Plant material
A cross between Florida-EP™ ‘113’ and Georgia Valencia was initiated in 2009 at the North Florida Research and Education Center (NFREC) near Marianna, FL. Florida-EP™ ‘113’ is a new runner type variety developed by the University of Florida (UF) peanut breeding program with high resistance to TSWV [22]. Georgia Valencia is a valencia type variety developed by the Georgia Agricultural Experiment Station and is highly susceptible to spotted wilt [23]. The Florida-EP™ ‘113’ was derived from a cross between NC94022 and ANorden [24], and NC94022 had very high resistance to spotted wilt. From F2 to F5 generations, seeds were bulk harvested without selection. In the F5 generation, a subset of 245 individuals was genotyped using two SSR markers on chromosome A01, which are GM 1694 and ARS 721 [20]. Individuals showing the heterozygous genotype at both marker loci were selected and planted to generate the F6 families at NFREC in 2015. Visually susceptible F6 plants were selected for DNA extraction and their seeds were harvested individually and planted to form the F7 generation in 2016 for phenotyping.
For the US peanut mini-core germplasm accessions, seeds of 107 accessions in the collection were planted with four replicates in a completely random design in 2012 at Plant Science Research and Education Unit (PSREU) near Citra, Florida. These accessions were again planted at North Florida Research and Education Center (NFREC) in April 2016 with two replicates in a randomized complete block design.
Phenotyping with visual rating and ELISA test
Each visually susceptible F6 plant was individually harvested and planted into a whole plot in the F7 for phenotyping with one plot corresponding to one susceptible plant in the F6 generation. Both visual rating and ELISA tests were used to phenotype the F7 lines. Plots were 0.9 m in width and 4.5 m in length, containing two rows with planting density one plant per 0.3 m. Visual rating was used to evaluate the infection severity of one whole plot before the plot was harvested. A scale of 1 to 10 was used to represent the percentage of infected plants (1, 2, 3, 4, 5, 6, 7, 8, 9, 10 equals 1–10%, 11–20%, 21–30%, 31–40%, 41–50%, 51–60%, 61–70%, 71–80%, 81–90%, and 91–100% respectively) [20].
The ELISA test was conducted with the TSWV PathoScreen Kit (Agdia, Inc., IN). For sample preparation, a maximum 10 roots from each plot were randomly collected, dried and stored in paper bags. Lateral roots in each sample were removed and the root crown was crushed with a hammer. A 0.1 g sub-sample of this crushed root crown sample was put into a 2 mL tube and grounded using a Mixer Mill Grinder (Retsch Inc, PA) at 30 times s− 1 for 1.5 min. General extract buffer of 1 mL was added into each tube after grinding and incubated overnight at 4 °C. These steps were followed according to the PathoScreen Kit User Guide. Root crown samples from spotted wilt-resistant parent Florida-EP™‘113’ were used as the negative control. The percentage of infected roots in all roots tested from one plot was calculated as infection rate for the plot.
The US peanut mini-core germplasm accessions were phenotyped using the visual rating method in both 2012 and 2016 just prior to harvesting, and the same scale and rule were followed as described above. The mini-core germplasm accessions were also mechanically inoculated in greenhouse and phenotyped by ELISA. Specifically, the accessions were grown in the greenhouse at a temperature of 25 to 30°C, and 60 to 90% relative humidity. Nine seeds per accession were sown in plastic seedling trays (7.87 cm × 7.87 cm × 5.92 cm per cell) containing all-purpose professional growing mix consisting of Canadian sphagnum peat moss, coarse perlite, vermiculite, and dolomitic limestone (Sun Gro Horticulture, Agawam, MA). Peanut plants at two- to three-leaf stage (7 to 9 days after planting [DAP]) were dusted with carborundum, and the TSWV inoculum (1 ml per plant) was applied by rubbing both surfaces of the leaf with a cotton swab. After inoculation, the sap and carborundum were rinsed from the seedlings with distilled water and the plants were kept in the greenhouse under the same environmental conditions as previously mentioned. Inoculated plants were observed daily for symptom development. Plants were considered to have localized infection when chlorotic rings or concentric rings developed only on the inoculated leaves, and without any symptoms on new leaves. The plants were considered to be systemically infected when the symptoms developed on new emerging leaves. The plants were monitored in the greenhouse for 40 days after inoculation. The percentage of infected plants was recorded at 40 days post inoculation (DPI). At 40 DPI, 0.2 g of roots was collected from every plant to assay by ELISA test using TSWV-specific antiserum (Agdia Inc, IN).
Genotyping with SSR markers
Genomic DNA was extracted using cetyltrimethyl ammonium bromide (CTAB) method. DNA quality and concentration was checked using 1% agarose gel and Quant-iT™ Picogreen dsDNA Assay Kit. Extracted DNA was diluted to 10 ng/μL for polymerase chain reaction (PCR) with SSR markers. PCR was done in 10 uL volume including 1 μL of 10 × PCR buffer, 1.25 μL of Magnesium Chloride (25 mM), 1 μL of dNTP (2 mM), 0.25 μL of Taq enzyme, 2 μL of forward and reverse primers (2 mM), 2 μL of DNA template (10 ng/μL), and 2.5 μL of distilled deionized water. The PCR was operated using a touchdown program with an initial denaturation at 95°C for 4 min; 10 cycles of amplification at 95°C for 30 s, 65°C for 30 s, 72°C for 1 min; 30 cycles of amplification at 95°C for 30 s, 55°C for 30 s, 72°C for 1 min; and a final extension at 72°C for 7 min. PCR products were separated using 6% polyacrylamide gel electrophoresis (PAGE) under 150 V for 2 h in 1X TBE buffer. The gels were stained with Ethidium Bromide (EB) for 10 mins before visualizing under UV light.
Data analysis
R (version 3.3.3) was used to conduct the ANOVA test and Tukey’s Honest Significant Test (TukeyHSD) on the phenotypic data sets. For phenotypic data from the mapping population, a one-way ANOVA test was conducted at significance level of P < 0.05. For phenotypic data from the mini-core germplasm, one-way ANOVA (RCBD) test was conducted to check whether the visual rating result was significant between resistant and susceptible genotypes, and was used for visual rating data in 2012 and 2016 separately because the visual rating was taken in two different places. Results were regarded as significant if P < 0.05. A two sample t-test was used to check whether the ELISA result was significant between resistant and susceptible genotypes.
Amplicon-seq
Amplicon-seq was used to develop SNP markers in the interval of interest. Primers were designed to be evenly distributed across this interval based on the diploid wild peanut genome sequence [12]; however, some specific regions with highly repetitive DNA or missing sequence information were avoided. Primer sequences were mapped to the genome reference by using Bowtie (−f –v 2 –I 100 –X 8000) [25]. Primers with multiple hits across the genome were eliminated. The length of each amplicon was approximately 7 kb. KOD Xtreme Hot Start DNA Polymerase (EMD Millipore, MA), which is an optimized PCR enzyme for the amplification of long DNA templates, was used for PCR reaction. The PCR was operated using a 2-step cycling with polymerase activation at 94°C for 2 min; 30 cycles of denaturation at 98°C for 10 s and annealing and extension at 68°C for 1 min. A total of eight samples were amplified and the PCR products were checked on 1% agarose gel. Subsequently, successfully-amplified amplicons from each sample were equally pooled and submitted to the Interdisciplinary Center for Biotechnology Research (ICBR) at the University of Florida for product-cleaning and library construction. Illumina MiSeq SE 1 × 300 was used for amplicon pool sequencing.
Raw sequences were trimmed with Trimmomatic [26] and the quality of trimmed data was checked by using FastQC [27]. The clean sequence reads were aligned to diploid peanut genomes [12] using aligner BWA MEM [28]. After alignment, SNP calling was conducted with three different software: GATK [29], freebayes [30] and Samtools [31] using parameters previously described [32]. Results from these three SNP callers were compared and genotyping results from newly-detected SNPs at the targeting interval were summarized.
Construction of linkage map and QTL analysis
QTL IciMapping v3.3 [33] was used for both linkage map construction with MAP function and QTL analysis with BIP function. For linkage map construction, the logarithm of odds (LOD) was set at 3 for grouping, and the nearest neighbor combined with two-opt algorithm (nnTwoOpt) was implemented as the algorithm for ordering. Sum of Adjacent Recombination Frequencies (SARF) was used for rippling with window size of five markers. For QTL analysis, inclusive composite interval mapping (ICIM) function was applied with additive method, using 1 cM as step and 0.001 probability in the stepwise regression. Individuals with less confident phenotype (number of tested roots for ELISA was less than 4)and with extreme influence on the QTL result were filtered out. The QTL results with LOD score above 3 was regarded as significant.
