Development of specific molecular markers to distinguish and quantify broomrape species in a soil sample
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Broomrapes (Orobanche and Phelipanche spp.) are destructive obligate plant parasites in Israel and in the Mediterranean basin. Conventional methods for parasitic weeds detection are difficult, since the parasite seeds are extremely small (dust-like seeds) and survive in the soil for several decades. Here, we report the development of specific molecular markers rbcL1 based on rbcL (large subunit of the ribulose-bisphosphate carboxylase) gene from Orobanche crenata and ITS100 based upon unique sequences in the internal transcribed spacer (ITS) regions of the nuclear ribosomal DNA of Phelipanche aegyptiaca. Genomic DNA was extracted from soil samples artificially infested with broomrape seeds or tissue of P. aegyptiaca, O. cumana and O. crenata and subjected to PCR analysis. rbcL1 marker, successfully differentiate between O. crenata and O. cumana, amplified a specific PCR products (1300 bp with O. crenata and 1000 bp with O. cumana). However, the rbcL1 marker failed to amplify soil samples with seeds or tissues of P. aegyptiaca or any soil-borne DNA. ITS100 marker and Real-Time PCR, allowed quantitative diagnostic of the parasite O. cumana in a soil sample; amplified a specific PCR products (100 bp). As expected the universal control primer (UCP-555) amplified a PCR product (555 bp), when genomic DNA extracted from soil samples with or without broomrape tissues. The development of an efficient, simple and robust molecular marker to detect and distinguish between broomrape species, has a significant insights on the assessment level of infestation and planning eradication program of the parasite in a field crop.
KeywordsPlant parasite Phelipanche aegyptiaca Orobanche spp. ribulose-1,5-bisphosphate carboxylase (rbcL) Internal transcribed spacer (ITS)
Results of this research were supported by the Chief Scientist of the Ministry of Agriculture and Rural Development - Israel, grant No.132-1499-10 (MEZAMALEKET). V.K.B is grateful to the ARO-Volcani Center, Agricultural Ministry of Israel for providing the Postdoctoral fellowship.
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Our manuscript is original research and it is not submitted to full or in parts to other journal for publication.
Conflict of interest
The authors declare that they have no conflict of interest.
Human and animal studies
Our study did not involve human participants and/or animal as experimental model.
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