DOI: 10.5061/dryad.xd2547dpk
title: The polymorphism of the broomrape generated by ISSR primers

description: 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 (&lt;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.
description: 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 <em>O. aegyptiaca</em> (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
 <em>rps2</em> sequences were amplified with the primer pairs
 <em>rps2</em> 2F (5’-AAATGGAATCCTAAAATGGC-3′) and
 <em>rps2</em> 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(Mg<sup>2+</sup>
 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.
description: # The polymorphism of the broomrape generated by ISSR primers
 [https://doi.org/10.5061/dryad.xd2547dpk](https://doi.org/10.5061/dryad.xd2547dpk) <br> ## Description of the data and file structure The 11 ISSR primers were obtained by screening and generated a total of 154 bands among 93 samples, of which 150 bands were polymorphic. ## Sharing/Access information No * ## Code/Software Lasergene DNASTAR (version 6.0) , DNAMAN (version 6.0), MEGA6.0, POPGENE version 1.32, and  Numerical Taxonomy Multivariate Analysis System version 2.10