Capsicum annuum with causal allele of hybrid weakness is prevalent in Asia.

Reproductive isolation, including hybrid weakness, plays an important role in the formation of species. Hybrid weakness in Capsicum, the cessation of plant growth, is caused by two complementary dominant genes, A from C. chinense or C. frutescens and B from C. annuum. In the present study, we surveyed whether 94 C. annuum accessions had B or b alleles by crossing with C. chinense having the A allele. Of the 94 C. annuum accessions, five had the B allele, three of which were native to Latin America and two were native to Asia. When combined with previous studies, the percentage of B carriers was 41% in Japan, 13% in Asia excluding Japan, 6% in Latin America, and 0% in Europe and Africa. In addition, 48 accessions of C. annuum from various countries were subjected to SSR analysis. Clades with high percentages of B-carriers were formed in the phylogenetic trees. In the principal coordinate analysis, most B-carriers were localized in a single group, although the group also included b-carriers. Based on these results, we presumed that the B allele was acquired in some C. annuum lines in Latin America, and B-carriers were introduced to the world during the Age of Discovery, as along with the b-carriers.

Introduction

Cross-breeding is commonly used in plant breeding to introduce new desirable traits from related species into cultivated species. However, reproductive isolation mechanisms, which are divided into prezygotic and postzygotic isolation, often prevent inter- or intra-specific crosses. Postzygotic isolation is further divided into several phenomena including seed abortion, hybrid weakness, and hybrid breakdown. Hybrid weakness is defined as the weak growth of F1 hybrids from parents showing normal growth and has been reported in several plant species [1-4]. Hybrid weakness is also called hybrid lethality or hybrid necrosis, depending on symptoms or species. In most cases, the interaction of two complementary genes causes hybrid weakness, which is consistent with the Bateson-Dobzhanzky-Muller model, and autoimmune responses are involved in these phenomena [1, 4–6].

The Capsicum genus comprises approximately 35 species, including five cultivated species: C. annuum, C. chinense, C. frutescens, C. baccatum, and C. pubescens. Although C. annuum is the most common commercial species, some useful traits (i.e., abiotic and biotic stress tolerance [7, 8] and multiple flowers [9]) have been found in C. chinense and C. frutescens, which are related to C. annuum. It is expected that these traits can be introduced into C. annuum by cross breeding. However, reproductive isolation often works between C. annuum and C. chinense or C. frutescens [10-15]. Therefore, understanding the mechanism and developing methods to overcome reproductive isolation are needed for cross-breeding in Capsicum.

C. annuum is native to Latin America and has been introduced worldwide since Christopher Columbus discovered the West Indies [16]. During the 16–17th centuries, Capsicum migrated to Europe and Africa from Latin America, and to Asia by Portuguese [16]. During the 17–18th centuries, Capsicum from Latin America was brought several times to Europe, the Middle East, and Asia by trade [16]. Additionally, it has been suggested that Capsicum has evolved in each country where Capsicum was introduced [17-19], although the evidence is weak at present.

Hybrid weakness, formerly called as “hybrid dwarfism,”, is observed in some reciprocal crosses of C. annuum × C. chinense or C. annuum × C. frutescens [11, 12]. F1 hybrids having hybrid weakness show cessation of growth approximately 40 days after germination and a hypersensitive response-like reaction, including H2O2 accumulation, programmed cell death, and upregulation of defense-related genes in leaves [20]. Capsicum hybrid weakness is caused by two complementary dominant genes, and it has been reported that C. annuum can exhibit the aaBB genotype, whereas C. chinense and C. frutescens exhibit only the AAbb genotype. However, extensive studies have not been conducted on C. chinense and C. frutescens [11, 13]. Yazawa et al. [11] reported that the distribution of C. annuum cultivars with the B allele prevails in East Asia. However, the C. annuum cultivars used in the geographic survey of causal alleles were native to Asia and Latin America [11]; thus, their global geographic distribution is unknown.

In the present study, we report the geographic and phylogenetic distribution of C. annuum with the B allele of hybrid weakness using cultivars from across the globe to uncover the evolutionary history of the causal gene of hybrid weakness in C. annuum.

Materials and methods

Plant materials

We used 94 C. annuum accessions (including 16 C. annuum var. glabriusculum accessions; S1 Table), one C. chinense accession, one C. frutescens accession, one C. baccatum accession, and one C. pubescens accession (Table 1). After the seeds were germinated on moistened filter papers placed in Petri dishes, all seedlings were transplanted to pots (9 cm diameter, 10 cm depth) filled with culture soil (Sakata Super Mix A, Sakata Seed Co., Tokyo, Japan). The seedlings were cultivated in a constant-temperature room (25°C, 12 h light and 12 h dark, light intensity of 85 μmol m-2 s-1). At 30 days after germination, plants were transferred to bigger pots (21 cm diameter, 15 cm depth) and placed in a greenhouse (natural day length; Osaka Prefecture University, Sakai, Osaka, Japan) where the temperature was maintained above 15°C. For additional fertilization, plants were supplied weekly with Otsuka-A prescription (OAT Agrio Co., Ltd., Tokyo, Japan) containing 18.6 mM nitrogen, 5.1 mM phosphorus, 8.6 mM potassium, 8.2 mM calcium, and 0.4 mM magnesium. For the test cross, we used 94 C. annuum accessions (S1 Table) and C. chinense PI 159236 (genotype AAbb). For SSR analysis, we used 48 C. annuum accessions and one accession each of C. chinense, C. frutescens, C. baccatum, and C. pubescens (Table 1).

Table 1

Plant materials used for SSR analysis.

Reference number a	Species	Accession	Cultivar name	Origin	B or b carrier
1	C. annuum var. glabriusculum	PI 631135	Chiltepe	Guatemala	b carrier
2	C. annuum var. glabriusculum	PI 631137	Tolito	Guatemala	b carrier
3	C. annuum var. glabriusculum	PI 311126		Nicaragua	b carrier
4	C. annuum var. glabriusculum	PI 439325		Nicaragua	b carrier
5	C. annuum	PI 201237	Chilpolce Mico	Mexico	b carrier
6	C. annuum	PI 645486		Columbia	b carrier
7	C. annuum	PI 438536	Chile verde	Belize	b carrier
8	C. annuum	PI 438538	Dzabalan	Belize	b carrier
9	C. annuum	PI 209112		Puerto Rico	b carrier
10	C. annuum	PI 585246		Ecuador	b carrier
11	C. annuum	PI 241660		Peru	b carrier
12	C. annuum	PI 241646		Peru	b carrier
13	C. annuum	PI 213915		Bolivia	B carrier
14	C. annuum	PI 224413		Trinidad and Tobago	B carrier
15	C. annuum	PI 497971	Ano Todo	Brazil	B carrier
16	C. annuum	PI 640449	Szechwan 4	Taiwan	b carrier
17	C. annuum	PI 640515	Cheongryong	South Korea	b carrier
18	C. annuum	PI 286419		Nepal	b carrier
19	C. annuum	PI 645534		Papua New Guinea	b carrier
20	C. annuum	PI 640751		Vietnam	b carrier
21	C. annuum	PI 470243		Indonesia	b carrier
22	C. annuum	PI 127442		Afghanistan	b carrier
23	C. annuum	PI 135873		Pakistan	b carrier
24	C. annuum	PI 177301		Syria	B carrier
25	C. annuum	PI 181734		Lebanon	b carrier
26	C. annuum	PI 640562	Kunja	South Korea	B carrier
27	C. annuum	Grif 9089		Greece	b carrier
28	C. annuum	PI 653659		Germany	b carrier
29	C. annuum	PI 439323		Netherlands	b carrier
30	C. annuum	PI 273415	Long Hot	Italy	b carrier
31	C. annuum	PI 249908	Pimentos Morrones	Portugal	b carrier
32	C. annuum	PI 645544		Tanzania	b carrier
33	C. annuum	PI 193471		Ethiopia	b carrier
34	C. annuum	PI 640770		Uganda	b carrier
35	C. annuum	JP 32511	Sapporo Onaga Nanban	Japan	B carrier [11]
36	C. annuum	JP 32520	Mie	Japan	B carrier [11]
37	C. annuum	JP 32523	Akashi	Japan	B carrier [11]
38	C. annuum	JP 32549	Yatsubusa	Japan	B carrier [11]
39	C. annuum	JP 32555	Zairai	Japan	b carrier [11]
40	C. annuum	JP 32562	Nikko	Japan	b carrier [11]
41	C. annuum	JP 32566	Fushimiamanaga	Japan	B carrier [11]
42	C. annuum	JP 82498	Takanotsume	Japan	B carrier [11]
43	C. annuum	JP 123787	Shosuke	Japan	B carrier [11]
44	C. annuum	JP 124339	Murasaki	Japan	b carrier [11]
45	C. annuum	PI 640723	Shishito	Japan	B carrier [11]
46	C. annuum		Miogi L3	Japan	b carrier [13]
47	C. annuum		Miogi L4	Japan	b carrier [13]
48	C. annuum		Enken-amanaga	Japan	b carrier [13]
49	C. chinense	PI 159236		USA	-
50	C. frutescens	PI 586675	Tabasco	USA	-
51	C. baccatum	PI 640882		Peru	-
52	C. pubescens	PI 593624	Chile de caballo	Guatemala	-

a Reference number corresponds to the number in Fig 6

Fig 6

Principal coordinate analysis in 48 accessions of C. annuum and 4 accessions of the other species based on 34 SSR markers.

Group A corresponds to that in Fig 5.

Test cross

Conventional crossing was conducted as follows: flowers of C. annuum accessions (S1 Table) used as maternal parents were emasculated one day before anthesis and pollinated with the pollen of C. chinense PI 159236, used as paternal parents. F1 seeds were collected from fully ripe fruits. After the F1 seeds were germinated on moistened filter papers placed in Petri dishes, all seedlings were transplanted to pots (9 cm diameter, 10 cm depth) filled with culture soil (Sakata Super Mix A, Sakata Seed Co.). The seedlings were cultivated in a constant-temperature room (25°C, 12 h light and 12 h dark, 85 μmol m-2 s-1). At 60 days after germination, the genotypes of each accession were determined as the aaBB genotype if all F1 hybrid plants did not obtain the first flower due to growth arrest, a characteristic of Capsicum hybrid weakness [20], and the aabb genotype if all F1 hybrid plants received the first flower and continued to grow.

DNA extraction

Total DNA of accessions for SSR analysis (Table 1) was extracted from individual leaves using the cetyltrimethylammonium bromide (CTAB) method [21] with minor modifications. Each leaf was ground in a mortar with liquid nitrogen. The ground leaf was mixed with CTAB isolation buffer (2% w/v CTAB, 1.4 M NaCl, 0.2% v/v β-mercaptoethanol, 20 mM EDTA, and 100 mM Tris-HCl, pH 8.0) preheated to 60°C, and the mixture was incubated at 60°C for 60 min. The suspension was extracted twice with chloroform/isoamyl alcohol (24:1) and centrifuged for 15 min at 500 g. The aqueous phase was transferred to a new tube, and nucleic acids were precipitated by the addition of isopropanol (2/3 volume) and centrifuged for 20 min at 1,000 g. The pellet was washed with 70% ethanol and dissolved in 50 μL of Tris-EDTA buffer (10 mM Tris-HCl and 1 mM EDTA, pH 8.0).

SSR analysis

34 SSR markers were selected from Sugita et al. [22] (Table 2 and S2 Table). PCR was performed with a final volume of 10 μL, which contained 2 mM each of dNTP, 10× reaction buffer, 0.2 mM each of forward and reverse primer pairs, 0.5 U of Taq polymerase, and 50 ng of DNA as template. PCR reaction cycles consisted of initial denaturation at 94°C for 2 min, followed by 35 cycles of 94°C for 30 s, 54°C for 1 min, 72°C for 30 s, and final extension for 2 min at 72°C. The PCR products were separated by electrophoresis on 8% polyacrylamide gels in Tris-borate-EDTA. Electrophoresis was performed at 55 mA for 90 min at 25°C. The gels were stained with ethidium bromide and visualized using a UV lamp. Polymorphic alleles were scored as present or absent by visual inspection.

Table 2

Polymorphic information of SSR markers in the present study.

SSR markers	Linkage group by Sugita et al [22]	Na	I	He	Ho
ge257-93pmA0742C	1	4	0.873	0.474	0.000
ge214-224pmc0769C	2	5	1.194	0.607	0.000
CAMS-358	3	4	0.879	0.463	0.000
CAMS-861	4	4	0.464	0.215	0.000
ge273-249pmc0783C	5	3	0.634	0.385	0.000
ge245-46pmA0870C	6	7	1.705	0.785	0.000
ge105-630pms0282C	7	6	1.073	0.556	0.000
Hpms 1–155	8	5	0.969	0.486	0.000
ge247-859pmA1091C	9	4	0.929	0.510	0.000
ge54-242pmA0133C	10	6	1.491	0.740	0.000
Hpms 2–2	11	5	0.858	0.453	0.000
CAMS-301	1	4	1.002	0.562	0.000
es723TC3943S	1	5	1.297	0.683	0.000
ge232-524pmr0557C	2	6	1.346	0.668	0.000
CAMS-492	2	3	0.829	0.531	0.000
es777CA516741S	3	4	0.834	0.464	0.000
ge279-375pmH0430W	3	5	1.259	0.669	0.000
es713BM064640S	4	6	1.486	0.714	0.000
PM12	4	6	1.343	0.679	0.000
CAMS-051	5	5	1.287	0.674	0.000
CAMS-020	5	2	0.540	0.355	0.000
CAMS-361	6	3	1.008	0.603	0.000
CAMS-351	6	5	1.221	0.624	0.000
PM27	7	5	1.469	0.741	0.000
CAMS-451	8	6	1.607	0.769	0.000
CAMS-405	8	5	1.263	0.668	0.000
CAMS-679	9	8	1.729	0.774	0.059
CAMS-844	9	6	1.420	0.714	0.000
CAMS-336	10	5	1.368	0.706	0.000
PM18	10	5	1.247	0.660	0.000
PM29	11	9	1.661	0.754	0.000
PM37	11	7	1.584	0.749	0.000
ge250-854pmr0454C	12	3	0.810	0.479	0.000
es743CA521534S	12	5	1.292	0.672	0.000

Na, number of different alleles; I, Shannon’s information index; He, Nei’s unbiased gene diversity index; Ho, observed heterozygosity.

Data analysis

GenAlEx 6.51 [23] was used to calculate the number of different alleles (Na), Shannon’s information index, observed heterozygosity (Ho), and expected heterozygosity (He) for each SSR marker. Power Marker version 3.25 [24] was used to calculate genetic distance and phylogenetic construction, and MEGA X [25] was used for viewing the dendrogram. For the phylogenetic analysis, genetic distance was calculated using Nei’s genetic distance [26], followed by phylogeny construction using the unweighted pair group method using arithmetic average (UPGMA) and neighbor-joining (NJ) methods. Branch support was assessed by bootstrap resampling with 1000 replicates. Principal coordinate analysis (PCoA) among populations was also conducted using GenAlEx 6.51 [27].

To infer the population structure of the pepper species used in this study, we used a model-based clustering algorithm implemented in the computer program Structure version 2.3.4 [28]. Analyses were based on an admixture ancestral model with correlated allele frequencies. The analysis was run for K = 1–10 with 10,000 Monte Carlo Markov Chain replicates after a burn-in of 10,000 replicates. For each value of K, 10 independent runs were performed to generate an estimate of the true number of subpopulations. The optimal K was selected using the Evanno method [29] estimated using Structure Harvester [30].

Results

Geographical distribution of hybrid weakness causal gene B-carrier in C. annuum

We surveyed whether 94 C. annuum accessions had the B allele of hybrid weakness by crossing with C. chinense having the A allele (S1 Table). Five accessions had B allele and the remaining 89 accessions had b allele. Among the B-carriers, three accessions were native to Latin America and two were native to Asia. The percentage of B-carriers was calculated for each geographic region (Latin America, Europe, Africa, Asia excluding Japan, and Japan), combining the present results with those of a previous study [11] (Tables 1 and 3, Fig 1). The percentage of B-carriers was 13% in all C. annuum accessions, 41% in those from Japan, 13% in those from Asia excluding Japan, 6% in those from Latin America, and 0% in those from Europe and Africa.

Table 3

Geographic distribution of b and B carriers.

		Yazawa et al. [11]			This study			Total
Region	b carrier	B carrier	Total	b carrier	B carrier	Total	b carrier	B carrier	Total
Latin America	26	1	27	34	3	37	60	4	64
Europe	-	-	-	17	0	17	17	0	17
Africa	-	-	-	16	0	16	16	0	16
Japan	19	14	33	1	0	1	20	14	34
Asia excluding Japan	33	6	39	21	2	23	54	8	62
All	78	21	99	89	5	94	167	26	193

Fig 1

Distribution and frequency of B or b allele related to hybrid weakness in C. annuum.

The data was based on this and a previous study [11]. The map is based on OpenStreetMap (https://www.openstreetmap.org/copyright).

Polymorphism and allelic diversity evaluated by SSR analysis

We analyzed 48 C. annuum accessions, including four C. annuum var. glabriusculum accessions (13 B-carriers and 35 b-carriers), one C. chinense accession, one C. frutescens accession, one C. baccatum accession, and one C. pubescens accession, using 34 SSR markers (Tables 1 and 2). The number of Na was 2–9, and the Shannon’s information index of the genetic variation index was 0.464–1.729. The He was 0.215–0.785. The Ho value was 0.059 for CAMS-679 but 0 for the other markers.

Genetic relationship analysis

Two phylogenetic trees were constructed using the UPGMA and NJ methods, based on analysis using 34 SSR markers (Figs 2 and 3). C. annuum accessions in Latin America, including C. annuum var. glabriusculum were close to those of other domestic species: C. chinense, C. frutescens, C. baccatum, and C. pubescens (Figs 2 and 3). C. annuum accessions did not form a region-specific clade on either UPGMA or NJ trees (Figs 2 and 3). The number of clades with a high percentage of B-carriers was two in the UPGMA tree and one in the NJ tree (Figs 2 and 3). Although PI 213915, PI 177301, and ‘Mie’ were B-carriers, they were not included in the clade with a high percentage of B-carriers in the UPGMA and NJ trees.

Fig 2

Phylogenetic UPGMA trees of 48 accessions of C. annuum with 4 other species based on 34 SSR markers.

B or b carriers are indicated as red or white circles, respectively. The branches in the clade with high percentage of B carriers are shown in red.

Fig 3

Phylogenetic NJ trees of 48 accessions of C. annuum with 4 other species based on 34 SSR markers.

B or b carriers are indicated as red or white circles, respectively. The branches in the clade with high percentage of B carriers are shown in red.

Population structure analysis

Population structure analysis was conducted based on the SSR analysis data. As the mean Delta K was maximum at K = 2, optimum K was 2 for population structure analysis (Fig 4). The accessions were divided into two groups (groups A and B) according to population structure (Fig 5). There were C. pubescens, C. frutescens, C. baccatum, C. chinense, and six accessions of C. annuum from Latin America, including C. annuum var. glabriusculum, and three accessions of C. annuum from Japan in group A, whereas there were 40 C. annuum accessions from various regions in group B. B-carriers were included in both groups.

Fig 4

Determination of optimal K by structure harvester.

K = 2 was considered as the optimal number of populations.

Fig 5

STRUCTURE analysis in 48 accessions of C. annuum and 4 other species based on 34 SSR markers.

B or b carriers were indicated as respectively red or white circles.

PCoA

PCoA was conducted based on the SSR analysis data to uncover phylogenetic relationships in two dimensions (Fig 6). The first two principal coordinate axes explained ~16.17% of the variation in the genetic distance matrix. The accessions of group A defined in the structural analysis also formed a group in the PCoA. C. annuum accessions from Latin America were distributed across a wide area of the graph. Most B-carriers were distributed in part of the area indicated as B-carrier group, although the area also included some b-carriers.

Discussion

In the present study, SSR analysis was conducted on 52 accessions of domesticated Capsicum species, using 34 markers covering all chromosomes. The Ho values were almost zero as heterozygous alleles were not detected for most markers, whereas He values ranged from 0.215 to 0.785 (Table 3). In general, Ho values lower than He values are due to the lack of crossbreeding with other accessions. Low Ho values in the domesticated Capsicum species have also been reported in other studies [17, 31]. Therefore, the low Ho value in the present study may suggest that the accessions supplied in the present study were fixed at almost all loci as all accessions supplied were domesticated species.

Accessions of C. annuum var. glabriusculum were closer to C. chinense and C. frutescens than the other accessions of C. annuum (Figs 2, 3, 5, and 6). C. annuum var. glabriusculum is considered the wild ancestor of C. annuum, and C. chinense and C. frutescens are closely related to C. annuum [32, 33]. Therefore, our results show that C. annuum evolved via the C. annuum var. glabriusculum after speciation from the common ancestors of C. annuum, C. chinense, and C. frutescens.

Accessions of C. annuum from various countries were investigated using SSR analysis. The accessions of C. annuum in Latin America had larger genetic diversity than those in other regions (Fig 6). A diversity center seems to be a region where plants are domesticated from wild species. It is assumed that C. annuum differentiated from a common ancestor of related species in Latin America [10, 32]. Moreover, it was found that C. annuum was domesticated in Mexico from C. annuum var. glabriusculum [34]. Therefore, our finding of a large diversity in Latin American lineages supports those of previous studies showing that Latin America is diversity-centric.

There was no particular tendency in the genetic structure by geographic origin, excluding Latin America, in C. annuum accessions (Figs 2, 3, 5, and 6). Similarly, a previous study showed that geographic origin had little effect on the genetic structure of C. annuum accessions, although C. annuum accessions from Turkey and central Europe formed a cluster [17]. Another study examined the phylogenetic relationship of nearly 10,000 lineages around the world, showing some effect of geographic origin on the genetic structure. However, the overlap was significantly higher than segregation in genetic structure among lineages with other regions [19]. Archaeological studies show that C. annuum existed more than 6,000 years ago [34, 35], although it is unknown when the species was established. It has only been about 500 years since C. annuum was introduced across the world during the Age of Discovery. Therefore, there may be less time for the lineages of C. annuum to change their genetic structure by geographic origin.

The accessions of B-carrier were in Latin America and Asia, and the percentage of B-carriers was particularly high in Japan (Fig 1). Here, we suggest a model showing the process of causal gene acquisition and the global spread of lines having B or b alleles (Fig 7). The common ancestor of C. annuum, C. chinense, and C. frutescens appears to have had the aabb genotype. In C. chinense and C. frutescens, only lines with the AAbb genotype have been reported, although no extensive studies have been conducted [11, 13]. All the lines in C. chinense and C. frutescens might have acquired the A gene after speciation from a common ancestor. However, parts of C. annuum acquired the B gene. No accessions of C. annuum var. glabriusculum, which is the wild ancestor of C. annuum, had the B allele in our survey. This suggests that C. annuum acquired the B allele since domestication or fewer lines have this allele. In the Age of Discovery, C. annuum was discovered in Latin America and was introduced globally by trade. Then, most lines brought to the world may be b-carriers. However, the lines brought to Asia, particularly Japan, included B-carriers. In fact, PI 213915 and PI 224413, the B-carriers in Latin America, were genetically close to the accessions in Japan (Fig 5). Therefore, these or similar lines were likely brought to Japan. Therefore, in Asia, C. annuum lines would have been bred based on the lines of both B- and b-carriers from Latin America. Perhaps the B allele might have functions that show suitable traits for the Asian environment or might be involved in traits preferred by Asians, as B-carriers exist at a high ratio in Japanese accessions.

Fig 7

Model on the process of causal gene acquisition and the spread of the lines of B- or b-carriers to the world.

It has been suggested that causal genes for hybrid necrosis may have been used in wheat breeding [36]. In many cases, hybrid weakness or necrosis involves an autoimmune response. Therefore, it is possible that the causal gene contributes to disease resistance [36, 37]. Similarly, Capsicum hybrid weakness has been shown to cause an autoimmune response [20], and B-allele of Capsicum hybrid weakness may have been used in the breeding process.

Our study suggests a history of B-allele involvement in Capsicum hybrid weakness in acquisition and spread. In the future, identification of causal genes A and B for hybrid weakness will advance research on speciation in Capsicum.

Origin and F1 phenotype of C. annuum as revealed by test crosses with C. chinense.

(XLSX)

Click here for additional data file.

Information on the primer sequences used in the present study.

(XLSX)

Click here for additional data file.

20 May 2022

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The Gateway to Astronaut Photography of Earth (public domain): http://eol.jsc.nasa.gov/sseop/clickmap/

Maps at the CIA (public domain): https://www.cia.gov/library/publications/the-world-factbook/index.html and https://www.cia.gov/library/publications/cia-maps-publications/index.html

NASA Earth Observatory (public domain): http://earthobservatory.nasa.gov/

Landsat: http://landsat.visibleearth.nasa.gov/

USGS EROS (Earth Resources Observatory and Science (EROS) Center) (public domain): http://eros.usgs.gov/#

Natural Earth (public domain): http://www.naturalearthdata.com/

3.  Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The manuscript is well written and I would like to congratulate the authors to the interesting manuscript and would like to recommend it for publication. the findings reported in the manuscript 'Capsicum annuum with causal allele of hybrid weakness is prevalent in Asia' will have great impact in the field of Capsicum sp. breeding.

Reviewer #2: In this manuscript, the authors reported their study of the presence of B or b allele for hybrid weakness in Capsicum annuum accessions from different countries. The work also inculded previous results which had been published in Japanese. Crossing between accessions with different genotypes were performed and SSR markers were analyzed. The experiments were nicely performed, and data were convincing. The manuscript is written in a clean and clear manner, and is easy to follow. I have only a few tiny suggestions.

1. Line 133, "tris" should be "Tris".

2. Line 142, "Mega" should be "MEGA".

3. Lines 84-86, I would suggest to revise it as "We used 94 C. annuum accessions (including 16 C. annuum var. glabriusculum accessions, Table S1), one C. chinese accession...". In this way, readers would not try to find information of other species from Table S1.

4. Line 107, ", Tokyo, Japan" can be removed. It has been introduced for Sakata Seed Co. before (line 88).

Reviewer #3: Reproductive isolation, including hybrid weakness, plays an important role in the formation of species.In this manuscript, authors surveyed whether 94 C. annuum accessions had B or b alleles by crossing with C. chinense having the A allele. Of the 94 C. annuum accessions, five had the B allele, three of which were native to Latin America and two were native to Asia. When combined with previous studies, the percentage of B carriers was 41% in Japan, 13% in Asia excluding Japan, 6% in Latin America, and 0% in Europe and Africa. In addition, 48 accessions of C. annuum from various countries were subjected to SSR analysis. Clades with high percentages of B -carriers were formed in the phylogenetic trees. In the principal coordinate analysis, most B -carriers were localized in a single group, although the group also included b -carriers. Based on these results, we presumed that the B allele was acquired in some C. annuum lines in Latin America, and B -carriers were introduced to the world during the Age of Discovery, as along with the b -carriers.

The manuscript has been well written. A good result about the B allele from C. annuum has been identified. It would be useful for the relative people to know.

(1)Line 61- 62: Capsicum migrated to Europe and Africa from Latin America and to Southeast Asia during the 17–18th centuries from Peru.

From my view, “pepper was introduced to Southeast Asia during the 16th centuries probably by Portuguese .”

For evidence:

In 1542, the Portuguese brought pepper to Goa, India, and pepper began to spread in South Asia. Then it spread to Malacca and spread in Southeast Asia. At the same time, the Spanish brought pepper to Luzon, the Philippines, which is another transmission point of pepper in Southeast Asia (watt, 2014).

It was first recorded in Japanese literature that chili was introduced in 1552. Balthazar Gago, a Portuguese missionary, gave it as a gift to daiyo Town, which was then known as Toyoda and Toyoda of Kyushu Island, 39 years earlier than China (Yamamoto, 2018).

During the compilation of Korean "history of Korea", it was introduced into the Korean "history of Korea" in 1602.

The earliest record of pepper in China can be found in Zunsheng eight notes by Gao Lian of the Ming Dynasty (1591): "pepper is born, white flowers, and the fruit looks like bald pen head. It tastes spicy and red, which is very impressive."

Reference:

Watt G. 2014. A dictionary of the economic products of India. Landon: Cambridge University Press：197

Yoshimoto Yamamoto (translated by Chen Xian-ruo ). 2018. World history of pepper: a journey across Europe, Asia, and Africa, a hot table revolution. Taiwan:Marco Polo Press：256

(2)Line 114-, “DNA extraction” Whose DNA will be extracted should be mentioned.

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Reviewer #1: Yes: Julia Meitz-Hopkins, PhD

Reviewer #2: No

Reviewer #3: No

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