Identification of seeds of Phelipanche ramosa, Phelipanche mutelii and Orobanche cumana in the soils from different agricultural regions in Bulgaria by molecular markers

Broomrapes are root holoparasites from Orobanchaceae family parasitizing other flowering plants. Several of the most aggressive broomrape species are widely spread in Bulgaria often causing serious yield losses of important crops. It is important to be able to detect the presence of broomrape seeds in the soil. In the present study, we combined a proven PCR-based assay for detection of broomrape seeds with methods to distinguish species based on nuclear ITS sequences to identify broomrape seeds isolated from soil samples and to study the population structure and the intraspecific variations within the three species. Fifty-six soil samples from 23 different regions in Bulgaria were studied. Based on molecular and bioinformatics analyses we found that 22 samples contained seeds of Orobanche cernua var. cumana and six samples, seeds of Phelipanche ramosa. Phylogenic and bioinformatics analyses surprisingly revealed that the isolated sequences from supposedly P. mutelii seeds diverge from those annotated in NCBI by other authors on 16 different nucleotide positions and formed two distant branches of the phylogenic tree. On the other hand, the isolated sequences were almost identical with P. rosmarina. Tajimas’ D-test revealed that O. cumana populations are currently stable. Regarding the Phelipanche representatives, based on the D-test we can hypothesize that P. mutelii/P. rosmarina populations are in a period of active expansion, while P. ramosa populations are contracting. All isolated sequences were deposited in NCBI Genbank database

Genetic diversity of Orobanche cumana and Orobanche cernua populations as revealed by variability of Internal Transcribed Spacers1/2 of ribosomal cistron and ribulose-bisphosphate carboxylase pseugene

Kirilova, Ina et al.-- Trabajo presentado en el Third Internacional Symposium on broomrape (Orobanche spp.) in Sunflower, celebrado en Córdoba (España) del 3 al 6 de junio de 2014.The sunflower broomrape - Orobanche cumana (Wallr) parasitizes on roots of sunflower plants and is a serious constraint on sunflower production, causing yield losses of up to 60%. The variability of Internal Transcribed Spacers1/2 of ribosomal cistron (ITS1/2) and ribulose-bisphosphate carboxylase pseugene (RbcL) in 32 samples of O. cumana and 4 samples O. crenata collected from different European locations were studied. The results showed that O. cumana can be differentiated from O. cernua, by single C/T transition located in ITS2 (rel. position 423). Rubisco large subunit in O cumana differs from O. cernua with two transversion: T/G (rel. position 15) and A/C (rel. position 84). The genetic diversity observed in O. cumana was lower than in O. cernua. When comparing the ITS and rbcL sequences isolated from O. cernua two single SNPs were found that can discriminate different origins. Both ITS1/2 and rbcL sequences isolated from O. cumana however were completely homogeneous, despite the fact that samples were collected form very distant locations: from Volgograd, Russia to the East to Spain to the West. This observation is in favor of hypothesis that the move of O. cumana from wild hosts on sunflower was a single act that occurred once and all invasive races are descendants from ancient Caucasus population. Probably genes related to O. cumana aggressiveness should be identified and used for molecular markers to determine genetic relationships within and among O. cumana populations.N