3 Biotech

, 9:9 | Cite as

Sequence-tagged site-based diagnostic markers linked to a novel anthracnose resistance gene RCt1 in chili pepper (Capsicum annuum L.)

  • Rukmini Mishra
  • Ellojita Rout
  • Jatindra Nath Mohanty
  • Raj Kumar JoshiEmail author
Original Article


Anthracnose, caused by Colletotrichum spp. is the most devastating disease of chili (Capsicum annuum) in the tropical and subtropical regions of the world. The present study aimed at molecular mapping and development of markers linked to a new gene for anthracnose resistance in the chili cultivar ‘Punjab Lal’. Phenotypic evaluation of F1, F2, and BC1F1 populations derived from a cross between ‘Punjab Lal’ and susceptible cultivar ‘Arka Lohit’ against a virulent isolate of C. truncatum revealed that anthracnose resistance in Punjab Lal is governed by a monogenic-dominant gene designated as RCt1. Forty-four (28 ISSRs and 16 AFLPs) out of 201 markers exhibited parental polymorphism and were used in bulk segregant analysis. Three ISSRs (ISSR411493, ISSR581485, and ISSR1121857) and one AFLP marker (E-ACA/M-CTG516) showed precise polymorphism between resistant and susceptible bulks, and were used for genotyping F2 and BC1 populations. The four putative fragments were converted into sequence-tagged site (STS) markers and southern blotting confirmed their association with the resistance locus. Molecular mapping revealed that the STS markers CtR-431 and CtR-594 were closely linked to the RCt1 locus in coupling at distances of 1.8 and 2.3 cM, respectively. Furthermore, both of these markers showed the presence of resistance-linked allele in seven genotypes including the highly resistant C. chinnese ‘PBC932’ and C. baccatum ‘PBC80’ while negatively validated in 32 susceptible genotypes. Therefore, CtR431 and CtR-594 could be recommended as efficient diagnostic markers to facilitate the introgression of RCt1 locus into susceptible chili variants towards the development of high-yielding anthracnose resistance genotypes in C. annuum background.


Capsicum Anthracnose Colletotrichum truncatum Resistance locus ISSR AFLP STS marker 



The study is funded by a research grant (FT/YS/LS-171/2013) from the Science and Engineering Research Board (SERB), Dept. Of Science and Technology (DST), Government of India. RM is grateful to SERB, DST, India for the financial support in the form of Young Scientist Fellowship. We also thank DST-FIST, Govt. of India, for the research infrastructure facilities provided to Centre of Biotechnology, Siksha O Anusandhan University.

Author contributions

RM and RKJ conceived and supervised the project. RM collected samples, isolated DNA, and performed molecular marker analysis and detection of linked markers. ER and JNM cloned the STS fragments and performed marker validation. RM and RKJ interpreted the data and prepared the manuscript. All authors read and approved the final manuscript.

Compliance with ethical standards

Conflict of interest

The authors have declared that there is no conflict of interest.

Supplementary material

13205_2018_1552_MOESM1_ESM.tif (1.6 mb)
Figure S1: Representative images depicting the screening of resistant and susceptible parents and F2 genotypes (Punjab Lal × Arka Lohit) of Capsicum annuum against anthracnose pathogen Colletotrichum truncatum. Inoculated matured red fruits of (A) Resistant parent ‘Punjab Lal’; (B) Susceptible parent ‘Arka Lohit’; (C–D) Resistance reaction in segregating F2 plants; (E–F) Susceptible reaction in the segregating F2 plants (TIF 1594 KB)
13205_2018_1552_MOESM2_ESM.tif (529 kb)
Figure S2: Bulk segregant analysis and amplification pattern of polymorphic ISSR and AFLP markers in F2 individuals either resistant or susceptible to chili anthracnose. (A) Amplification with Ca-ISSR41 [(GACA)4]; (B) Amplification with Ca-ISSR112 [(GT)8C]; (C) Amplification with Ca-ISSR58 [(CA)8T]; (D) Amplification with AFLP marker E-ACA/M-CTG. M, molecular weight marker; R, Resistant genotype—Punjab Lal; S, susceptible genotype—Arka Lohit, RB, bulk DNA from ten resistant F2 individuals; SB, bulk DNA from ten susceptible F2 individuals. 1–10, F2 individuals resistant to anthracnose; 11–20, F2 individuals susceptible to anthracnose. Please note that 18 F2 individuals were amplified using Ca-ISSR112 and E-ACA/M-CTG markers (TIF 528 KB)
13205_2018_1552_MOESM3_ESM.tif (102 kb)
Figure S3: DNA sequence of the Capsicum annuum resistance-specific markers ISSR41 [(GACA)4]-1493. The underlined region represents the position of the specific ISSR primers. The arrow with the shaded region indicates the position of specific primers for amplifying the sequenced tagged sites marker CtR-431 (TIF 102 KB)
13205_2018_1552_MOESM4_ESM.tif (118 kb)
Figure S4: DNA sequence of the Capsicum annuum resistance-specific markers ISSR112 [(GT)8C]-1857. The underlined region represents the position of the specific ISSR primers. The arrow with the shaded region indicates the position of specific primers for amplifying the sequenced tagged sites marker CtR-594 (TIF 117 KB)
13205_2018_1552_MOESM5_ESM.tif (101 kb)
Figure S5: DNA sequence of the Capsicum annuum resistance-specific markers ISSR58 [(CA)8T]-1485. The underlined region represents the position of the specific ISSR primers. The arrow with the shaded region indicates the position of specific primers for amplifying the sequenced tagged sites marker CtR-496 (TIF 100 KB)
13205_2018_1552_MOESM6_ESM.tif (55 kb)
Figure S6: DNA sequence of the Capsicum annuum resistance-specific AFLP marker E-ACA/M-CTG-516. The underlined region represents the position of the specific AFLP primers. The arrow with the shaded region indicates the position of specific primers for amplifying the sequenced tagged sites marker AFLP-376 (TIF 55 KB)
13205_2018_1552_MOESM7_ESM.docx (37 kb)
Supplementary material 7 (DOCX 37 KB)


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Copyright information

© King Abdulaziz City for Science and Technology 2019

Authors and Affiliations

  • Rukmini Mishra
    • 1
  • Ellojita Rout
    • 1
  • Jatindra Nath Mohanty
    • 1
  • Raj Kumar Joshi
    • 1
    • 2
    Email author
  1. 1.Functional Genomics Laboratory, Centre for BiotechnologySiksha O Anusandhan UniversityBhubaneswarIndia
  2. 2.Department of BiotechnologyRama Devi Women’s UniversityBhubaneswarIndia

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