Marker-Assisted Evaluation of Two Powdery Mildew Resistance Candidate Genes in Korean Cucumber Inbred Lines

Two genes, CsLRR-RPK2 (CsGy5G015660) and CsaMLO8 (Csa5G623470), have been considered as powdery mildew (PM) resistance genes in cucumbers. In this study, we evaluated the involvement of the alleles of these two genes in PM resistance in 100 commercial Korean cucumber inbred lines. To achieve this, we developed cleaved amplified polymorphic sequences (CAPS) and InDel markers from CsLRR-RPK2 and CsaMLO8. Genotyping analysis indicated that the CsLRR-RPK2-CAPS marker showed a stronger correlation with the PM-resistant phenotype, with an 84% consistency compared to the CsaMLO8-InDel marker. The use of the CsaMLO8-InDel marker showed a 70% consistency between phenotype and genotype results. It was proposed that the CsLRR-RPK2-CAPS marker successfully eliminated PM-susceptible inbred lines, since both genotype and phenotype results were 100% identical. Furthermore, the present study revealed that the introduction of one of these alleles is probably enough to confer PM resistance in cucumbers. However, seven PM-resistant inbred lines harbored either CsaMLO8 or CsLRR-RPK2 alleles, indicating that there is another PM-resistant resource(s) besides CsaMLO8- and CsLRR-RPK2–originated resistance in the commercial Korean inbred lines. Our results provide reliable evidence confirming two PM-resistant candidate genes for the detection of PM resistance resources in cucumber inbred lines.

Development of a Co-Dominant Cleaved Amplified Polymorphic Sequences Assay for the Rapid Detection and Differentiation of Two Pathogenic Clarireedia spp. Associated with Dollar Spot in Turfgrass

Dollar spot is one of the most destructive diseases in turfgrass. The causal agents belong to the genus Clarireedia, which are known for causing necrotic, sunken spots in turfgrass that coalesce into large damaged areas. In low tolerance settings like turfgrass, it is of vital importance to rapidly detect and identify the pathogens. There are a few methods available to identify the genus Clarireedia, but none of those are rapid enough and characterize down to the species level. This study produced a co-dominant cleaved amplified polymorphic sequences (CAPS) test that differentiates between C. jacksonii and C. monteithiana, the two species that cause dollar spot disease within the United States. The calmodulin gene (CaM) was targeted to generate Clarireedia spp. specific PCR primers. The CAPS assay was optimized and tested for specificity and sensitivity using DNA extracted from pure cultures of two Clarireedia spp. and other closely related fungal species. The results showed that the newly developed primer set could amplify both species and was highly sensitive as it detected DNA concentrations as low as 0.005 ng/µL. The assay was further validated using direct PCR to speed up the diagnosis process. This drastically reduces the time needed to identify the dollar spot pathogens. The resulting assay could be used throughout turfgrass settings for a rapid and precise identification method in the US.

Cleaved amplified polymorphic sequences (CAPS) marker for identification of two mutant alleles of the rapeseed BnaA.FAD2 gene

Two mutants of winter rapeseed (Brassica napus L. var. oleifera) with an increased amount of oleic acid in seeds were created by chemical mutagenesis (HOR3-M10453 and HOR4-M10464). The overall performance of the mutated plants was much lower than that of wild-type cultivars. Multiple rounds of crossing with high-yielding double-low (“00”) cultivars and breeding lines having valuable agronomic traits, followed by selection of high oleic acid genotypes is then needed to obtain new “00” varieties of rapeseed having high oleic acid content in seeds. To perform such selection, the specific codominant cleaved amplified polymorphic sequences (CAPS) marker was used. This marker was designed to detect the presence of two relevant point mutations in the desaturase gene BnaA.FAD2, and it was previously described and patented. The specific polymerase chain reaction product (732 bp) was digested using FspBI restriction enzyme that recognizes the 5′-C↓TAG-3′ sequence which is common to both mutated alleles, thereby yielding band patterns specific for those alleles. The method proposed in the patent was redesigned, adjusted to specific laboratory conditions, and thoroughly tested. Different DNA extraction protocols were tested to optimize the procedure. Two variants of the CAPS method (with and without purification of amplified product) were considered to choose the best option. In addition, the ability of the studied marker to detect heterozygosity in the BnaA.FAD2 locus was also tested. Finally, we also presented some examples for the use of the new CAPS marker in the marker-assisted selection (MAS) during our breeding programs. The standard CTAB method of DNA extraction and the simplified, two-step (amplification/digestion) procedure for the CAPS marker are recommended. The marker was found to be useful for the detection of two mutated alleles of the studied BnaA.FAD2 desaturase gene and can potentially assure the breeders of the purity of their HOLL lines. However, it was also shown that it could not detect any other alleles or genes that were revealed to play a role in the regulation of oleic acid level.

Rapid and easy detection of the five most common mutations in BRCA1 and BRCA2 genes in the Polish population using CAPS and ACRS-PCR methods

In this publication we present a fast method of diagnosing the most common polymorphisms of BRCA1 and BRCA2 genes in Poland – C61G [c.300T>G], C64R [c.190T>C], 4153delA [c.4035delA], 3819del5 [c.3700_3704delGTAAA], and C5972T [c.5744C>T]. Our procedure is based on the use of the cleaved amplified polymorphic sequences (CAPS) and artificially created restriction site (ACRS) PCR techniques. The precise selection of appropriate primer sequences and restriction enzymes enabled specific cuts of DNA fragments. The final quantity and size of the obtained products depend on the presence or the absence of the mutations. The obtained results are unambiguous and do not have to be confirmed by sequencing. The methods of detection of the C61G, C64R, 4153delA, 3819del5, and C5972T mutations in the BRCA1 and BRCA2 genes described by us do not require a sequencing process, which is more expensive, time-consuming and associated with numerous errors. The technique developed by us enables the use of simple electrophoresis for accurate detection of the presence or absence of a specific mutation. Our procedures are fast, precise and unambiguous.

Genetic variability assessment in ‘Muscat’ grapevines including ‘Muscat of Alexandria’ clones from selection programs

Genetic variability is needed to face environmental changes and pathogen constrains. In addition, the search for intravarietal variability contributes to the avoidance of genetic erosion, preserving clones that are adapted to particular conditions. Variability is also important to diversify grapevine-derived products. In this work, we have analyzed the genetic variability of ‘Muscat germplasm’ including samples from neglected vineyards from Alicante and Valencia provinces, accessions of the germplasm collections of ‘Colección de Vides de El Encín’ (Alcalá de Henares, Madrid) and ‘La Casa de las Vides’ (Agullent, Valencia), accessions supplied by nurseries of Valencia province, and ‘Muscat of Alexandria’ clones selected using differential ampelographic characteristics in selection programs (La Marina, Alicante). Fifteen microsatellites (SSRs) were used to study intervarietal variability. The SSR fingerprinting allowed the identification of some accessions, variants, and synonymies. Amplified Fragment Length Polymorphisms (AFLPs) markers and Microsatellite-AFLPs were used to determine the variability attended in ‘Muscat of Alexandria’ accessions. A CAPs (Cleaved Amplified Polymorphic Sequences) marker, recently developed for the discrimination of ‘Muscat’ flavor genotypes using the SNP1822 G>T, was assessed and showed that all the analyzed accessions were ‘Muscat’ flavored. The variation found among the analyzed germplasm is very interesting because variants within ‘Muscat of Alexandria’, ‘Muscat Italia’, and ‘Muscat d’Istambul’ have been identified. In addition, intravarietal genetic variation was found among the analyzed accessions in ‘Muscat of Alexandria’ from selection programs.

Allelic variation at the EF-G locus among northern Moroccan six-rowed barleys

A germplasm panel of 52 six-rowed barley landraces from northern Morocco was analysed by a Cleaved Amplified Polymorphic Sequences (CAPS) assay of a fragment of the elongation factor G (EF-G) gene. Forty-nine of these accessions carried allele A, and the other three carried allele D. The latter all originated from a narrow region close to the border with Algeria, whereas the former were represented across the whole collection area. Since six-rowed D allele carriers are present in North Africa, along with both two-rowed cultivated and wild barleys, it is likely that the European six-rowed barley varieties carrying the D allele have Moroccan parentage.