QTL mapping of bacterial leaf blight based on genotype by sequencing approach

Introduction

bacterial leaf blight problem

Ralstonia solanacearum causes bacterial wilt (BW) in solanaceous crops including peppers, tomatoes, and eggplants. This soil-borne pathogen infects plants via cracks or wounds in root parts invading the xylem vessels to block the water and mineral flow and eventually causing host wilting to death. The destructive symptoms of this disease made it ranked second out of the top 10 important pathogens in terms of economic and scientific consequences (Hayward 1991, MANSFIELD 2012).

QTL or gene cloning effort for identify responsible loci conferring resistance

R. solanacearum causing BW in solanaceous plants are well-studied especially in model crop tomato in terms of quantitative trait loci (QTL) and genes that was demonstrated to have significant effect to disease resistance (Carmeille 2006, Wang 2012). These researches revealed the polygenic nature of BW resistance in tomato and several major loci such as Bwr-12 and Bwr-6 and minor loci (Wang 2000, Carmeille 2006, Wang 2012) that are comparable to the resistance of Arabidopsis thaliana that has single major loci called RRS1-R (Kim 2016). However, the researches for BW are comparably lacking for another important solanaceous crop, pepper.

With recent technological and analytical advances, the genome sequences of pepper have been revealed allowing resequencing approach to our target accessions to observe base-level polymorphism compared to the reference (Kim 2014). Moreover, with barcoding technology, it allowed high-throughput genotyping methodology called genotype-by-sequencing (GBS) for genome-wide association analysis and high-resolution QTL mapping with low cost and less laborious procedure (Kang 2015). This rapid advances in next generation sequencing (NGS) technology provide chances to develop the fundamental genomic/genetic resources for under-developed crops for knowledge based breeding such as genome assisted breeding .

Here, we applied GBS to map the responsible loci providing resistance bacterial wilt disease of pepper using recombinant inbred line (RIL) population.

Materials and methods

# Plant material (population)

Inoculation and evaluation of resistance to BW

Inoculation and evaluation of resistance to BW for pepper plants were performed at National Institute of Horticultural & Herbal Science, RDA. For pathogen inoculations, R. solanacearum isolate WR-1 was cultured on NB medium at 28 degree of Celcius for 48 h and a bacterial suspension was adjusted to \(1\times 10^{7}\) to \(1\times 10^{8}\) CFU/mL (A 600 = 0.3 to 0.4). Each RIL and the parents were sown in a 72-cell plastic tray filled with sterilized soil, and the seedlings were inoculated at the 6 to 8 leaf stage using soil-drenching (3 to 5 ml/ plant) inoculation methods with 3 replications. After 10 days post inoculation (dpi) in the growth chamber, each line was evaluated visually for occurrence of bacterial wilt ranging from 1 (most resistant) to 5 (most susceptible).

# NGS libarary (GBS)

# Bioinfo

Results

Preparation population and phenotyping

We developed 169 F7 recombinant inbred lines (RIL) of cross between YCM334 and Taean. The F7 population were grown in green house. After days from artificial infection by method, leaf symptoms were phenotyped according to bacterial leaf blight resistance scoring scheme. The distribution of phenotype scores were result suggesting the phenotype may associated with segregation distortion of genotype.

GBS analysis

For the genome-wide genotyping of 169 RILs we used genotype by sequencing (GBS) strategy using restriction enzyme, ApeKI (Elshire 2011). The ApeKI recognition sites were in silico checked using reference genome sequence of Capsicum annuum cv. CM334 (version 1.5) (Kim 2014). As the GBS libraries are constructed with size selection of which distribution ranges from 200 to 2000 (bp), we also selected in silico restricted fragments with same ranges. The restricted fragments cover whole genome in dense distribution (Figure S1). The GBS libraries were sequenced with Illumina Hiseq4000 in paired-end (Table S1). The parent lines, YCM334 and Taean, were sequenced three times for each as technical replications (Table S2). After barcode deconvolution, we mapped the reads to the sequences of in silico selected ApeKI fragments using BWA software (Li 2009).

ApeKI restriction site distribution and potential for marker development.

## Segregation distortion values in Chromosomes

synteny plot between pepper and tomato and check if tomato chromosome 6 is matched with pepper chromosome 7.

# Mapping

# Genomic regions disection

# Candidate genes

# Conclusion