TYPE Original Research

PUBLISHED 18 September 2023

DOI 10.3389/fpls.2023.1151765

Development of a speed
OPEN ACCESS breeding protocol with flowering
EDITED BY
Christopher John Lambrides,
The University of Queensland, Australia
gene investigation in pepper
REVIEWED BY
Kenny Paul,
(Capsicum annuum)
Reliance Industries, India
Marina Ceran,
Institute of Field and Vegetable Crops, Hayoung Choi, Seungki Back, Geon Woo Kim,
Serbia
Kyeongseok Lee, Jelli Venkatesh, Hyo Beom Lee,
*CORRESPONDENCE
Byoung-Cheorl Kang
Jin-Kyung Kwon and Byoung-Cheorl Kang*
bk54@snu.ac.kr
Department of Agriculture, Forestry and Bioresources, Research Institute of Agriculture and Life
RECEIVED 26 January 2023 Sciences, Plant Genomics and Breeding Institute, College of Agriculture and Life Sciences, Seoul
ACCEPTED 14 August 2023
National University, Seoul, Republic of Korea
PUBLISHED 18 September 2023

CITATION
Choi H, Back S, Kim GW, Lee K,
Venkatesh J, Lee HB, Kwon J-K and Pepper (Capsicum spp.) is a vegetable and spice crop in the Solanaceae family with
Kang B-C (2023) Development of a speed many nutritional benefits for human health. During several decades, horticultural
breeding protocol with flowering gene
investigation in pepper traits, including disease resistance, yield, and fruit quality, have been improved
(Capsicum annuum). through conventional breeding methods. Nevertheless, cultivar development is a
Front. Plant Sci. 14:1151765.
time-consuming process because of the long generation time of pepper. Recently,
doi: 10.3389/fpls.2023.1151765
speed breeding has been introduced as a solution for shorting the breeding cycle in
COPYRIGHT
© 2023 Choi, Back, Kim, Lee, Venkatesh, long-day or day-neutral field crops, but there have been only a few studies on speed
Lee, Kwon and Kang. This is an open-access breeding in vegetable crops. In this study, a speed breeding protocol for pepper was
article distributed under the terms of the
developed by controlling the photoperiod and light quality. Under the condition of a
Creative Commons Attribution License
(CC BY). The use, distribution or low red (R) to far-red (FR) ratio of 0.3 with an extended photoperiod (Epp) of 20 h (95
reproduction in other forums is permitted, ± 0 DAT), the time to first harvest was shortened by 75 days after transplant (DAT)
provided the original author(s) and the
copyright owner(s) are credited and that compared to that of the control treatment (170 ± 2 DAT), suggesting that Epp with
the original publication in this journal is FR light is an essential factor for flowering in pepper. In addition, we established the
cited, in accordance with accepted
speed breeding system in a greenhouse with a 20 h photoperiod and a 3.8 R:FR ratio
academic practice. No use, distribution or
reproduction is permitted which does not and promoted the breeding cycle of C. annuum for 110 days from seed to seed. To
comply with these terms. explain the accelerated flowering response to the Epp and supplemented FR light,
genome-wide association study (GWAS) and gene expression analysis were
performed. As a result of the GWAS, we identified a new flowering gene locus for
pepper and suggested four candidate genes for flowering (APETALA2 (AP2),
WUSCHEL-RELATED HOMEOBOX4 (WOX4), FLOWERING LOCUS T (FT), and
GIGANTEA (GI)). Through expression analysis with the candidate genes, it
appeared that Epp and FR induced flowering by up-regulating the flowering-
promoting gene GI and down-regulating FT. The results demonstrate the effect of
a combination of Epp and FR light by genetic analysis of flowering gene expression.
This is the first study that verifies gene expression patterns associated with the
flowering responses of pepper in a speed breeding system. Overall, this study
demonstrates that speed breeding can shorten the breeding cycle and accelerate
genetic research in pepper through reduced generation time.

KEYWORDS

pepper, speed breeding, photoperiod, far-red light, genome-wide association study,

gene expression analysis

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Choi et al. 10.3389/fpls.2023.1151765

1 Introduction amaranth and rice genotypes, but there was no impact on soybeans.
Therefore, crop-specific LED lighting schemes will need to be developed
Pepper is a great source of vitamin C and b-carotene, and the (Jähne et al., 2020). Previous studies on speed breeding primarily
only plant genus that synthesizes capsaicinoids (Srivastava and focused on field crops, but research for vegetable crops is scarce. Liu
Mangal, 2019). Pepper capsaicinoids are used in food ingredients et al. (2022) developed a speed breeding scheme for hot pepper through
due to their unique taste, and also have physiological and light environment modification. They investigated the growth and
pharmacological health benefits including anticancer, anti- development of hot pepper varieties (‘Xiangyan 55’ and ‘Xiangla 712’)
inflammatory, and anti-obesity properties (Aza-Gonzá lez et al., under various light intensities (240, 300, 360, and 420 PPFD),
2011). Owing to these nutritional benefits, producing high-quality photoperiods (14 h, 16 h, 18 h, and 20 h), and R:FR ratios (2.1, 2.7,
cultivars is one of the major goals of pepper breeding. 3.8, and 6.3) (Liu et al., 2022). They reported that light intensities of 300
The long generation time of pepper is a bottleneck in breeding and 420 PPFD, a photoperiod of 20h, and an R:FR ratio of 3.8 were
programs; it takes 7-8 years to develop a cultivar. To mitigate this effective in shortening the flowering time for hot peppers (Liu et al.,
issue, there have been various technologies to shorten the generation 2022). However, they did not confirm the effect of combining all the
time. The most well-known approach is shuttle breeding, which uses conditions of light intensity, photoperiod, and supplementary FR. Since
two distinct fields with different climates (Ortiz et al., 2007). Shuttle the plant’s response to the speed breeding conditions in growth
breeding enables only two generations per year by growing breeding chambers or plant factories can be different from in actual
populations at two locations where plants can produce seeds (Ortiz greenhouse conditions, further study for applying comprehensive
et al., 2007). For a successful shuttle breeding approach, two fields speed breeding in the greenhouse needs to be done. Moreover, the
with different environmental requirements are needed for testing and genetic and molecular basis of speed breeding has not been
generation advancement, which can be laborious and expensive. fully elucidated.
Doubled haploid (DH) technology can generate homozygous lines Flowering time is controlled by the interaction of endogenous and
rapidly by bypassing the process of inbreeding (Gerald et al., 2013). exogenous signals such as photoperiod, temperature, and plant
However, each step of DH is strongly genotype-dependent, and this hormones (Srikanth and Schmid, 2011). Depending on the
method is laborious, expensive, and requires high skills to obtain a environmental signals, the pattern of flowering gene expression is
successful product (Gerald et al., 2013). Genome-editing technologies diverse (Cho et al., 2017; Ding et al., 2020). Previous studies on
can also generate desired plants by directly editing target genes (Araki Arabidopsis, a model plant, have revealed many flowering-associated
and Ishii, 2015). However, this approach is difficult to applying to genes and flowering gene expression patterns according to
certain species and requires skilled technicians (Araki and Ishii, environmental controls (Mouradov et al., 2002). In contrast, there
2015). To overcome these issues, Watson et al. (2018) proposed a are few studies on the expression patterns of flowering genes in pepper
simple but effective technology, ‘speed breeding’. under environmental change (extending day length, adding FR, etc.).
The response of crops to continuous light conditions has been the Moreover, although flowering genes are conserved between species,
subject of scientific investigation for many years. In the 1980s, NASA they are likely to have very different functions in each species. For
conducted an experiment in which crops, including wheat, soybean, example, SHORT VEGETATIVE PHASE (SVP) acts as a major
lettuce, and potato, were grown under constant light conditions. It was flowering repressor together with FLOWERING LOCUS C (FLC) in
observed that the total biomass of these crops strongly depended on the Arabidopsis (Li et al., 2008), while its homolog, CaJOINTLESS, acts as a
amount of light provided to the plants (Wheeler et al., 1996). O'Connell major flowering activator in pepper (Cohen et al., 2012). Therefore, a
(2007) focused specifically on exploring how wheat plants respond to genetic study is required for each particular species. Furthermore, it is
continuous light and investigated the potential implications of necessary to analyze the changes in flowering gene expression in a
continuous light on wheat growth and development. O'Connor et al. controlled environment specific to each crop speed breeding system.
(2013) described a speed breeding technique that utilized controlled This genetic analysis would identify the cause of accelerated flowering
environment conditions, continuous light, and a single seed descent time under speed breeding.
breeding strategy in a greenhouse to reduce the generation time of full- In this paper, we established a speed breeding system suitable
season maturity peanut cultivars from 145 to 89 days, potentially for pepper by shortening the generation time of pepper via
accelerating the development of new varieties. Inspired by previous extending the day length to 20 h, controlling light quality by
works, Watson et al. (2018) coined the term ‘speed breeding’ and supplementing FR, and adjusting the R:FR ratio. We then
developed protocols for shortening the generation time of long-day field demonstrated the effectiveness of this protocol on a genetic and
crops by extending the photoperiod. They achieved up to 6 generations molecular basis. Moreover, we applied the speed breeding system in
per year for wheat (Triticum aestivum), barley (Hordeum vulgare), the greenhouse and established a speed breeding protocol for C.
chickpea (Cicer arietinum), and pea (Pisum sativum) compared to the 2 annuum, enabling us to shorten the breeding pipeline in less than
to 3 generations of traditionally grown crops. Although speed breeding three years. To identify the genetic loci associated with flowering
is a powerful alternative for reducing generation time in long-day plants, time, we performed a genome-wide association study (GWAS) and
this method has rarely been applied to short-day plants. Recently, Jähne investigated the expression levels of candidate genes for
et al. (2020) demonstrated a protocol for short-day crops soybean comparative analysis of the flowering gene expression patterns
(Glycine max), rice (Oryza sativa), and amaranth (Amaranthus spp.), b e tw e en t h e s p e e d b r e e d i n g sy s t e m an d the no r m a l
adjusting the photoperiod to 10 h and adjusting the light intensity and environmental growing conditions. It is expected that this study
quality. Far-red (FR) light allowed flowering time reduction in some will have a significant impact on breeding and genetic research by

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shortening the breeding cycle, and the GWAS-identified candidate 2.2 Extending the photoperiod experiment
genes for loci associated with shortened flowering time provide
valuable information about the genetic control of flowering time For each treatment, a multi-room chamber (Hanbaek, Bucheon,
in pepper. Republic of Korea) was programmed to have a day and night cycle
of 12/12 h, 16/8 h, 20/4 h, and 24/0 h. In all the treatments, the
temperature was set at 26°C during the day and 20°C during
2 Materials and methods the dark (Table 1). The light source was a fluorescent lamp, and
the light intensity was adjusted to 66.2 µmol·m-2·s-1 at bench height
2.1 Plant materials (Table 1). The seeds were sown in a 72-hole seedling tray, and the
seedlings were grown for 20 days in a walk-in chamber under a 16/
For setting the speed breeding system for pepper, Capsicum 8-hour day/night cycle and temperature conditions of 25/20°C.
annuum ‘MicroPep Red’ (‘MR’) from germplasm of the Subsequently, individual seedlings were transplanted into pots with
Horticultural Crops Breeding and Genetics Laboratory (Seoul a diameter of 9.2 cm and a height of 8.8 cm. The pots were filled
National University, Republic of Korea) was used as the plant with approximately 90% of a commercial substrate called ‘Barokuh’
material of this study. ‘MR’ has a rapid generation time and short (SeoulBio, Eumseong, Republic of Korea). The composition and
plant height compared to other cultivars. ratio of the ‘Barokuh’ substrate were as follows: zeolite 4%, perlite
For GWAS, 220 C. annuum accessions were used (Lee et al., 7%, pumice 6%, coco peat 68%, peat moss 14.73%, fertilizer 0.201%
2020). The 220 accessions comprising the GWAS population were (containing N, P, K, Ca, Mg, Fe, Cu, Zn, B, and other elements),
grown in a greenhouse at the RDA-GenBank (Jeonju, Republic of wetting agent 0.064%, and pH adjuster 0.005%. The physical
Korea) in 2015, 2016, and 2017. Over three years, six plants per properties of the substrate included a moisture content range of
accession were randomly planted, and three plants per accession 40-60%, an air-filled porosity range of 30-50%, and a bulk density of
were evaluated for flowering time (Lee et al., 2020). 0.15-0.25 Mg/m3. The chemical properties of the substrate were

TABLE 1 Growth conditions for extending the photoperiod, far-red (FR) light intensity, pepper-specific speed breeding system, and application of a
speed breeding system in a greenhouse (GH).

Light intensity
(µmol·m-2·s-1)
Photoperiod (h) Temperature (°C) Red Far-red R:FR
Treatment Light source White
(day/night) (day/night) (R: (FR: ratio
(W: 400-
600- 700-
700 nm)
700 nm) 780 nm)
12/12 h 12/12 26/20 Fluorescent lamp 66.2 15.3 2.7 5.7

16/8 h 16/8 26/20 Fluorescent lamp 66.2 15.3 2.7 5.7

20/4 h 20/4 26/20 Fluorescent lamp 66.2 15.3 2.7 5.7

24/0 h 24/0 26/20 Fluorescent lamp 66.2 15.3 2.7 5.7

W45 20/4 27/24 Warm-white LED 45.0 22.2 3.0 7.3

W45+LFR 20/4 27/24 Warm-white LED +FR LED 45.2 22.3 18.8 1.2

W45+MFR 20/4 27/24 Warm-white LED +FR LED 45.3 23.0 38.7 0.6

W45+HFR 20/4 27/24 Warm-white LED +FR LED 45.2 23.3 78.2 0.3

Ctl 12/12 26/20 Fluorescent lamp 66.2 15.3 2.7 5.7

Fluorescent lamp
Ctl+FR 12/12 26/20 66.2 17.0 56.5 0.3
+FR LED

Epp 20/4 26/20 Fluorescent lamp 66.2 15.4 2.7 5.8

Fluorescent lamp
Epp+FR 20/4 26/20 66.2 17.0 57.1 0.3
+FR LED

Normal GH Natural light Suwon farm condition Natural light 152.8 55.1 40.3 1.4

Natural light
Epp GH 20/4 Suwon farm condition +LED 319.7 151.3 31.8 4.8
(400-700 nm)

Natural light
Epp+FR GH 20/4 Suwon farm condition +LED 450.7 283.2 74.7 3.8
(400-772 nm)

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characterized by a pH range of 5.5-7.0 and an electrical conductivity ‘MR’ plants at the 1-2 leaf stage (with a minimum length of 1 cm)
(EC) of 0.65 ds/m. Throughout the cultivation period, no additional were transplanted. Various parameters were recorded, including the
fertilizers were applied. Each pot was watered once daily in the duration of the transition phase, the timing of the first flower
morning using approximately 150-200 ml of tap water. Eight plants appearance, the onset of first fruit production, the ripening of the
of ‘MR’ at two leaves (at least 1cm) were transplanted for testing the fruits, the number of fruits produced, and the distance from the
effect of extended photoperiod on flowering time and phenotyped shoot apical meristem to the soil surface. The observed data
for number of days to transition (floral meristem appearance) and pertaining to these parameters have been organized and presented
flowering from days after transplanting (DAT). in Tables 2, 3. Specifically, the parameter labeled as ‘Transition’
denotes the critical moment when the floral meristem becomes
visible, signaling the emergence of the first flower bud. ‘First flower’
2.3 FR light intensity experiment represents the precise point in time when the initial flower blossoms
on the plant. ‘First fruit’ characterizes the developmental stage when
The FR light intensity experiment was conducted in the walk-in the plant generates its first fruit. ‘First ripening’ captures the
chamber at Seoul National University Farm (Suwon, Republic of significant stage at which the first fruit reaches full maturity,
Korea). This walk-in chamber was programmed to run a 20 h acquiring a vibrant red coloration referred to as the mature
photoperiod and 4 h dark period with a temperature of 27°C red stage.
during the day and 24°C during the night (Table 1). The relative
humidity was set to 70%. Warm-white (W) and FR LEDs were used
to test the effect of FR light on accelerating the transition from the 2.5 Application of a speed breeding system
vegetative to the reproductive stage. Light intensity (400-700nm) was in the greenhouse
set at 45 µmol·m-2·s-1 (W LEDs; W45) in all four treatments: W45
treatment (non-FR), W45+LFR (low FR), W45+MFR (medium FR), For applying the pepper-specific speed breeding system in the
and W45+HFR (high FR) (Table 1). The light intensity of FR light in greenhouse, a system was devised in the greenhouse at Seoul
LFR, MFR, and HFR was 18.8, 38.7, and 78.2 µmol·m-2·s-1 each National University Farm (Suwon, Republic of Korea). The ‘MR’
(Table 1 and Supplementary Figure 1). The ‘MR’ seeds were directly seeds were directly sown in the 72-hole seedling tray and grown in
sown in the 72 hole seedling tray and grown in each treatment of the each treatment of the greenhouses during the fall season in Suwon,
walk-in chamber without undergoing a separate seedling growth Republic of Korea. The basic cultivation conditions, such as pot
process. The basic cultivation conditions, such as pot size, amount of size, amount of media, composition of the media, and water
media, composition of the media, and water management, remained management, remained consistent with those described in the
consistent with those described in the “Extending the photoperiod “Extending the photoperiod experiment.” However,
experiment” section. Ten plants of ‘MR’ were grown in each approximately 85 days after transplantation in the greenhouse,
treatment and plant height (distance from the shoot apical an additional granular fertilizer (Inovatec, Jeongeup, Republic of
meristem to the soil) and time to floral meristem were recorded. Korea) was applied at a rate of approximately 5 pellets per pot.
The fundamental cultivation conditions, including pot size,
amount of media, composition of the media, and water
2.4 Pepper-specific speed breeding system management, remained consistent with those described in the
“Extending the photoperiod experiment”. For the normal
Plants were grown in a multi-room chamber (Hanbaek, greenhouse (Normal GH) condition, the natural light and
Bucheon, Republic of Korea). For all the treatments: Control temperature in Suwon Farm were used. For the treatments of
(Ctl), Control+Far-red light (Ctl+FR), Extended photoperiod extended photoperiod in GH (Epp GH) and extended photoperiod
(Epp), and Extended photoperiod+Far-red light (Epp+FR), each with supplemented FR in GH (Epp+FR GH), the 20 h photoperiod
chamber room was programmed at 26°C during the day and 20°C at and 4 h dark period were used (Table 1). Three plants for each
night (Table 1). In Ctl and Ctl+FR treatments, each chamber room treatment were analyzed. The light spectrum of LED (Bissol,
was set to have a 12 h photoperiod and 12 h dark period (Table 1). Seoul, Korea) was 400-700 nm for Epp GH and 400-772 nm for
In the case of the Epp and Epp+FR treatments, the day and night Epp+FR GH (Table 1). The ratio of red to FR was adjusted to 4.8
lengths were set to 20 h and 4 h (Table 1). In Ctl+FR and Epp+FR in Epp GH and 3.8 in Epp+FR GH (Table 1).
treatments, FR light was supplemented with R:FR=0.3 (Table 1 and
Supplementary Figure 2). The light intensity of the fluorescent lamp
for all the treatments was adjusted to 66.2 µmol·m-2·s-1 (Table 1). 2.6 Genomic DNA extraction
The seeds were sown in the 72 hole seedling tray, and the seedlings
were cultivated for 20 days in a walk-in chamber with a day/night Total genomic DNA (gDNA) was extracted from three young
cycle of 16/8 hours and temperature settings of 25/20°C. The basic leaves of plants using a modified cetyltrimethylammonium bromide
cultivation conditions, such as pot size, amount of media, (CTAB) method (Porebski et al., 1997). The extracted DNAs were
composition of the media, and water management, remained quantified with a NanoDrop 1000 spectrophotometer (NanoDrop
consistent with those described in the “Extending the photoperiod Technologies, Wilmington, DE, USA). The gDNA concentration
experiment” section. In each experimental treatment, a total of 10 was measured and diluted to 20 ng·mL–1.

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TABLE 2 Development of Capsicum annuum ‘MicroPep Red’ (‘MR’) under the treatments of extending the photoperiod experiment, far-red (FR) light
intensity experiment, pepper-specific speed breeding system, and application of speed breeding in greenhouse (GH).

Days after transplant to **

Experiment Treatment First First First
Transition
flower fruit ripening
12/12 h 28.9±2.5 a
43.6±3.7 a
– –

16/8 h 28.9±2.5 a
43.1±4.3 a
– –
Extending the photoperiod
experiment
20/4 h 28±0 a
37.5±2.1 b
– –

24/0 h 23.6±3.6 b
36.5±2.1 b
– –

W45 25.9±3.4 a
– – –

W45+LFR 23.9±4.4 ab – – –
FR light intensity experiment
(day/night: 20/4 h)
W45+MFR 21.7±4.3 bc
– – –

W45+HFR 18.6±0.5 c
– – –
a a a a
Ctl 36±2 56±2 113±5 170±2
Pepper-specific speed breeding system b b a b
Ctl+FR 29±5 50±6 106±8 163±2
(day/night
- Ctl: 12/12 h
Epp 24±5 c 45±5 c
88±6 b 130±3 c
- Epp: 20/4 h)
d
Epp+FR 19±4 36±2 d 53±8 c 95±0 d
a a
Application of speed breeding in GH Normal GH 28± 51±0 71±0 a 128±0 a

(day/night b b
Epp GH 21±0 28±0 41±0 b 84±0 b
- Normal: natural light
- Epp: 20/4 h) Epp+FR GH 7±0 c
21± c
28±0 c 71±0 c

*Means ± SD with the same letter in the stages of each experiment are not significantly different at the P = 0.05 level according to Duncan’s multiple range test.
**Days after germination in the FR light intensity experiment.

2.7 GWAS and candidate The digested DNA was ligated to adapters and the libraries were
gene identification amplified using ‘TA’ primers (Lee et al., 2020). The libraries were
pooled in five tubes, and the pooled libraries were sequenced using
The GWAS accessions were genotyped using the genotyping- an Illumina HiSeq2000 sequencing system (Illumina, San Diego,
by-sequencing (GBS) method based on the PstI/MseI and EcoRI/ CA, United States) at Macrogen (Seoul, South Korea). Filtered raw
MseI restriction enzymes as previously described (Lee et al., 2020). reads of GWAS population accessions were aligned to the C.

TABLE 3 Plant height and number of fruits of Capsicum annuum ‘MicroPep Red’ (‘MR’) from far-red (FR) light intensity experiment, pepper-specific
speed breeding system, and application of speed breeding in greenhouse (GH).

Plant height
Experiment Treatment No. of fruit
(cm)
W45 9.3±0.6 a –

W45+LFR 9.2±0.9 a
–
FR light intensity experiment
(day/night: 20/4 h)
W45+MFR 10.6±0.9 b
–

W45+HFR 16.4±1.0 c
–
a
Ctl 8.6±0.5 1.7±0.8 a
Pepper-specific speed breeding system b
Ctl+FR 21.3±0.8 2.8±1.2 a
(day/night
- Ctl: 12/12 h
Epp 9.5±0.5 c 2.7±1.0 a
- Epp: 20/4 h)
b
Epp+FR 21.7±1.1 3.0±1.7 a

Application of speed breeding in GH Normal GH 2.9±0.2 a 3±0 a

(day/night b a
Epp GH 2.9±0.2 3±0
- Normal: natural light
- Epp: 20/4 h) Epp+FR GH 5.5±0.4 b
27.8±3.7 b

*Means ± SD with the same letter in the stages of each experiment are not significantly different at the P = 0.05 level according to Duncan’s multiple range test.

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annuum ‘Dempsey’ reference genome (Lee et al., 2022) using the flowering of the ‘MR’. The qRT-PCR was performed using the
Burrows-Wheeler Aligner (Hong et al., 2020). Alignment primers AP2_qRT (Borovsky et al., 2015), WOX4_qRT, FT_qRT,
procedures yielded various types of variants, from which single and GI_qRT. The qRT-PCR was performed in a 20 mL reaction
nucleotide polymorphisms (SNPs) were exclusively extracted. volume containing 2 mL of 5x diluted cDNA, 2 mL of 10mM dNTPs,
These SNPs underwent a filtering process conditioned on 2 mL of 10x reaction buffer, 0.5 mL of SYTO 9 (Thermo Fisher
parameters of a quality score (QUAL) < 30, a mapping quality Scientific Korea, Seoul, Republic of Korea), 0.5 mL of 10 pmol
(MQ) < 30.00, a Strand Odds Ratio (SOR) > 4.000, and a depth of primers, and 0.4 mL of R Taq (Takara Bio). A Rotor-Gene 6000 real-
coverage (DP) < 3 to ensure data reliability. A further filtering step time PCR thermocycler (Corbett Research, Sydney, Australia) was
followed, operating under a Minor Allele Frequency (MAF) cutoff used with the following PCR amplification conditions: 95°C for
of 0.05% and a call rate threshold of 50%. This filtering procedure 5 min; 45 cycles of 95°C for 30 s, 58°C for 30 s, and 72°C for 30 s.
resulted in the acquisition of 221,487 SNPs for utilization in the The relative expression levels of candidate genes were normalized
GWAS. Prior to conducting the GWAS, an exploratory principal against CaUBIQUITIN (DQ975458.1) using the primer UBQ_qRT
component analysis (PCA) of the genotypic data earmarked for (Borovsky et al., 2015). The reliability and reproducibility were
GWAS revealed that two principal components accounted for over ensured with three independent replicates per plant. Statistical
95% of the population variance. This insight dictated the analytical analysis was conducted by Tukey’s honest significant difference
strategy for the subsequent GWAS, integrating the two principal (HSD) test for pairwise comparisons and significant differences of
components to consider population structure appropriately. The means (Tukey, 1977) using R program.
GWAS based on the compressed mixed linear model (CMLM) was
conducted using the R Package Genomic Association with default
settings (Wang and Zhang, 2021). The significance threshold was 3 Results
set after the Bonferroni multiple-test correction, setting the
significant threshold level (Bonferroni, 1936). Candidate gene 3.1 A photoperiod extended to 20 h was
prediction for flowering time was performed based on the the best condition for accelerating the
‘Dempsey’ reference genome (Lee et al., 2022). flowering time

To find out the best photoperiod for the flowering of the pepper,
2.8 Sequence analysis of GWAS as shown in Figure 1, pepper plants were grown under various
candidate genes conditions with 12 h, 16 h, 20 h, and 24 h of photoperiod. As the
photoperiod extended, the ‘MR’ variety demonstrated a reduction in
PCR was performed using 5 mL of 5x GXL buffer, 100 ng of the number of days required for transition, indicating an accelerated
template DNA, 10 pmol of each primer, 2 mL of 10mM dNTPs, and emergence of floral meristem (Table 2). Flowering time was
0.5 mL of Taq polymerase (PrimeStar GXL; Takara, Shiga, Japan). shortened by 6-7 days under the 20/4 h and 24/0 h conditions
Primers were designed to amplify all the coding sequences of compared to the 12/12 h and 16/8 h conditions (Table 2). However,
candidate genes. The amplified products were resolved in 1% the plants under 24 h showed physiological disorders with wrinkled
agarose gel (Lonza, Lockland, ME, USA) and gel eluted and and yellowing leaves (Supplementary Figure 3). Thus, it was
purified using a PCR Clean-up Kit (Cosmogenetech, Seoul, demonstrated that extending the day length to 20 h effectively
Korea). The amplicons were sequenced at Macrogen (Macrogen, promotes the flowering time and is the best photoperiod for
Seoul, Korea) and analyzed using SeqMan (Ver 5.00, DNASTAR speeding up the flowering of ‘MR’ (Figure 1 and Table 2).
Inc., Madison, WI, USA).

3.2 A lower R:FR ratio promoted the

2.9 Expression analysis of GWAS candidate flowering of Capsicum annuum
genes using quantitative real-time PCR
In the above experiment, it was found that extending the day
For gene expression analysis, the young leaves (1 cm) of ‘MR’ length to 20 h could promote the flowering time of pepper. To
grown in the pepper-specific speed breeding system were sampled further shorten the flowering time of ‘MR’, diverse R:FR ratios were
when the fourth and the sixth leaf were initiated. The leaves were tested. The plants under higher FR light treatment showed
frozen immediately in liquid nitrogen after sampling. Total RNA accelerated floral induction (Figure 2 and Table 2). FR light
was extracted from the leaves using the MG Total RNA extraction stimulation facilitated floral differentiation, as evidenced by a
kit (MGmed, Seoul, Republic of Korea). Total RNA (1mg) was used reduction in the number of days required for the transition of the
for cDNA synthesis by reverse transcription (RT) PCR using ‘MR’ variety (Table 2). However, it is important to note that FR light
AccuPower RT PreMix (Bioneer, Daejeon, Republic of Korea). also induced undesired stem elongation. As the intensity of FR light
For qRT-PCR, three biological replicates were used for each increased, there was a significant promotion of stem elongation
sample. Based on the exon sequencing results of the ‘MR’, (Table 3). Overall, this study indicates that supplemented higher FR
primers for qRT-PCR have been designed. The identified was effective for shortening the time to floral induction of ‘MR’,
variations in the exons have the potential to explain the rapid although unwanted stem elongation was observed.

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FIGURE 1
The growth and development of Capsicum annuum ‘MicroPep Red’ (‘MR’) in extending the photoperiod experiment. (A) ‘MR’ at 39 days after
transplant (DAT) showed transition stage (appearance of flower buds) under the 12/12 h and 16/8 h of light/dark cycle. Concurrently, ‘MR’ showed
flowering under the 20/4 h and 24/0 h of light/dark cycle. (B) Representative graph depicting the development stages of ‘MR’ under the various
photoperiod conditions.

3.3 The combination of extended photoperiod and supplementing FR light. A 20 h photoperiod and an
photoperiod and FR light as a pepper- R:FR ratio of 0.3 was adopted from the previous experiment, which was
specific speed breeding system shortened effective in promoting transition and flowering. Compared to other
the generation time effectively treatments (Ctl, Ctl+FR, Epp), transition, flowering, fruit, and ripening
time were significantly reduced under Epp+FR treatment (Figure 3 and
Through extending the photoperiod experiment and the FR light Table 2). Specifically, compared to Ctl (56 ± 2 DAT), the combination
intensity test, it was found that increasing the light period to 20 h and of extended photoperiod and FR light (Epp+FR) shortened the
higher FR light intensity could promote the flowering of ‘MR’. flowering time significantly (36 ± 2 DAT) (Figure 3 and Table 2). In
Therefore, to shorten pepper generation time dramatically, a pepper- addition to transition and flowering, fruit setting and ripening times
specific speed breeding system was devised by combining extended were also significantly decreased in Epp+FR, suggesting that the

FIGURE 2
The appearance of flower buds in far-red (FR) light intensity experiment. (A) Capsicum annuum ‘MicroPep Red’ (‘MR’) at 23 days after germination showed
vegetative shoot apical meristem under W45. (B) ‘MR’ at 23 days after germination showed inflorescence meristem under W45+HFR (R:FR = 0.3).

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Choi et al. 10.3389/fpls.2023.1151765

generation time of pepper can be remarkably reduced due to a significantly shortened under Epp+FR GH compared to Normal
combination of extended photoperiod and supplemented FR, which GH and Epp GH (Figure 4). Days to flowering was 51, 28, and 21
can be a speed breeding system suitable for pepper (Figure 3). In DAT under Normal, Epp, and Epp+FR GH, respectively
addition, there were significant differences in stem length among the (Table 2). Days to first fruit and ripening were 28 and 71 DAT
treatments (Ctl, Ctl+FR, Epp, and Epp+FR). Plants grown under FR under Epp+FR GH (Table 2). Concurrently, the corresponding
light (Ctl+FR and Epp+FR) had higher stem lengths as compared to plants under Normal GH had only reached floral induction, while
those grown under other conditions without FR light (non-FR) the plants under Epp+FR GH had already reached the first fruit
(Figure 3 and Table 3). stage (Figure 4 and Supplementary Figure 4). Unlike the stem
elongation under the higher R:FR of the FR light intensity
experiment (Figure 3 and Table 3), the ratio of R:FR at Epp+FR
3.4 Application of a pepper-specific speed GH did not cause stem elongation (Figure 4 and Table 3). In
breeding system in a greenhouse addition to speeding up the flowering, Epp+FR condition
accelerated the breeding cycle of upregulated the number of fruits, which is important to harvest
Capsicum annuum more seeds for breeding peppers (Table 3).

Employing the results of the experiments in the chambers,

speed breeding conditions with extended light and supplemented 3.5 Genome-wide association study
FR light were applied in the greenhouse. Since it was confirmed provided candidate flowering-time genes
that the plant height was elongated when the FR light was higher for Capsicum annuum
than the red light in ‘MR’, an R:FR ratio of 3.8 was set in the
greenhouse to prevent over-growth (Liu et al., 2022). Days to Through previous experiments, it was confirmed that pepper
transition, flowering, first fruit, and first ripening were generation time is different depending on the day length and the

FIGURE 3
Growth and development of Capsicum annuum ‘MicroPep Red’ (‘MR’) in pepper-specific speed breeding system. (A) ‘MR’ at 100 days after transplant
(DAT) under the Ctl (12/12 h and 26/20°C of light/dark cycle), Ctl+FR (12/12 h, 26/20°C of light/dark cycle, and supplemented far-red (FR) light), Epp
(20/4 h and 26/20°C of light/dark cycle), Epp+FR (20/4 h, 26/20°C of light/dark cycle, and supplemented FR). (B) Representative graph depicting the
development stages of ‘MR’ under the treatments (Ctl, Ctl+FR, Epp, and Epp+FR).

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presence or absence of FR. In particular, growth and development genes for sequencing and expression analysis (Supplementary
were promoted the most in the experimental group in which the Tables 3–5).
photoperiod was prolonged and FR was supplemented. To
demonstrate that different day lengths and light quality can be a
genetic signal for the transition from vegetative to reproductive 3.6 Gene expression of FT and GI showed
stage, as well as simply increasing photosynthesis, GWAS analysis significant differences due to growth
was performed to determine the genetic loci associated with conditions in the pepper-specific speed
flowering time. breeding system
The phenotypic distribution of the flowering time using the
GWAS population is illustrated in Figure 5A. This figure depicts the Candidate gene expression patterns were analyzed in young leaves
average flowering time over three years (2015, 2016, and 2017) for of ‘MR’ under Ctl and other treatments (Ctl+FR, Epp, and Epp+FR).
each C. annuum accession. Notably, the ‘MR’ accession exhibited an The gene expression patterns of AP2 and WOX4, which play an
average flowering time of 68 ± 1.7 days under the normal important role in floral development in Arabidopsis (Costanzo et al.,
greenhouse (traditional sunlight). The GWAS of the flowering- 2014; Borovsky et al., 2015), did not show significant differences among
time traits was performed using a compressed MLM model environments (Supplementary Tables 4, 5). On the other hand, the
(CMLM) as implemented in FarmCPU program with 221,487 expression levels of FT and GI were significantly different depending on
high-quality SNPs and 220 C. annuum accessions. We identified growth condition (Supplementary Tables 4, 5). The FT gene found near
5 genome-wide significant SNPs (−log10 P > 7.158562). These SNPs the SNP on chromosome 5 had the highest expression in the Ctl
were located on chromosomes 2, 3, 4, 5, and 12 (Figures 5B, C). The (Figure 6). GI was highly expressed in Epp+FR, which extended
boxplots drawn with the genotypes of the 5 significant SNPs against photoperiod and supplemented FR light, and its expression level was
the flowering-time phenotypes revealed that 3 SNPs on the lowest under Ctl conditions (Figure 6). These gene expression
chromosomes 2, 4, and 12 were significantly associated with results indicate that not only was flowering accelerated by increasing
phenotype variation (Figures 5D-H). The gene annotation dataset the amount of photosynthesis and photosynthetic efficiency due to
was investigated to find significant flowering-related genes in the extended photoperiod and adding FR (Zhen and van Iersel, 2017;
vicinity of the SNPs. The APETALA2 (AP2), WUSCHEL-RELATED Watson et al., 2018), but also the prolonged photoperiod or FR light
HOMEOBOX4 (WOX4), FLOWERING LOCUS T (FT), and can promote or inhibit the expression of flowering-related genes to
GIGANTEA (GI) genes were identified and selected as candidate change the flowering phenotype.

FIGURE 4
Growth and development of Capsicum annuum ‘MicroPep Red’ (‘MR’) from application of speed breeding in greenhouse (GH). (A) ‘MR’ at 99 days after
transplant (DAT) under the Normal GH (Suwon farm condition), Epp GH (20/4 h of light/dark cycle), and Epp+FR GH (20/4 h of light/dark cycle and
supplemented FR). (B) Representative graph depicting the development stages of ‘MR’ under the treatments (Normal GH, Epp GH, and Epp+FR GH).

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A B C

D E F G H

FIGURE 5
Genome-wide association analyses (GWAS) for flowering time. (A) Histogram for flowering time phenotype of pepper GWAS population. The
histogram was drawn using the average flowering time of three-year data (2015, 2016, and 2017) in GWAS population. The red dotted line is the
probability density curve following a normal distribution. (B) The 221,487 filtered single nucleotide polymorphisms (SNPs) detected from the 220
individuals of the GWAS population were used for detection of candidate genes. The GWAS were conducted using the R package Genomic
Association with default settings. Blue line represents the threshold for GWAS significance after a Bonferroni correction. Five genome-wide
significant SNPs (−log10P > 7.158562) were identified on chromosomes 2, 3, 4, 5, and 12. (C) Quantile-quantile (QQ) plot of the data derived through
FarmCPU model shown in the Manhattan plot. (D–H) The box plot showed the phenotypic variation of each SNP in the chromosomes 2, 3, 4, 5, and
12. The boxplot displays the phenotype variation in the GWAS population with the three different genotypes (-1: reference, 0: hetero, 1: alternative
genotype) of 5 SNPs over the Bonferroni correction. Statistical significance was determined by ANOVA test (*P < 0.05, **P < 0.01, ***P < 0.001). The
3 SNPs in the chromosomes 2 (**), 4 (***), and 12 (**) showed significance in the phenotype variation among the 5 SNPs.

4 Discussion light intensities. FR light did not affect the flowering time of
soybean, but early flowering was observed under higher FR light
Developing strategies for the rapid generation of homozygous in rice and amaranth. The flowering responses of plants can vary
lines is a dream for plant breeders. Watson et al. (2018) followed a new depending on environmental factors, such as various day-length
breeding approach, speed breeding, to shorten the generation time. In and specific light spectrums and different plant species (Watson
this approach, the authors exposed the plants to prolonged et al., 2018; Jähne et al., 2020); thus, species-specific breeding
photoperiods, which resulted in a significantly shorter generation protocols need to be developed for each species, cultivar,
time for field crops such as wheat, barley, pea, and chickpea. However, and accession.
this protocol is limited to long-day or day-neutral plants and does not In this study, a speed breeding protocol for pepper was
consider other environmental controls such as light quality. developed by extending the photoperiod to 20 h, controlling
Extended daylight can clearly be beneficial for the flowering of proper temperature, and supplementing FR light. Interestingly,
some plant species and confirming the best day length is the key to this system demonstrated that prolonged photoperiod with FR
success in speeding up the flowering. Extending the photoperiod, lighting was the best condition for shortening the transition,
however, may also have some negative effects, because plants use the flowering, fruit, and ripening time of pepper. This protocol
photoperiod as a signal for many other physiological processes. For shortened the flowering time to that of half of the plants grown
example, tomato plants under continuous light showed leaf in normal conditions. The time to first harvest was shortened by 75
chlorosis and epinasty (Pham et al., 2019). These effects are DAT, which can shorten the breeding and research cycle of pepper
largely on a species-specific basis. Continuous photosynthesis and remarkably. The effectiveness of the developed speed breeding
accumulation of carbohydrates produced by continuous light may system was observed not only in C. annuum ‘MicroPep Red’, the
also result in negative feedback (Demers and Gosselin, 2000). These main variety used in this study but also in another species, C.
responses of plants to extended photoperiod and continuous light chinense ‘Habanero’. Supplementary Figure 5 illustrates that the
are very complex and are largely species or even cultivar specific flowering time was significantly promoted in the extended
(Velez-Ramirez et al., 2011). There is still much that is unknown photoperiod+far-red light (Epp+FR) treatment compared to the
about the mechanisms of plant responses to continuous light and control and extended photoperiod (Epp) treatments for ‘Habanero’.
the causes of the negative effects (foliar chlorosis, limited growth, These findings imply that the speed breeding system developed in
productivity, leaf injury, etc.) (Sysoeva et al., 2010). this study holds potential for application in other species within the
Recently, Jähne et al. (2020) proposed a protocol for short-day Capsicum genus.
crops, such as soybean, rice, and amaranth, modulating optimal FR light was the key to this protocol and finding the best ratio of
light conditions for each crop by adjusting blue, green, red, and FR R:FR was essential. In the FR light intensity experiment, the lower R:

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A B

C D

FIGURE 6
Quantitative RT-PCR results of candidate genes. Relative mRNA level of (A) AP2, (B) WOX4, (C) FT, and (D) GI in the Ctl, Ctl+FR, Epp, and Epp+FR at
4th and 6th leaves. Data for each group are means ± SE of three independent replicates. The P-values for significant differences between each
treatment are specified in Supplementary Tables 4, 5.

FR ratio was effective for shortening flowering time in pepper, and 2001). These studies suggest that flowering-related genes can
the best R:FR ratio was 0.3. However, FR light can cause abnormal promote or inhibit flowering depending on the light conditions,
plant morphology of tall plants (Franklin, 2008). Plants with therefore it is necessary to confirm gene expression under speed
abnormally elongated stems are not suitable for growing in breeding, and relevant studies are lacking to date. To identify the
growth chambers and plant factories that have a limited height. genetic loci associated with flowering responses to day length and
Furthermore, breeders may face difficulties in growing and light and verify the gene expression patterns in the speed breeding
phenotyping plants due to lodging associated with elongated system, GWAS and gene expression analysis were performed.
stems. To obtain an environment that can both promote Multiple GWAS studies have elucidated the genetic structure
flowering and maintain optimal stem growth in pepper, light related to key characteristics of pepper (Capsicum spp.), such as
quality and duration should be optimized. It is possible that the capsaicinoid content, fruit weight, and shape (Lozada et al., 2022a).
use of additional blue light along with Epp and FR light conditions Recently, Lozada et al. (2022b) performed a multi-locus GWAS
(Epp+FR) could prevent the enhanced shoot growth due to blue analysis to investigate quantitative trait loci (QTL) associated with
light-mediated inhibition of hypocotyl growth regulated by agronomic traits, including flowering time, in pepper (Capsicum
cryptochromes (Franklin, 2008). spp.). Flowering time exhibited a strong correlation with the first
For the application of the developed speed breeding system in fruit date trait, and shared QTL on chromosomes P1, P6, and P7
the greenhouse, we established an LED-controlled system. We set were identified. Relevant candidate genes were found to be involved
the photoperiod at 20 h and the R:FR ratio at 3.8 for accelerating the in biological functions such as defense response, metabolic
flowering time. We shortened the duration of seed to seed by 57 processes, oxidation-reduction, phosphorylation, and gene
DAT with Epp+FR GH compared with Normal GH. When we silencing (Lozada et al., 2022b). In this study, we identified five
applied this Epp+FR GH system in breeding the multiple disease significant SNPs that were located in different positions compared
resistant C. annuum and producing mature seeds of F1 plants, it to previous GWAS studies. Among them, AP2 (located on
took only 110 days and is predicted to be 880 days for developing chromosome 2), WOX4 (located on chromosome 4), FT (located
BC5F3 lines. on chromosome 5), and GI (located on chromosome 12) were
The differences in plant responses to light can be explained by selected as candidate genes. Notably, AP2 is the same locus as
the expression of flowering-related genes. For example, in the case CaAP2 introduced in a previous paper (Borovsky et al., 2015). As
of Arabidopsis, FR light can promote FT expression (Cerdá n and CaAP2 acts as a flowering repressor in pepper, it was expected that
Chory, 2003; Halliday et al., 2003; Valverde et al., 2004) and AP2 gene expression would be lowered in the Epp+FR treatment;
flowering (Goto et al., 1991; Reed et al., 1993; Bagnall and King, however, CaAP2 expression did not change significantly depending

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Choi et al. 10.3389/fpls.2023.1151765

on the environment. A similar result was observed for the CaWOX4 Data availability statement
gene, a member of the WOX gene family. The WOX4 protein
contains the distinct WUS-box motif (van der Graaff et al., 2009), The datasets presented in the study are deposited in the NABIC
which is essential for shoot stem-cell population maintenance or repository (https://nabic.rda.go.kr/nolog/NV-0799-000001/
differentiation, lateral organ formation, floral patterning, and snpVcfView.do), accession number NV-0799.
embryogenesis (Kamiya et al., 2003; Haecker et al., 2004).
Contrary to AP2 and WOX4, significant differences were found
in the expression of GI and FT under different treatment conditions. Author contributions
Expression of GI, a flowering promoter in Arabidopsis (Mizoguchi
et al., 2005), was significantly increased in the experimental Conceived and designed the experiments: HC, SB, HL, B-CK.
conditions in which FR was applied with Epp. It was expected Performed the experiments: HC, SB, GK, KL, B-CK. Analyzed the
that FT gene expression would be enhanced in the FR condition data: HC, GK, B-CK. Wrote the paper: HC, JV, J-KK, B-CK.
since it is a representative flowering promoter and considered to be
a florigen in Arabidopsis (Corbesier et al., 2007); however,
expression of CaFT was inhibited in Epp+FR. This is because the Funding
functions of the flowering gene homologs in different plant species
can be very different (Borovsky et al., 2015). Therefore, it can be This work was supported by Korea Institute of Planning and
inferred that the FT gene in C. annuum functions to delay flowering. Evaluation for Technology in Food, Agriculture and Forestry
To clarify the role of these candidate genes, further functional (IPET) through Technology Commercialization Support Program
studies are required to better understand the molecular mechanisms (821012-03) and Digital Breeding Transformation Technology
by which these genes are involved in flower transition. Development Program (322062-3), funded by Ministry of
In conclusion, our results demonstrate the usefulness of the new Agriculture, Food and Rural Affairs (MAFRA).
speed breeding protocol developed for pepper. Pepper speed
breeding can shorten the crop period to half that normally
required for pepper, and thus shorten the crossing and inbreeding Conflict of interest
phase of breeding programs and enable breeders and scientists to
save cost and time in their research programs. The shortened The authors declare that the research was conducted in the
breeding cycle, made possible by speed breeding, leads to absence of any commercial or financial relationships that could be
decreased resource requirements linked to extended cultivation construed as a potential conflict of interest.
periods, labor expenditure, and maintenance expenses.
Furthermore, the shortened breeding time empowers breeders
and scientists to rapidly assess and evaluate different genetic Publisher’s note
combinations, facilitating prompt decision making and optimal
allocation of their research resources. In addition, breeding All claims expressed in this article are solely those of the authors
technologies such as marker-assisted or genomic selection can be and do not necessarily represent those of their affiliated
incorporated into this speed breeding system to accelerate the organizations, or those of the publisher, the editors and the
development of pepper cultivars. The developed speed breeding reviewers. Any product that may be evaluated in this article, or
protocol exhibits exceptional flexibility, making it highly adaptable claim that may be made by its manufacturer, is not guaranteed or
for farm implementation. By incorporating this protocol into their endorsed by the publisher.
farming operations, growers have the opportunity to innovate their
cultivation techniques, leading to expedited yields and heightened
overall efficiency. Furthermore, we located a new flowering gene
locus for C. annuum and revealed the changes in gene expression Supplementary material
during the speed breeding treatments. The significant associations
identified herein provide a basis for further GWAS and mapping The Supplementary Material for this article can be found online
efforts to determine causal genetic variants and to clarify how the at: https://www.frontiersin.org/articles/10.3389/fpls.2023.1151765/
associated genes affect flowering transition in pepper. full#supplementary-material

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