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. 2021 Dec 14;27(12):2757–2765. doi: 10.1007/s12298-021-01109-9

Development and application of KASP markers associated with Restorer-of-fertility gene in Capsicum annuum L.

Zhenghai Zhang 1, Dongliang An 1, Yacong Cao 1, Hailong Yu 1, Yanshu Zhu 1, Yajie Mei 1, Baoxi Zhang 1, Lihao Wang 1,✉
PMCID: PMC8720122  PMID: 35035134

Abstract

Fertility restoration of cytoplasmic male sterility (CMS) in Capsicum annuum is controlled by multiple alleles of Restorer-of-fertility (Rf) genes. The isolation of additional Rf genes should therefore enrich the knowledge of CMS/Rf systems and accelerate their exploitation in hybrid seed production. In this study, the fertility restorer gene CaRfm of ‘0601 M’, a non-pungent bell pepper, was genetically mapped to a 1.2-cM region flanked by KASP markers S761 and S183. CaRfm was then physically mapped to a 128.96-Kb interval predicted from 24 recombinants with two co-segregated markers, S423 and S424. CaPPR6 encoding a pentatricopeptide repeat (PPR) protein was suggested as the most likely candidate gene for the CaRfm locus on the basis of sequence alignment as well as genotyping of tightly linked markers. In addition, molecular markers S1597 and S1609, which are immediately adjacent to CaRfm at 15.7 and 57.8-Kb respectively, were developed and applied to marker-assisted selection. The results provided friendly markers for breeding pepper restorer lines and laid the foundation for elucidating the male fertility restoration mechanism.

Supplementary Information

The online version contains supplementary material available at 10.1007/s12298-021-01109-9.

Keywords: Genetic mapping, KASP markers, Restorer-of-fertility, Sweet pepper

Introduction

Peppers (Capsicum spp.) belong to the Solanaceae family are native to the tropical and subtropical Americas and comprise 35 species (Carrizo García et al. 2016). Five cultivated species of Capsicum, C. annuum L., C. baccatum L., C. chinense Jacq., C. frutescens L., and C. pubescens Ruiz and Pav, are among the world’s most consumed spices and vegetables (Wang and Bosland 2006; Bosland and Votava 2012). Worldwide, the pepper planting area was 3.72 million hectares (ha) and 42.3 million tons of peppers were produced (FAOSTAT 2021). In China, the total planted area of pungent and non-pungent Capsicum varieties exceeds 2.1 million ha with an annual production of 64 million tons (Zou et al. 2020). F1 hybrid cultivars have greater stress tolerance and yield ability than open-pollinated varieties, which account for more than 80% of the pepper seed market in China (Wang et al. 2020). The use of male sterile lines facilitates efficient commercial hybrid seed production by eliminating manual emasculation, thereby reducing labor costs, and ensuring the purity of hybrid seeds.

Nuclear male sterility and cytoplasmic male sterility (CMS) systems are present in Capsicum (Jindal et al. 2019). CMS is a maternally inherited trait controlled by mitochondrial genes. The application of CMS to hybrid seed production requires a male sterile line [(S) rfrf], a maintainer line [(N) rfrf], and a male fertility restoration line [(S/N) RfRf] and is referred to as the three-line system. A strong male fertility restorer line harboring a Restorer-of-fertility (Rf) gene is needed to ensure the fertility of F1 hybrids. The selection and/or breeding of male fertility restoration line is an important breeding goal in the CMS/Rf system.

Rf genes, which are responsible for male fertility restoration in CMS/Rf systems, have been isolated from maize (Cui et al. 1996), rice (Komori et al. 2004; Wang et al. 2006; Fujii and Toriyama 2009; Itabashi et al. 2011; Hu et al. 2012; Tang et al. 2014; Huang et al. 2015; Liu et al. 2016), wheat (Castillo et al. 2014, 2015), barley (Ui et al. 2015), sugar beet (Matsuhira et al. 2012; Kitazaki et al. 2015), rapeseed (Uyttewaal et al. 2008), cotton (Zhang et al. 2005; Yin et al. 2006), radish (Koizuka et al. 2003), onion (Kim et al. 2015), Chinese cabbage (Zhang et al. 2017), and chili pepper (Jo et al. 2010, 2016; Wu et al. 2019; Cheng et al. 2020; Wei et al. 2020; Zhang et al. 2020). Most of the Rf genes have been found to encode pentatricopeptide repeat (PPR) proteins. Other types of identified Rf genes include Rf2 (encoding aldehyde dehydrogenase) in maize (Cui et al. 1996), Rf2 (encoding glycine-rich protein) and Rf17 (encoding acyl-carrier protein synthase) in rice (Fujii and Toriyama 2009; Itabashi et al. 2011), and Rf1 (encoding peptidase) in sugar beet (Kitazaki et al. 2015). Research on plant male fertility restoration genes is essential for CMS utilization in hybrid breeding and contributes to the understanding of nuclear-cytoplasmic interactions.

Fertility restoration in chili pepper is a quantitatively or qualitatively inherited trait controlled by single or multiple genes (Peterson 1958; Novak et al. 1971; Wang et al. 2004; Jo et al. 2010, 2016; Wu et al. 2019; Cheng et al. 2020; Wei et al. 2020; Zhang et al. 2020). Chili pepper restorer lines have different restoration abilities, with their F1 hybrids endowed with complete (Wang et al. 2004; Jo et al. 2010, 2016; Wu et al. 2019; Cheng et al. 2020; Wei et al. 2020; Zhang et al. 2020) or partial (Lee et al. 2008a) restoration. In some studies, the F1 crossed by sterile and restore lines has normal fertility, with different fertility levels observed among fertile F2 plants (Wang et al. 2004). In other studies, the fertility restoration of F2 plants by some fertility restorer lines was equal to that of F1 plants (Jo et al. 2010, 2016; Cheng et al. 2020; Zhang et al. 2020). These conflicting results indicate that the genetic control of fertility restoration in chili pepper is diverse and complex and that some fertility restorer lines of chili pepper may have major and minor (partial) Rf genes.

Following the publication of the chili pepper genome, several Rf genes have been isolated and characterized in chili pepper. Jo et al. (2010) cloned the PePPR1 using a homology-based approach that included a petunia Rf sequence as a reference and then isolated CaPPR6 from C. annuum ‘Chungyang C’ using comparative mapping and chromosome-walking techniques (Jo et al. 2016). Wu et al. (2019) identified Capana06g002967 and Capana06g002969 as Rf candidate genes in a genome-wide association study combining functional annotation and expression analysis. Cheng et al. (2020) fine-mapped the CaRf gene in C. annuum ‘B702’ based on a conjoint analysis of recombinants and genome collinearity. Zhang et al. (2020) fine-mapped the CaRf032 (CA00g82510) from C. annuum ‘IVF2014032’ by genome resequencing and recombinant analysis. Wei et al. (2020) used bulk segregant RNA sequencing to predict the NEDD 8 conjugating enzyme gene (Capana06g002866) as the Rf gene of C. annuum ‘R1’. In addition, Barchenger et al. (2018) identified 552 PPR domains in the C. annuum ‘Zunla-1’ genome. Twelve PPRs (11 clustered on chromosome 6, and one located on chromosome 1) were identified as candidate Rf genes. These studies indicate that multiple Rf genes may be present in the chili pepper genome and that different restorer lines may have genotype-specific Rf genes. This situation hinders the development of a universally applicable marker for the fertility restorer genotype in chili pepper.

Many different types of molecular markers for chili-pepper fertility restorer genes have been developed. Such markers include sequence-characterized amplified region (SCAR) (Gulyas et al. 2006; Lee et al. 2008a; Jo et al. 2010; Wu et al. 2012), random amplified polymorphic DNA (Zhang et al. 2000), simple sequence repeat (SSR) (Cheng et al. 2020), insertion–deletion (InDel) (Cheng et al. 2020), cleaved amplified polymorphic sequence (CAPS) (Kim et al. 2006; Lee et al. 2008a, 2008b; Min et al. 2009; Jo et al. 2016; Ortega et al. 2020), and Kompetitive Allele-specific PCR (KASP) (Wei et al. 2020; Zhang et al. 2020) markers. According to various studies, the marker CRF-SCAR (Lee et al. 2008a), derived from the marker CRF3S1S (Gulyas et al. 2006), has the highest rate of accurate detestation of the fertility restorer trait reported as 89.1% (Jo et al. 2010), 88.7% (Jo et al. 2016), and 79.2% (Zhang et al. 2020) in different natural populations. The CaPPR6 co-segregated marker Mo1Cod1-CAPS has been found to have a relatively higher rate of 88.0% and 92.2% in 50 and 51 chili pepper groups, respectively (Jo et al. 2016). In contrast, the accuracy rate of KASP markers co-segregating with CaRf032 was only 64.4% in 101 chili pepper groups, as opposed to 77.2%, 79.2, and 70.3% for CRF1S3S, CRF-SCAR, and Co1Mod1-CAPS, respectively (Zhang et al. 2020). These results further illustrate that multiple Rf genes are present in the chili pepper genome and that different restorer lines may have genotype-specific Rf genes. The development of universal markers or less genotype-specific ones is thus the most effective way to improve the accuracy rate of Rf markers.

In this study, we fine mapped the fertility restoration locus CaRfm of non-pungent bell pepper 0601 M to a 128.96-Kb region on chromosome 6 of the CM334 reference genome (Kim et al. 2014). On the basis of sequence alignment and molecular marker analysis, we identified CaPPR6 of chili pepper Chungyang C (Jo et al. 2016) as a strong candidate gene for CaRfm and developed multiple tightly linked markers of CaRfm for use in marker-assisted selection for CMS/Rf system breeding.

Materials and methods

Plant materials

The CMS line 77013A (AP) was crossed with restorer line 0601 M (RP) to develop an F2 segregating population. The 77013A was developed in the Institute of Vegetables and Flowers (IVF, Beijing, China) (Wang et al. 2004), and 0601 M was developed by self-pollination of a farm variety provided by Taiyuan Yinong horticulture seedling co. Ltd. As the recurrent parent, the C. annuum maintainer line 77,013 (BP) was crossed with the fertile progeny of F2 to develop near-isogenic restorer lines containing the restorer gene from 0601 M. In addition, seven backcrossed, higher-generation individuals were crossed with AP to check their restoration ability on the basis of phenotype and to confirm the Rf candidate region according to the genotypes of closely linked markers. We used 101 accessions (Table S1) from National Mid-term Genebank for Vegetables, Beijing, China. (Gu et al. 2019), and the segregating populations of chili pepper cultivar ‘Xiyangyang’ (Qingzhou Tiancheng Agricultural Development Co., Shandong, China), ‘Weishiyihao’(Dong Fang Chia Tai Seed Co., Beijing, China), Kcdian Hot (South Korea), and KmaA (South Korea) to validate the molecular markers for CaRfm of 0601 M developed in this study.

All plants were grown in a greenhouse with day/night temperatures of 26–32/16–22 °C under natural light during April to June at Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences. Phenotyping of male-fertile and male-sterile individuals was performed by visual observation of the presence or absence of pollen grains and fruits (if set fruits) with or without seeds according to Zhang et al. (2020). Segregation was assessed by performing chi-square tests using SPSS Statistics 17.0 (SPSS Inc., Chicago, IL, USA).

Molecular marker development and genotyping

Primers design and KASP assays were performed according to Zhang et al. (2020). SSR detection and primer design were carried out using the web-based version of BatchPrimer3 (https://wheat.pw.usda.gov/demos/BatchPrimer3/) (You et al. 2008) following standard guidelines. InDels and structural variations (SVs) were detected by SAMtools (Li et al. 2009) from the genome resequencing date of AP and RP parent lines. PCR primers for these two types of markers were designed using Primer3web (version 4.1.0) online software (Untergrasser et al. 2012). Primers details are provided in the supplementary information (Table S2).

PCR amplifications were performed in a 20 μl reaction volumes consisting of 10 μl of 2 × GoTaq Green Master Mix (Promega, USA), 1 μl template DNA (100 ng), 1 μl of each primer (10 μM), and 7 μl ddH2O. PCR cycling conditions were as follows: a hot start at 94 °C for 5 min, followed by 35 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s, with a final extension of 72 °C for 10 min. The products were electrophoresed on 7% polyacrylamide gels. The gels were then stained with 0.12% silver nitrate for 5 min, followed by band development in 1.5% sodium hydroxide solution and the fresh addition of 1.5 ml of 37% formaldehyde before use.

Genetic linkage analysis and fine mapping of CaRfm

A genetic linkage analysis of CaRfm using 180 F2 individuals derived from AP and RP was carried out by SNP genotyping with a KASP assay. The SNPs between parents were generated from the genome sequencing date of AP and RP lines using Illumina. Plant genome resequencing, sequence alignment, and SNP detection were performed by OE Biotechnology (Beijing, China). Sequencing reads were aligned to the CM334 reference genome v1.55 (Kim et al. 2014) using Burrows-Wheeler Aligner software (Li and Durbin 2009), and SNPs were detected using SAMtools (Li et al. 2009). Allele-specific primers were designed using the BatchPrimer3 (https://wheat.pw.usda.gov/demos/BatchPrimer3/), the KASP assay was developed on a Roche LightCycler® 480 (Roche Diagnostics, Rotkreuz, Switzerland) in 4-μl reaction mixtures consisting of 2 μl template DNA (5 ng μl−1), 2 μl of 2 × KASP Master Mix, and 0.055 μl primer mixture according to Zhang et al. (2020). Genetic maps were constructed using JoinMap v4 (Van Ooijen 2006) with a minimum LOD score of 3 and a maximum recombination fraction of 0.3.

To fine map CaRfm, recombinant screening was carried out on 4, 961 plants from different segregating populations (568 F2, 682 BC4F2-7, 958 BC4F2-27, 754 BC4F2-33, 190 BC6-17, 998 BC7-3, 498 BC7-10, and 313 BC7-15) with newly developed polymorphic markers (34 KASP, four InDel, one SV, one SSR, and one derived cleaved amplified polymorphic sequences) located within the candidate interval region. We initially screened for candidate genes from the coding DNA sequence of CM334 (Kim et al. 2014).

Cloning of the CaPPR6 sequence from maintainer and restorer plants

PCR amplifications were performed according to Zhang et al. (2020) using primers 5'-ACCTGCAGATAAACTTAACAAACACT-3′ and 5′-GTGAAGAAACCCTATAACATAGTAGCTT-3′. PCR products were purified with a TIANgel Midi DNA Purification kit (Tiangen, Beijing, China), cloned into an pEASY-BLUNT vector (Tiangen), and transformed into competent cells of Escherichia coli strain TOP10 (Tiangen). Sequencing of the confirmed inserts was performed by Sangon Biotech Shanghai Co. (Shanghai, China) using vector primers M13-20 and M13-26. A multiple sequence alignment was generated using DNAMAN v6 software.

Verification and application of CaRfm-linked markers

The candidate region of CaRfm was further verified by genotyping co-segregated markers in six populations derived from crosses AP with BC10-4, BC10-7, BC10-8, BC10-9, BC10-13, and BC10-14. The detection accuracy of the co-segregated marker was assessed in 38 maintainer and 63 restorer breeding lines used by Zhang et al. (2020) and the segregating populations of chili pepper cultivar ‘Xiyangyang’, ‘Weishiyihao’, Kcdian Hot, and KmaA.

Results

Genetic analysis of the male fertility restoration locus

Male-fertility (MF) and Male-sterility (MS) phenotypes were clearly distinguishable in observed anthers during flowering. The fertility of the BP and RP parents was normal, and the male-sterile parent AP displayed complete and stable sterility. All F1 plants were fertile, whereas 180 F2 progeny segregated into 139 MF and 41 MS plants, which showed no significant deviation from the expected 3:1 Mendelian ratio (χ2 = 0.4741, p = 0.4911) (Table 1). All BC1RP plants exhibited fertility, while the BC1BP population was separated into 27 male-fertile plants and 43 male-sterile plants, fitted to a 1:1 ratio (Table 1). These results indicate that the male fertility restoration trait of 0601 M is controlled by a single dominant gene, which we named CaRfm.

Table 1.

Genetic analysis of male fertility and sterility in segregating populations developed from cytoplasmic male sterility lines

Populations* Number of plants Expected Ratio χ2 Probability**
Fertile Sterile Total
F1 (AP × RP) 117 0 117 / / /
F2 (AP × RP) 139 41 180 3:1 0.4741 0.4911
BC1RP (F1 × RP) 72 0 72 1:0 / /
BC1BP (F1 × BP) 27 43 70 1:1 3.6571 0.0558
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* AP, The cytoplasmic male sterility line 77013A; RP, the restorer line 0601 M; BP, the maintainer line 77,013; ** Probability > 0.05 were considered to confirming this expected ratio

Genetic linkage map and fine mapping of CaRfm

First, we used 11 KASP markers to develop a linkage map based on 180 individuals of an F2 population. As a result, CaRfm was preliminarily mapped to a 1.2-cM interval flanked by markers S761 and S183 (Fig. 1a).

Fig. 1.

Fig. 1

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Genetic mapping of the CaRfm locus of Capsicum annuum ‘0601 M’ using the CM334 reference genome (v1.55). a Genetic linkage map of the CaRfm locus based on 11 KASP markers. b Recombinants between phenotypes and marker types in the segregating population. Ten marker types were obtained among the 24 recombinants; types 1 to 10 consisted of two, one, one, seven, two, one, two, one, four, and three recombinants, respectively. Yellow, red, and green rectangles indicate BP, RP, and F1 marker types, respectively. BP, maintainer line 77,013; RP, restorer line 0601 M; MS, male-sterile; MF, male-fertile

To narrow the CaRfm region, recombinant individuals were screened from segregating populations (F2, BC4F2-7, BC4F2-27, BC4F2-33, BC6-17, BC7-3, BC7-10, and BC7-15) using four flanking markers: S112, S761, S183, and S1036. We identified 24 recombinants—12 male-fertile and 12 male-sterile out of 4,961 individuals. We genotyped these 24 recombinants using 15 newly developed markers (13 KASP, one SSR, and one SV) located between the four flanking markers, which allowed us to delimit the CaRfm candidate region to the interval between S409 and S425 at a physical distance of 128.96-Kb (Fig. 1b).

We observed that two markers, S423 and S424, co-segregated with CaRfm in the 24 recombinant plants (Fig. 1b). We then identified two potential candidate genes, CA06g00210 and CA06g00220, predicted to encode PPR proteins and annotated within the interval of the CM334 reference genome (Kim et al. 2014); however, CA06g00210 was only expressed at low levels in flower buds, and CA06g00220 was not significantly expressed in the parental lines (data not shown).

Candidate genes screening and sequence alignment

No candidate genes were found in the fine mapping interval. So, a BLAST search against the National Center for Biotechnology Information (NCBI) database aligned the sequence delimited by the CaRfm-linked markers to the CaPPR6-containing sequence KU997025.1 (CaPPR6-SEQ). This 574, 716-bp CaPPR6-SEQ sequence, which was cloned by chromosome walking, contains the chili pepper Rf gene CaPPR6 (Jo et al. 2016). We subsequently aligned the whole CaPPR6-SEQ sequence against the CM334 reference genome. As a result, CaPPR6-SEQ could be delineated into four sections (A, B, C, and D). Sections A and D, at the two ends of CaPPR6-SEQ sequence, were reversed and forward compared with their arrangement on the CM334 assembly (Fig. 2b). Sections B and C, did not overlap with the CaRfm candidate region (Fig. 2a, b). Sections B and C, located between sections A and D, were closely matched to the CM334 genomic physical locations of 2,221,405– 2,387,187 and 1,259,830–1,403,516, respectively (Fig. 2a, b). Furthermore, CaPPR6 and the co-segregated marker Cod1Mod1-CAPS were located in section B (Fig. 2a, b).

Fig. 2.

Fig. 2

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Candidate gene screening based on sequence alignment and molecular marker analysis. a Physical locations of the high-density markers in the fine-mapping interval, and newly developed markers of the two-insert fragment. b Relative position of markers on the CaPPR6-containing sequence KU997025.1 compared with their locations on the candidate sequence of v1.55 CM334. Red markers and lines indicate the candidate region of CaRfm. c The molecular markers on the two segments (2,221,405– 2,387,187 and 1,259,830–1,403,516, respectively) of the reference genome (CM334, v1.55) aligned with the sections B and C of CaPPR6-containing sequence KU997025.1 (CaPPR6-SEQ), respectively

We next constructed a set of 25 polymorphic, high-density markers for CaPPR6-SEQ. This set consisted of 6 markers, which were newly developed from resequencing data of the parents, on sections B and C, and the 19 markers used in the fine mapping of CaRfm (Fig. 2b). The latter 19 markers were also used to genotype the six segregating populations obtained by crossing AP with six higher-generation backcrossed fertile plants (BC10-4, BC10-7, BC10-8, BC10-9, BC10-13, and BC10-14). Seven markers on the first sequence fragment (section A) were homozygous for the allele of the recurrent parent (the maintainer line 77,013) in all individuals in the test populations. Two markers, D6-45 and S1199 located at the end of the fourth sequence fragment (section D), were observed to co-segregate with the fertility phenotype in AP × BC10-4, AP × BC10-7, AP × BC10-8, and AP × BC10-13 populations only partially co-segregated in AP × BC10-9 and AP × BC10-14 populations. Furthermore, six markers located in the middle (sections B and C) of PPR6-SEQ co-segregated with the fertility phenotype of the individuals in the six AP × BC10 segregating populations (data not shown). These results suggested that CaPPR6 (Jo et al. 2016) was a strong candidate gene for CaRfm in non-pungent bell pepper 0601 M. We successfully cloned the full-length sequence of CaPPR6 from BP and RP including 113 SNPs (Fig. S1), but due to unsuccessful primer design or PCR amplification, we did not develop KASP markers for the SNPs.

Validation of KASP markers and their application in breeding

In this study, 14 molecular markers were confirmed to co-segregate with CaRfm. In addition, two markers, S1585 and S1609 located at either end of the CaRfm-containing fragment (Fig. 2c), were selected and verified in 38 maintainer and 63 restorer breeding lines. We obtained a prediction accuracy of 67.3%, which was close to that obtained using Co1Mod1-CAPS located near CaPPR6 (Table S1).

Two other markers, S1597 and S1609, were found to co-segregate with the fertile/sterile of an F6 generation population of 332 individual plants of C. annuum ‘Xiyangyang’ (Table 2). A 3:1 Mendelian ratio of fertile to sterile phenotypes was observed consistent with the genotypes of the molecular markers (Table 2). In addition, 160 plants of eight homozygous fertile individuals were planted, and all plants were found to be fertile (data not shown). The S1597 genotype–phenotype association were further validated in four segregating population of chili pepper cultivar, which showed that the marker genotype and phenotype were completely consistent (Table 3; Fig. 3). These results indicate that markers S1597 and S1609 can be applied in CMS/Rf systems breeding.

Table 2.

Genetic analysis and genotypes of molecular markers in an F6 population of Capsicum annuum ‘Xiyangyang’

Phenotypes Genotypes of S1597 Genotypes of S1609 Total Expected ratio (Fertile: Sterile) χ2 Probability*
C:C C:G G:G C:C C:T T:T
Fertile 83 163 / 83 163 / 246 3:1 0.1446 0.7038
Sterile / / 86 / / 86 86
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* Probability > 0.05 were considered to confirming this expected ratio

Table 3.

Validation of S1597 genotype–phenotype association in the segregating population of chili pepper cultivar

Populations Fertile Sterile Total
C:C C:G G:G
F7 Xiyangyang 27 61 36 124
F2BC5 Weishiyihao 0 15 4 19
F4 Kcdian Hot 6 13 2 21
F4 KmaA 12 6 1 19
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Fig. 3.

Fig. 3

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Genotypes of KASP marker S1597 in the segregating population of chili pepper cultivar ‘Xiyangyang’, ‘Weishiyihao’, Kcdian Hot, and KmaA. Blue and green triangles indicate individuals homozygous for the FAM-labeled dominant male fertility restorer allele and HEX-labeled recessive male fertility restorer allele, respectively. Red triangles correspond to heterozygous individuals, and black dots represent the non-template control (NTC)

Discussion

Many researchers have reported that the distal region of chromosome 6 of C. annuum is a hotspot region of the Rf locus, which controls male fertility restoration, in different restorer lines (Jo et al. 2016; Barchenger et al. 2018; Wu et al. 2019; Cheng et al. 2020; Zhang et al. 2020; Wei et al. 2020). The existence of this hotspot region may complicate the isolation of genotype-specific Rf genes.

In addition to a limited number of restorer lines for sweet-pepper breeding programs, genetic mapping is seriously lacking (Kumar et al. 2007; Lin et al. 2015). To date, genetic mapping of Rf genes in Capsicum has only been performed using chili pepper (Wang et al. 2004; Jo et al. 2010, 2016; Cheng et al. 2020; Wei et al. 2020; Zhang et al. 2020). In the present study, we identified that the strong, stable fertility restoration ability of the sweet pepper line 0601 M is controlled by a single dominant gene, designated as CaRfm (Table 1). In addition, we genetically mapped CaRfm to the terminus of chromosome 6 (Fig. 1a), a region in which a few candidates have been identified by in silico techniques (Barchenger et al. 2018), homology analysis (Jo et al. 2010), and fine mapping strategies (Jo et al. 2016; Cheng et al. 2020; Zhang et al. 2020).

CaRfm was further mapped to a 128.96-Kb interval bounded by four markers, two of which co-segregated (Fig. 1b). But no candidate genes were detected in this fine-mapping region. Misassembled sequences may therefore exist within the candidate region of the reference genome, an issue that has been raised in other studies as well (Cheng et al. 2020; Zhang et al. 2020). The candidate region was anchored to the NCBI sequence KU997025.1 that contains CaPPR6, a chili-pepper Rf candidate gene isolated by Jo et al. (2016). These results provide new information on chili pepper reference genome improvement and suggest that the Rf of C. annuum between pungent (chili pepper) and non-pungent (sweet pepper) varieties may have no difference.

The pepper genome contains multiple candidate Rf genes (Barchenger et al. 2018), and different restore lines may have different restorer genes (Jo et al. 2010, 2016; Wu et al. 2019; Cheng et al. 2020; Wei et al. 2020; Zhang et al. 2020). A molecular marker co-segregating with a defined Rf gene may not be applicable to other restorer lines because of the existence of different Rf genes or the lack of marker polymorphism among lines. A higher detection accuracy, 89.1% (Jo et al. 2016), has been reported for the Rf-linked marker CRF-SCAR (Lee et al. 2008a). It was considered that the PPR gene containing regions, in general, are highly repetitive. The CRF-SCAR might be located in the PPR-clustered region, although it was not a tightly linked marker of reported restorer genes, but it showed higher accuracy. The use of markers not closely linked to a gene, however, may result in the loss of target traits during genetic transfer. Molecular markers co-segregating with specific restorer genes are more effective for the transfer of restorer traits. KASP assays are a cost-effective, high-throughput option for plant genotyping (Semagn et al. 2014). KASP-based assays have recently been developed for fine mapping and molecular marker analyses of genes for disease resistance (Rehrig et al. 2014; Holdsworth & Mazourek 2015; Mahasuk et al. 2016; Wang et al. 2018; Zhao et al. 2020), abiotic stress (Liu et al. 2016) and genetic diversity (Du et al. 2019) in the genus Capsicum. In this study, the CaRfm-linked markers S1597 and S1609 developed from the restorer line 0601 M were successfully applied to marker-assisted selection in chili pepper segregating population of ‘Xiyangyang’, ‘Weishiyihao’, Kcdian Hot, and KmaA (Table 2; Fig. 3). We have therefore developed useful and efficient KASP markers for CMS/Rf systems breeding in peppe.

Conclusions and future prospectives

The Restorer-of-fertility gene CaRfm of non-pungent bell pepper 0601 M was physically mapped to a 128.96-Kb interval. Analysis of aligned sequences supported CaPPR6 as the most likely candidate gene for CaRfm. Markers tightly linked to CaRfm were developed and successfully applied to the molecular markers-assisted selection. To illustrate the restoration ability and mechanism of the CaPPR6, much more work needed to be carried out, such as the restore ability analysis of CaPPR6 to different CMS genes, gene function verification and fertility restoration mechanism of the CaPPR6.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (PDF 413 KB) (412.7KB, pdf)

Acknowledgements

This work was supported by the Beijing Natural Science Foundation (6212029), the Central Public-Interest Scientific Institution Basal Research Fund (IVF-BRF2020005), and the Science and Technology Innovation Program of the Chinese Academy of Agricultural Sciences (CAAS-ASTIP-IVFCAAS). We thank Dr. Huang Zejun for constructive advice on fine mapping the CaRfm gene. We thank Liwen Bianji, Edanz Group China (www.liwenbianji.cn/ac), for editing the English text of a draft of this manuscript.

Authors' contributions

ZHZ designed the study and developed the KASP markers. DLA, YSZ, and YJM carried out the genetic mapping. YCC and HLY performed the phenotypic investigation. BXZ and LHW participated in the design of the study and revision of the manuscript. All authors read and approved the final version of the manuscript.

Funding

This work was supported by the Beijing Natural Science Foundation (6212029), the Central Public-Interest Scientific Institution Basal Research Fund (IVF-BRF2020005), and the Science and Technology Innovation Program of the Chinese Academy of Agricultural Sciences (CAAS-ASTIP-IVFCAAS).

Declarations

Conflicts of interest

The authors declare that they have no Conflicts of interest/Competing interests.

Ethics approval

The experiments in this study comply with the current laws of China.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

References

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