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Mapping of Quantitative Trait Loci Controlling Resistance to Dark Leaf Spot (alternaria Brassicicola) Disease in Brassica Oleracea L.

M. A. U. Doullah
Associate Professor
Department of Plant Pathology and Seed Science, Sylhet Agricultural University, Sylhet-3100, Bangladesh

K. Okazaki
Professor
Department of Agrobiology, Niigata University, Niigata, Japan

Abstract/ Executive Summary:

Dark leaf spot and black rot are important diseases in Brassicas worldwide. New sources of resistance to the diseases are urgently needed for sustainable management. Quantitative trait loci (QTL) controlling resistance to Alternaria brassicicola was mapped using cleaved amplified polymorphic sequences (CAPS) and sequence-related amplified polymorphism (SRAP) analysis with disease rating of F₃ families from a susceptible broccoli Green commet P09 × resistant cabbage Reiho P01. For dark leaf spot disease, 3rd/4th leaves of 30-day old plants were inoculated by detached leaf inoculation method. A total of 38 CAPS and 60 SRAP primer pairs were screened to assess parental polymorphism against dark leaf spot resistance. Ninety-two markers were distributed in 10 linkage groups (LGs) covering 320.5 cM, with average 3.56 cM interval between markers. The QTL effect was detected significantly in the interval between IPI - FLC3 on LG 7 for dark leaf spot disease resistance explained 43.8 % of the phenotypic variation with LOD score of 4.27. This is the first QTL detected for resistance to A. brassicicola. One non-significant QTL on LG 2 in the interval between CAM – GSA1 (LOD = 2.02) was also detected for resistance to the dark spot disease. The QTL, which was mapped to LG 7, could be useful for marker-assisted selection of dark leaf spot resistance breeding.

Keywords: Genetic analysis, mapping, QTL, Alternaria leaf spot, resistance, Brassica oleracea L.

Location: Biotechnology laboratory and Department of Plant Pathology and Seed Science of Sylhet Agricultural University

Start Date:

End Date:

Program Area: Variety and Species

Commodity: Mustard

Objectives:

To map the resistance loci.
To find markers linked to these loci that could be helpful in marker-assisted selection programs for breeding of dark leaf spot resistant varieties.

Methodology:

Plant materials: Doubled hybrid (DH) broccoli line ‘Green Commet (GC) P09’ (Brassica oleracea subsp. italica) and cabbage DH line ‘Reiho P01’ (B. oleracea subsp. capitata) were selected. The ‘GC P09’ was susceptible to Alternaria brassicicola and ‘Reiho’ was tolerant. Seedlings were grown to 20-day old in greenhouse and were transferred to plastic pot (12 cm). F₁ progeny followed by F₂and F₃was developed from the cross between ‘GC P09’ and ‘Reiho P01’ where ‘GC P09’ served as mother parent. Young leaves from each parent and 94 individual of F₂plants were collected for DNA extraction and the plants were allowed to grow for bud self-pollination to generate F₃ seeds for evaluation of resistance to the disease. Fungal isolate: Fungus A. brassicicola ‘Akakura’ isolate was used in this study. Plant inoculation: Third or fourth true leaf from 30-day old plant of parents and F₃ population as a representative of F₂ individual were inoculated by detached leaf inoculation test for dark leaf spot disease. Isolation of genomic DNA: Healthy leaves harvested from the parents and 94 F₂ individual were used for genomic DNA extraction. Total genomic DNA was isolated according to Cetyltrimethyl ammonium bromide (CTAB) method. Analysis of CAPS: Primer sequences used in CAPS (Cleaved Amplified Polymorphic Sequences) analysis. The primer sequences were designed based on the structural gene sequences published in the NCBI and TAIR databases. Annealing temperature and extension time for PCR were set according to the primer sequence and gene size. The amplicons were digested with one of four restriction enzymes (AfaI, AluI, MspI or MobI) and were separated on 8-15% polyacrylamide gel according to gene size. Restriction enzyme was chosen according to their digestion of PCR amplicons against respective primer. Polyacrylamide gel was prepared. The digestion was checked by 2% agarose gel before running polyacrylamide gel. An example of segregation pattern of F₂ progenies (GC P09 × Reiho P01) in 13% polyacrylamide gel against CAPS primer is shown. Analysis of SRAP: Polymorphic detection by the sequence-related amplified polymorphism (SRAP) method was conducted with minor modifications. The first five cycles of PCR were performed at 95°C for 30 seconds for denaturing, 35°C for 30 seconds for annealing and 72°C for 2 minutes for extension. The annealing temperature was raised to 50°C for another 35 cycles. The success of the amplification was checked by electrophoresis of the PCR products in a 1% agarose gel. 10 × loading buffer (Takara Biomedicals, Japan) was added to the PCR products and mixed well prior to loading (2μl) in the agarose gel. Amplified DNA fragments were loaded onto a native 8% polyacrylamide gel that was made and separated at a power of 100 V for 1.5 hours and 250 V for 3 hours. The gel was subsequently stained with a Gelstar solution (0.1μl/10ml). An example of segregation pattern of F₂ progenies (GC P09 × Reiho P01) in 8% polyacrylamide gel against SRAP primer pairs is shown. Construction of Map: For making map, CAPS and SRAP markers were used. Linkage analyses were performed using JOIN MAP program, version 3.0. The Kosambi mapping function was used to convert recombination frequencies into genetic (map) distances. QTL analysis: The QTLs for A. brassicicola resistance were analyzed using a composite interval-mapping analysis with Map QTL version 2.0 (Van Ooijien et. al. 2000) and QTL Cartographer version 1.16. A forward-backward stepwise regression was performed to choose co-factors before performing QTL detection. A 1,000-permutation test was performed with QTL Cartographer to estimate the appropriate significance threshold for analysis. A minimum logarithm of the odds ratio (LOD) threshold of 2.4, corresponding to a genome-wise significance level of 0.10, was chosen.

Source of Information: Int. J. Sustain. Crop Prod. 10(1):11-18, 2015

Article Link (Online Journal):

Funding Source:

1. Government

Budget:

Total Budget (Tk.) :

Achievement/Findings:

Quantitative trait loci (QTL) controlling resistance to A. brassicicola was mapped using cleaved amplified plymorphic sequences (CAPS) and sequence-related amplified polymorphism (SRAP) analysis with disease rating of F₃ families from a susceptible broccoli ‘GC P09’ × resistant cabbage ‘Reiho P01’. Detached leaf inoculation test was used for dark leaf spot disease. A total of 38 CAPS and 60 SRAP primer pairs were screened to assess parental polymorphism against the disease resistance where 92 markers were distributed in 10 linkage groups (LGs) covering 320.5 cM, with average 3.56 cM interval between the markers. One QTL resistance to A. brassicicola was detected significantly in the interval IPI - FLC3 on LG 7 based on the LOD threshold of 4.27 explaining up to 43.8% of the phenotypic variation. This is the first QTL detected resistance to A. brassicicola.

Knowledge Management: Journal