Strong host resistance to root-knot nematode (RKN;
Root-knot nematode (RKN) (
Thirteen polymorphic markers defined the introgressed region from TxAG-6 when mapped in two populations, Gregory x Tifguard and NemaTAM x GP NC WS14 (
Seventy-eight RILs from Gregory x Tifguard F7:9 were used for genotyping with polymorphic SSR or SNP markers. Gregory, the susceptible female parent, is a virginia-type cultivar developed from a cross between two elite parents (
DNA was extracted using the DNeasy plant mini-kit (Qiagen, Valencia, CA). PCR amplifications for SSR markers were carried out in a GeneAmp® PCR System 9700 (Applied Biosystems, Foster City, CA) using a touchdown program, starting with 95 C for 5 min, followed by 6 cycles of 95 C for 30 s, 64 C (dropping 1 C /cycle) for 30 sec and 72 C for 30 sec, followed by 30 cycles of 95 C for 30 sec, 58 C for 30 sec and 72 C for 30 sec; final extension was performed at 72 C for 7 min. Forward primers were labeled with FAM, HEX, or TAMRA fluorophores (Supplemental table 1). PCR products were run on 1.5% agarose gels to check for amplification and contamination. These PCR products were then diluted 40 times using sterile water. The final mixture for fragment analysis in each well of a 96-well semi-skirted plate was constituted by 1 μl of the diluted PCR product, 9 μl of Hi-Di formamide (Applied Biosystems, Foster City, CA) and ∼ 0.20 μl of the ROX™ dye-labeled size standard. SSR markers were genotyped on an ABI3730XL Capillary DNA Sequencer (Applied Biosystems, Foster City, CA). Genotypes were called using the Gene Mapper 4.0 software (Applied Biosystems, Foster City, CA) and manual correction.
For SNP markers, KASP assays (KBioscience Ltd., Hoddesdon, UK) were developed for a set of 25 markers that were polymorphic between parents (Supplemental table 2). In this study, ‘GKAM' (Groundnut KASP Assay Markers) nomenclature (
Thermocycling and endpoint genotyping for the KASP assays were performed on a Roche LC480 (Roche Applied Science Indianapolis, IN) using two oligos 5′-labeled with FAM and HEX fluorophores. Each 5 μl volume reaction contained 2.5 μl of KASP Genotyping Mix, 0.07 μl of primer assay mix, 1.63 μl of water and 0.8 μl of genomic DNA template (5 ng/μl). The following thermal cycling program was used: hot start or activation at 95 C for 15 min, followed by 9 cycles of 94 C for 20 sec and 61 C for 60 sec, the annealing temperature dropped at the rate of 0.6 C/cycle, followed by 27 cycles at 94 C for 10 sec and 55 C for 60 sec and 2 cycles of 94 C for 20 sec and 57 C for 60 sec. Pre- or post-melt cycles were at 30 C for 1 sec and cooling to 25 C during plate reading. If the signals did not separate sufficiently, three additional cycles of 94 C for 20 sec and 57 C for 60 sec were performed. Roche LC480 allowed for endpoint genotyping based on dual color hydrolysis probes (FAM and HEX) and automated scatterplot analysis.
Phenotyping for nematode resistance of RIL 46, 48 and parents was performed using a greenhouse screening method as described previously (
A total of 109 SSRs (24% of screened primer pairs) and 24 SNP markers were polymorphic between Gregory and Tifguard and mapped to 17 linkage groups, with 31 markers (23%) distributed on linkage group A09 at a map distance of 7.9 cM (
Linkage group A09 populated with 31 markers and allele distribution among RILs. Solid black bar represents Gregory haplotype; Bars with diagonal hatch marks indicate Tifguard haplotype. Grey bar represents genomic region with unassigned haplotype designation.
To test nematode resistance levels conferred by the two introgressed regions on linkage group A09, recombinant lines between these two regions were identified by revisiting the genotyping data. Two RIL lines (17 and 46) retained alien alleles in the second introgressed region and exhibited a haplotype from susceptible parent Gregory in the first introgressed region (
To further analyze the effect of the recombination, two inoculation trials, with 10 replicates per trial, were performed. Although a standard greenhouse inoculation test usually consists of 3 to 5 replicates (
Gall rating and number of eggs/gram root from nematode inoculation study. A) Gall rating; B) Eggs/gram root. Gall ratings are based on the following scale: 0 = no gall, 1 = 1 to 2, 2 = 3 to 10, 3 = 11 to 30, 4 = 31 to 100, 5 = more than 100 galls per root system. Eggs/gram root was transformed by square root. Different letters indicate significant differences at P<0.05 for mean separation by Fisher's LSD test.
SNP marker AdSNP 584 detects a simple SNP, i.e., two alleles at a single locus in one of the two sub-genomes. It is located at the second introgressed region that has an “A” in Gregory and a “G” in Tifguard (Supplemental table 2). KASP analysis shows that signals from homozygotes and heterozygotes separate perfectly in the scatter plot with this marker (
Marker AdSNP584 demonstrates optimum signal separation for genotyping. Green cluster: resistant individuals (GG); red cluster: heterozygous individuals (AG); blue cluster: susceptible individuals (AA).
This work was supported by The Peanut Foundation, The National Peanut Board, Georgia Peanut Commission, and The Agriculture and Food Research Initiative competitive grant # 2010-85117-20550 of the USDA National Institute of Food and Agriculture.
First, second, third and last authors: Research Professional, MS student, PhD student, and Professor, Department of Horticulture and Institute of Plant Breeding, Genetics & Genomics, University of Georgia Tifton Campus, Tifton, GA 31793; Fourth and fifth authors: Research Plant Pathologist and Research Geneticist, USDA-ARS, P.O. Box 748, Tifton, GA 31793.