The viral disease induced by
To speed up the process of introgression of disease resistance genes to elite breeding materials, molecular markers have been developed and utilized in major crop plants. Accordingly, molecular markers have become essential tools for marker-assisted selection in breeding programs, including those aimed to improve resistance to TYLCV, and also for map-based cloning to isolate the genes controlling traits (Gonzalez-Cabezuelo and Lozano 2012). Most tomato cultivars are susceptible to TYLCV; however, several resistance sources have been identified in wild tomato species, such as
The accessions used in this genetic analysis were kindly provided by Prof. Wang Fu (College of Horticulture, Qingdao Agricultural University, China). The F1 progeny were produced by crossing a susceptible line
Fresh leaves of plants were sampled before and after infiltration with TYLCV. DNA was extracted from young leaves using the cetyltrimethylammonium bromide protocol (Doyle and Doyle 1987). DNA was dissolved in 1×TE buffer, separated by electrophoresis through 1% agarose gels in 1×tris-acetate ethylenediaminetetraacetic acid buffer, and quality was tested by staining with ethidium bromide and visualization under the ultraviolet light.
According to previous reports, Solyc06g051170, Solyc06g051180, and Solyc06g051190 are part of the same gene (Verlaan
Primers for the sequence-characterized amplified region (SCAR) marker were developed to amplify the region including the deletion in the susceptible line (Table 1). PCR was performed in a total volume of 25 μl containing 10× PCR reaction buffer, 0.25 mM dNTP, 0.2 U
Single nucleotide polymorphism (SNP) was detected using the high-resolution melting (HRM) method. HRM was carried out in 20-μl reaction mixtures containing 10×PCR reaction buffer, 0.25 mM each dNTP, 5 pmol each primer, 0.2 U
The two parental lines of
Three SNPs between susceptible and resistant lines were detected in the
A SCAR marker was developed using the obtained sequences of the parental plants for Solyc06g051190 (Verlaan
TYLCV transmitted by the whitefly (
In this study, we developed codominant HRM markers in different regions of
In our study, the phenotype of one BC1F1 and three F2 plants did not match the genotype revealed using our markers. Even though these lines showed susceptible phenotypes, their disease severity was lower than that of the susceptible parent ‘A39’. In addition, when the recombinant plants showed the susceptible phenotype, the marker genotypes of the plants were all heterozygous. This discrepancy may be due to the resistance mechanism of
Taken together, our results show that the
This research was supported by Golden Seed Project (213002-04-4-CG900), Ministry of Agriculture, Food and Rural Affairs (MAFRA), Ministry of Oceans and Fisheries (MOF), Rural Development Administration (RDA) and Korea Forest Service (KFS), Republic of Korea, and a grant (710001-07) from the Vegetable Breeding Research Center through Agriculture, Food and Rural Affairs Research Center Support Program, Ministry of Agriculture, Food and Rural Affairs.
S: susceptible, R: resistant.
HRM: high-resolution melting, SCAR: sequence-characterized amplified region.
M: size marker, S: genotype of susceptible line ‘A39’, R: genotype of resistant line ‘A45’, H: genotype of heterozygous F1 plants, HRM: high-resolution melting, SCAR: sequence-characterized amplified region.
Primer sequences for high-resolution melting (HRM) and the sequence-characterized amplified region (SCAR) analysis.
|Marker||Sequence (5′-3′)||Type||Product size (bp)|
|Ty3-SCAR1||GCTCAGCATCACCTGAGACA||SCAR||519 (R)/269 (S)|
Segregation for resistance to
|Population||Expected ratio of R:S||Observed number of plantsz)||χ2|
z)R: resistant, S: susceptible.
Co-segregation analysis of
z)R: resistant, S: susceptible.
y)Number of lines did not show the co-segregated results.