Development of chloroplast microsatellite markers for giant ragweed (Ambrosia trifida)

Premise Plant invasions are increasing globally, and extensive study of the genetic background of the source and invading populations is needed to understand such biological processes. For this reason, chloroplast microsatellite markers were identified to explore the genetic diversity of the noxious weed Ambrosia trifida (Asteraceae). Methods and Results The complete chloroplast genome of A. trifida was mined for microsatellite loci, and 15 novel chloroplast primers were identified to assess the genetic diversity of 49 Ambrosia samples. The number of alleles amplified ranged from two to six, with an average of 3.2 alleles per locus. Shannon's information index varied from 0.305 and 1.467, expected heterozygosity ranged from 0.178 to 0.645, and the polymorphism information content value ranged from 0.211 to 0.675 (average 0.428). The cross‐species transferability of the 15 microsatellite loci was also evaluated in four related Ambrosia species (A. artemisiifolia, A. maritima, A. psilostachya, and A. tenuifolia). Conclusions The novel chloroplast microsatellite markers developed in the current study demonstrate substantial cross‐species transferability and will be helpful in future genetic diversity studies of A. trifida and related species.

loci. Simple sequence repeats (SSRs) were identified using MISA software (Thiel et al., 2003). Mononucleotide repeats were excluded from our search, and we applied a threshold based on minimum length criteria (unit size/minimum repeat time): six for di-, four for tri-, and three for tetra-, penta-, and hexanucleotide repeats, respectively. MISA allowed the identification and localization of perfect as well as compound microsatellites. Compound SSRs were considered repeats disrupted by a non-repetitive length of 100-bp sequence. Based on the flanking regions of the repeat regions, we designed primers for 15 cpSSR loci using the BatchPrimer3 version 1.0 web program (You et al., 2008). The parameters for designing PCR primers were as follows: (1) primer length ranging from 18 to 23 bp; (2) PCR product size range of 100-300 bp; (3) melting temperature between 50°C and 70°C, with 55°C as the optimum annealing temperature; and (4) a GC content of 40-70%, with an optimum of 50% (Table 1).
After primer design, amplification efficiency and polymorphism were evaluated using 29 A. trifida DNA samples from two different historical populations collected in Europe and North America. Crossamplification in the genus Ambrosia was assessed in five individuals each of A. artemisiifolia, A. maritima L., A. psilostachya DC., and A. tenuifolia Spreng. DNA samples were taken from the herbarium collection of the Finnish Museum of Natural History (Appendix 1). Leaf samples were rinsed with deionized water and 70% ethanol, and total genomic DNA was isolated using the E.Z.N.A. Plant Kit (Omega Bio-tek, Norcross, Georgia, USA). Laboratory work was carried out in a separate DNA laboratory at the University of Helsinki. Blank samples were processed together with herbarium material during DNA extractions. Final concentrations were measured with a Qubit 2.0 Fluorometer (Thermo Fisher Scientific, Waltham, Massachusetts, USA). PCR amplification was carried out in a 20-μL volume containing 20 ng of genomic DNA, 0.2 mM of dNTPs (Thermo Fisher Scientific), 2 μL 1× PCR buffer, 20 pM of each primer, and 0.25 units DyNAzyme DNA polymerase (Thermo Fisher Scientific). All reactions were performed in a MasterCycler ep96 (Eppendorf, Hamburg, Germany) with the following settings: 2 min of initial denaturation at 94°C; 35 cycles of denaturation for 30 s at 94°C, annealing for 1 min at 55°C, and extension for 2 min at 72°C; followed by a final extension for 5 min at 72°C. Amplification products were separated on 1.5% agarose gels (GE Healthcare, Chicago, Illinois, USA) using a 0.5× TBE buffer (220 V, 0.5 h) and stained with SYBR Safe DNA gel stain (Thermo Fisher Scientific) to validate successful amplifications.
After initial testing with three A. trifida and four Ambrosia species samples (A. artemisiifolia, A. maritima, A. psilostachya, and A. tenuifolia), the amplifications were repeated using fluorescently labeled primers applying the PCR protocol described above using all 49 Ambrosia samples. PCR products were analyzed on an ABI 3730XL DNA sequencer (Applied Biosystems, Foster City, California, USA) using a GeneScan 500 LIZ Size Standard (Applied Biosystems). The scoring of electropherograms was carried out with Geneious Prime (Biomatters Ltd., Auckland, New Zealand). All primers amplified successfully across the test individuals and 10 proved to be polymorphic, whereas five gave monomorphic patterns (Table 1). The polymorphic markers were used to evaluate the genetic diversity of 49 Ambrosia samples. The number of alleles, effective number of alleles, and Shannon's information index were calculated with GenAlEx 6.5 (Peakall and Smouse, 2012), while expected heterozygosity and the polymorphism information content were calculated using iMEC (Amiryousefi et al., 2018). The number of alleles per locus ranged from two to six, the effective number of alleles ranged from 1.198 to 2.324, Shannon's information index ranged from 0.305 and 1.467, and the expected heterozygosity ranged from 0.178 to 0.645, while the polymorphism information content ranged from 0.211 to 0.675 (Table 2). The 10 polymorphic loci were also successfully amplified in 20 individuals (five individuals each) in the following four related species: A. artemisiifolia, A. maritima, A. psilostachya, and A. tenuifolia (Table 3, Appendix 1).

CONCLUSIONS
We used the recently sequenced plastid genome of A. trifida (Sablok et al., 2019) to develop and characterize 15 chloroplast microsatellite markers; these were then used to identify high genetic diversity among the analyzed giant ragweed samples. Overall population genetic variation was similar to that detected in common ragweed (A. artemisiifolia) in North America and in the Rhône-Alpes region (Genton et al., 2005). Given the high level of polymorphism detected with the developed cpSSR primer set in the A. trifida population, the markers developed here should be suitable for further studies investigating the origin of invasive populations in Europe and studying the dynamics of invasion and modes of dispersal. Due to the high rate of cross-amplification, the developed polymorphic cpSSR primers will likely be useful in intra-or interspecific genetic studies of the genus Ambrosia. Grant (no. 322635 to H.S.). The authors thank Elina Laiho for laboratory assistance and performance of DNA extractions, Aija Ronkainen for assistance in editing herbarium data, and Julie Marie Uusinarkaus for editing the manuscript.

DATA AVAILABILITY
All sequence information was deposited in the National Center for Biotechnology Information (NCBI) GenBank database (accession numbers MN385584-MN385593 and NC_036810.2; Table 1).  Locality and voucher information are provided in Appendix 1.