Genetic Mapping and Marker Development for the Powdery Mildew Resistance Gene PMR1 in Pepper (Capsicum annuum)

(1)

(PC) Genetic Mapping and Marker Development for the Powdery Mildew

Resis-tance Gene PMR1 in Pepper (Capsicum annuum)

Jinkwan Jo

1

, Jelli Venkatesh

1

, Koeun Han

1

and Byoung-Cheorl Kang

1

*

1_{Department of Plant Science, Plant Genomics and Breeding Institute, and Vegetable Breeding Research Center, College of Agriculture and Life Sciences, Seoul National University, Seoul 151-921, Republic of Korea}

*Corresponding author Byoung-Cheorl Kang [email protected] +82-2-880-4563

Abstract

Powdery mildew (Capsicum annuum) is a major fungal disease caused by Leveillula taurica in greenhouse. Although powdery mildew resistance has a complex mode of inheritance, resistant cultivars have been steadily developed. Therefore, our objective was to map the resistance gene to powdery mildew resistance using a breeding line, C. annuum ‘VK515 R’. Based genetic analysis of an F_2:3families derived from across between the resistant parent ‘VK515 R’ and a susceptible parent ‘VK515 S’, it was found that the powdery mildew resistant is controlled by a single dominant gene, PMR1. Molecular mapping revealed that the

PMR1 locus is located at 1 cM genetic interval between CZ2_11628, and HRM4.1.6 markers on pepper chromosome 0. These results provide a solid foundation for map-based cloning of the PMR1 gene and

development of markers for breeding powdery mildew resistant cultivars. This is the first report showing the localization of the resistance gene to powdery mildew in pepper.

Materials and Methods

Plant materials

• Resistant parent C. annuum ‘VK-515 R’ • Susceptible patent C. annuum ‘VK-515 S’

• Resistant control is the commercial cultivar C. annuum ‘PM Singang’ • Susceptible control is the commercial cultivar C. annuum ‘Bukang’

• VK515’ 102 F_2:3 families were derived from a ‘VK515 R’ × ‘VK515 S’ cross

Inoculum preparation and disease infection

• Infectious ‘Bukang’ and ‘VK515 S’ plants were kept around F_2:3 plants grown in plastic trays (50 cell trays) at one-tray intervals.

• Disease resistance scoring

Localization of the PMR1 gene on a pepper linkage map

• Genotyping was performed with the Fluidigm® EP1TM system and 48.48 Dynamic Array Chip

Genotyping-by-sequencing (GBS)

• CLC genomics workbench • Disestion: PstI and MseI

• Sequencing: Illumina Hiseq 2000

Genetic mapping of PMR1 locus

• CarthaGene Software and MapChart 2.3 software

Comparison of GBS SNP data between C. annuum and C. chinense

• Genome: C. annuum L_Zunla-1, C. chinense v.1.2

References

• Kang JH, Yang HB, Jeong HS, Choe P, Kwon JK, Kang BC (2014) Single nucleotide polymorphism marker discovery from transcriptome sequencing for marker-assisted backcrossing in Capsicum. Kor J Hortic Sci 32:535-543.

• Kim DH, Park JH, Lee JS, Han KS, Han YK, Hwang JH (2009) Effect of temperature, relative hu-midity on germination and development of powdery mildew (Leveillula taurica) on pepper and its inoculation method. Res Plant Dis 15:187-192.

Acknowledgement

• This research was supported by the Golden Seed Project (213002-04-4-CG900), Ministry of

Agriculture, Food and Rural Affairs (MAFRA), Ministry of Oceans and Fisheries (MOF),

Ru-ral Development Administration (RDA), and Korea Forest Service (KFS), Republic of Korea.

Marker enrichment

Genotyping was performed with the Fluidigm® EP1TM system and PMR1 locus were localized on

chromosome 0. Therefore, more markers were tried to be added around the PMR1 locus using genomic information of Capsicum. A total 8 markers were developed.

Marker validation using GBS

To validate closely markers, genotyping by sequencing (GBS) was performed using twelve VK515.

Genetic mapping of the powdery mildew resistance gene

A total of nine polymorphic markers were further developed in 102 VK515 F_2:3 families based on genotyping analysis, three recombinants with ZL1_10691, one recombinants with each of the markers CZ2_11628 and HRM4.1.6 markers were identified from 102 F_2:3 population (Figs 2, 3). Among the nine markers used, six markers, ZL1_1826, HPGV_1313, HPGV_1344, HPGV_1412, KS and HRM2_A4, were perfectly co-segregated with powdery mildew resistant phenotype and found to be at a genetic distance of 0 cM from the PMR1 locus. The PMR1 locus was delimited to a 1 cM region be-tween CZ2_11628 and HRM4.1.6 markers on chromosome 0 (Fig 3).

Comparison of the physical position of the PMR1 region

The GBS SNP data from C. chinense and C. annuum were aligned. A total of 59 SNP markers were detected from the PMR1 region from C. chinense, whereas as 92 SNP markers detected from C.

an-nuum. Among 59 SNP markers were detected from C. chinense, a total 35 SNP were validated by

BLAST searches against C. annuum and 14 SNP markers showed collinearity with C. chinense (Fig 3). These results provide a solid foundation for map-based cloning of the PMR1 gene and development of markers for breeding powdery mildew resistant cultivars.

Objectives

• To develop molecular markers linked to the PM resistance in Capsicum

• To conduct fine mapping of the PM resistance gene

Results and Discussion

Inheritance analysis of the PMR1 gene

Segregating analysis of the 102 ‘VK515’ F_2:3 families showed 24 resistant, 48 segregating, and 30 sus-ceptible phenotypes, which fit to a 1:2:1 ratio. Phenotype analysis were performed in the 1,932 F₃ popu-lation derived from F₂ individuals (Fig 1). Out of 897 F₃ showed 652 resistant and 245 susceptible plants which fit a 3:1 ratio. This results suggested that resistant to powdery mildew is controlled by a single dominant gene (Table 1).

Population No. of plants Phenotype Expected ratio χ2 P-value R H S VK515 R 20 20 VK515 S 20 20 ‘VK515’ F₂ ₁₀₂ ₂₄ ₄₈ ₃₀ _1:2:1 _1.06 _0.589 ‘VK515’ F₃ population 897 652 245 3:1 2.56 0.110

Table 1. Segregation analysis of powdery mildew resistance in VK515 families

R, Resistant; H, heterozygote; S, susceptible

Figure 3. Comparative genetic linkage and physical maps of the powdery mildew resistance gene PMR1. A)

Physical location of linked scaffolds in C. chinense v.0.0. B) Physical location of linked markers in Zunla-1 genome chromosome 0. C) Genetic map of VK515 F_2:3 families.

Figure 2. Analysis of molecular markers. A) PCR analysis of ZL1_1826 SCAR marker and CZ2_11628 CAPS

marker. B) Normalized HRM curves of SNP based markers, HRM4.1.6 and HRM2_A4. RR: genotype of resistant line, rr: genotype of susceptible line, Rr: genotype of heterozygous F₁ plants.

Figure 1. Comparison of phenotype of resistant, susceptible parental lines and commercial pepper cultivar in-fected with L. taurica. A) ‘VK515 R’ resistant parental line, B) ‘VK515 S’ susceptible parental line, C) ‘PM Singang’

resistant cultivar, D) ‘Bukang’ susceptible cultivar. E) Inoculum preparation where ‘Bukang’ seedlings were sur-rounded by infectious old ‘Bukang’ plants, F) VK515 F_2:3 families surrounded by ‘Bukang’ plants with disease.