As a holoparasitic weed, broomrape has seriously threatened the production of economically important crops, such as melon, watermelon, processed tomato, and sunflower, in Xinjiang Province in recent years. However, the distribution and genetic diversity of broomrape populations in Xinjiang are not clear at present, which hinders their prevention and control process. In the present study, 93 samples from different geographic regions of Xinjiang were collected to identify the species based on ITS and plastid rps2 regions, and the samples were used to analyze the genetic diversity based on ISSR markers. The results showed that broomrape is not monophyletic in Xinjiang and consists of two major clades (Orobanche cf. aegyptiaca and O. cernua) and three subclades (O. cf. aegyptiaca var. tch, O. cf. aegyptiaca var. klz, O. cernua.var. alt) by phylogenetic analysis based on ITS and rps2. Furthermore, the results of the genetic diversity analysis indicated that the 11 selected primers produced 154 repeatable polymorphic bands, of which 150 were polymorphic. The genetic diversity of the samples was 37.19% within populations and 62.81% among the populations, indicating that the main genetic differentiation occurred among the populations. There was less gene exchange between populations, with a gene flow index (Nm) of 0.2961 (<1). The UPGMA dendrogram indicated that most populations with similar geographical conditions and hosts were clustered first, and then all samples were separated into two major groups and seven subclusters. This is the first study to clarify genetic diversity and population distribution of broomrape in Xinjiang. Our results provide a theoretical basis for the prevention, control, and breeding of broomrape-resistant varieties.

DNA extraction and PCR amplification

Total DNA was extracted from all samples following the cetyl-trimethylammonium bromide (CTAB) method described by Xin and Chen (Xin and Chen, 2012). The purity of the DNA was determined by 1.0% agarose gel electrophoresis and absorption at 260 nm, and the ratios A260/А280 and A260/А230 were used to determine the presence of contaminants such as proteins, polyphenolic compounds, sugars, and lipids. The samples were diluted to 50 ng/μl for PCR amplification.

The ITS sequences of the partial samples were amplified with primer pair QR-F (5’- CCTGCAAAAGCAGACCGT-3′) and QR-R (5’-CGCAATCGAAGGCACGAG-3′), which were designed based on the ITS alignment of O. aegyptiaca (AY209327.1). Other ITS sequences were amplified with the primer pair ITS1 (5’-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTC CGCTTATTGATATGC-3′) (Kwon et al., 2012). PCR amplification was carried out on a DNA engine dyad® Peltierp thermal cycler (Bio-Rad) using a 50 μL reaction mixture containing 100 ng DNA, 25 μL master PCR mix, and 2 μL of each primer (10 μm/L). The PCR conditions were as follows: 94°C for 4 min, followed by 30 cycles of 94°C for 1 min each, 56°C or 60°C for 1 min, and 72°C for 1 min, followed by 10 min at 72°C. The PCR products were purified using a universal DNA purification kit (TIANGEN, Beijing), according to the manufacturer’s instructions, cloned and inserted into pMD18-T (TaKaRa Biotech, Dalian), and sequenced. All rps2 sequences were amplified with the primer pairs rps2 2F (5’-AAATGGAATCCTAAAATGGC-3′) and rps2 2R (5’-AAACAAATTGTTGGA ATTCC-3′) (Park et al., 2007). The reaction conditions were as follows: 94°C for 4 min, followed by 35 cycles of 94°C for 1 min each, 55°C for 1 min, and 72°C for 1 min, followed by 10 min at 72°C. The PCR products were sequenced using the above method.

ISSR assay

A total of 100 ISSR primers from the University of British Columbia were synthesized at the BGI of Beijing and used for the study, of which 11 primers showed a high percentage of polymorphism (Table S1). The PCR amplification was carried out using a DNA engine dyad® Peltierp thermal cycler (Bio-RAD), using 20μL reaction mixture containing 50 ng DNA, 2.2 μL 10 × PCR buffer(Mg²⁺ Plus), 1.0μL of dNTP(2.5 mM), 0.4μL primer(10μm/L) and 0.2μl of Taq DNA polymerase(5U/μL, TaKaRa Biotech, Dalian). The PCR conditions were as follows: 94°C for 4 min, followed by 35 cycles of 94°C for 1 min, 50.3-55°C for 30 s, 72°C for 90 s, and a final extension of 10 min at 72°C.

The PCR products were mixed with 5 μL Roti-Load-DNA loading buffer (TaKaRa Biotech, Dalian), resolved on a 1.5% agarose gel in 0.5× Tris-borate-EDTA (TBE) buffer, and electrophoresis was carried out with a constant voltage of 120 V for 90 min, visualized under UV light, and documented using a gel documentation and image analysis system (Universal Hood, Bio-Rad). The experiment was repeated three times, and the bands appeared consistently in all three gels, which were scored and used for the analysis.