Figure 7(a) and Figure 7(c)). This is a codominant marker that can recognize heterozygous samples.

Sixteen non-red fruit cultivars of C. chinense, the second major pepper species, were genotyped by the PCR marker for ccs-del (Figure 8(a)). Among 16 cultivars, 13 showed normal allele bands and a cultivar PI 485593 (lane 13) exhibited the 4430 bp large deletion specific band. No amplification was observed in two cultivars PI 224448 and CGN 22854 (lanes 10 and 16), suggesting that these cultivars had unknown mutation in the Ccs gene. When using the dCAPS marker for ccs-3, it was found that a cultivar PI 670898 (lane 14) had ccs-3 (Figure 8(b)). In Figure 8(b), PI 485593 (lane 13) that was revealed to have the 4430 bp deletion did not show band, which is as expected since the forward primer used

for dCAPS was included in the deleted region. These results indicated that the mutated alleles of the Ccs genes generating non-red fruit color, ccs-del and ccs-3, were shared in two major Capsicum species, C. annuum and C. chinense.