Development of race-specific molecular marker for Xanthomonas campestris pv. campestris race 3, the causal agent of black rot of crucifers

Race-specific molecular markers were established to distinguish Xanthomonas campestris pv. campestris (Xcc) race 3, the causal agent of black rot disease of crucifers. The available genome sequences of Xcc races were aligned and identified three DNA fragments specific to Xcc race 3. The identified race-specific DNA fragments namely XccR3-49, XccR3-52, and XccR3-55 were used for designing the race-specific primers to detect and identify Xcc race 3. The specificity of race-specific primers was tested against the genomic DNA extracted from Xcc (races 1–7), Xcc strains, Xc pathovars, and other bacterial species. XccR3-49, a specific sequence characterized amplified region (SCAR) primer set, gave a single band with 867 bp length for Xcc race 3 only. The remaining two markers XccR3-52 and XccR3-55 showed polymorphic amplification with amplicon sizes of 1889 and 2109 bp for Xcc race 3, respectively. Additionally, the SCAR primer set detected Xcc race 3 rapidly and efficiently in artificially infected cabbage leaves with bio-PCR. This result showed that the newly developed race-specific markers can successfully and efficiently detect and identify Xcc race 3 from Xanthomonas campestris pv. campestris races, Xanthomonas species/pathovars, as well as other plant pathogenic bacteria (Pseudomonas syringae pv. maculicola and Erwinia carotovora subsp. carotovora). Up to now, this is the first report describing the race-specific marker for the detection of Xcc race 3.

Identification of the B, Q, and native Brazilian biotypes of the Bemisia tabaci species complex using Scar markers

The objective of this work was to develop sequence-characterized amplified region (Scar) markers to identify the B, Q, and native Brazilian biotypes of the sweet potato whitefly [Bemisia tabaci (Hemiptera: Aleyrodidae)]. Random amplified polymorphic DNA (RAPD) amplification products, exclusive to the B and Brazilian biotypes, were selected after the analysis of 12,000 samples, in order to design a specific Scar primer set. The BT-B1 and BT-B3 Scar markers, used to detect the B biotype, produced PCR fragments of 850 and 582 bp, respectively. The BT-BR1 Scar marker, used to identify the Brazilian biotype, produced a PCR fragment of 700 bp. The Scar markers were tested against the Q biotype, and a flowchart was proposed to indicate the decision steps to use these primers, in order to correctly discriminate the biotypes. This procedure allowed to identify the biotypes that occur in field samples, such as the B biotype. The used set of primers allowed to discriminate the B, Q, and native Brazilian biotypes of B. tabaci. These primers can be successfully used to identify the B biotype of B. tabaci from field samples, showing only one specific biotype present in all cultures.

Development of SCAR Primers for PCR Assay to Detect Diplodia seriata

The aim of this study was to develop primer pairs for Diplodia seriata identification, one of the most common fungal species associated with grapevine decline in Castilla y León (Spain). Genetic variability of selected isolates of D. seriata was estimated. A molecular marker was generated from a random amplified polymorphic DNA (RAPD) fragment. PCR products of around 1200 bp were obtained with OPE20 primer. The PCR products were cloned and sequenced. The sequences were compared and a fragment of 1207 bp was used to design primer pairs. Two primer pairs were selected (DS3.8 S3-DS3.8 R6 and DS3.8 S3-DS3.8 R4) that amplified a single DNA product of 634 bp and 233 bp, respectively, with D. seriata isolates. No amplification was obtained for any of the 57 isolates of other species. The designed SCAR primer pairs allowed a rapid detection of D. seriata, and were able to detect 0.1 pg of the target DNA. Detection was specific and sensitive for D. seriata. The established protocols detected these fungi in naturally infected grapevines after DNA purification. Diplodia seriata was detectable without DNA purification and isolation in 62.5% to 75% of reactions. The detection of this pathogen in wood samples has great potential for use in pathogen-free certification schemes.

SCAR molecular markers of the B biotype and two non-B populations of the whitefly, Bemisia tabaci (Hemiptera: Aleyrodidae)

Random amplified polymorphic DNA (RAPD) analyses were performed with random primer H16 for the B biotype and two non-B populations of Bemisia tabaci collected from Zhejiang (China). The specific sequence fragments containing 446, 390 and 1317 nucleotides were amplified for the B biotype, ZHJ-1, ZHJ-2 populations, respectively. The three specific fragments were cloned and sequenced, and three pairs of SCAR primers were designed according to the sequences determined. With improvement of the conditions of the polymerase chain reaction (PCR), the specific fragments of B biotype, ZHJ-1 and ZHJ-2 populations, namely 439, 366 and 1238 nucleotides, respectively, were amplified with the sequence characterized amplified region (SCAR) primer of the corresponding population, while specific fragments of the other populations of B. tabaci or Trialeurodes vaporariorum could not be amplified.

Development of SCAR Markers Linked to a Phytophthora fragariae Resistance Gene and Their Assessment in European and North American Strawberry Genotypes

Two dominant sequence characterized amplified region (SCAR) markers (linked at 3.0 cM, coupling phase) were constructed for the strawberry (Fragaria ×ananassa Duch.) gene Rpf1. This gene confers resistance to red stele root rot, caused by the soil-born fungus Phytophthora fragariae Hickman var. fragariae. The SCAR markers were developed originally from the sequence of RAPD OPO-16C(438) that is linked in repulsion phase to the Rpf1 allele. This SCAR primer set produced multiple bands in the resistant test progeny and in some of the susceptible progeny; therefore, new SCARs were developed based on the sequence differences among these bands. These new SCARs were linked in coupling phase to the Rpf allele and mapped to the same location as the original RAPD OPO-16C(438). The SCAR markers, as well as some additional RAPD markers known to be linked to Rpf1, were shown to be highly conserved in linkage to the gene based on examination of 133 European and North American Fragaria L. sp. cultivars and breeding selections. These flanking RAPD and SCAR-PCR markers can be used in breeding programs for the selection of red stele (Rpf1) resistance.