Characterization of microsatellite markers for Moricandia moricandioides (Brassicaceae) and related species

Premise of the Study Polymorphic microsatellite markers were developed to study population structure and mating patterns of the monocarpic herb Moricandia moricandioides (Brassicaceae). Methods and Results Illumina MiSeq sequencing was used to develop a panel of 15 polymorphic microsatellite markers that were tested across 77 individuals from three populations on the Iberian Peninsula. All markers were polymorphic in at least two studied populations, and the number of alleles ranged from one to 11 per locus. The levels of observed and expected heterozygosity ranged from 0.000 to 1.000 and from 0.153 to 0.865, respectively. Nine and 11 loci were successfully amplified in the congeneric species M. arvensis and M. foetida, respectively. Conclusions The 15 microsatellite markers will be useful for population genetic studies of the genus Moricandia. These markers will serve as a useful tool for exploring population structure and mating patterns of M. moricandioides.


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Moricandia moricandioides (Boiss.) Heywood (Brassicaceae) is a monocarpic herb inhabiting semi-arid areas in the Iberian Peninsula (Sobrino Vesperinas, 1993). Moricandia moricandioides displays a particular floral morphology with parallel, dark pink petals (Gómez et al., 2016). Their nearly zygomorphic flowers are commonly visited by a highly specialized assembly of insects, mostly composed of long-tongued bees (Gómez et al., 2016). The species has been used as a model species in several ecological studies related to the analysis of food webs and the interaction between aboveground and belowground insect species (e.g., González-Megías and Müller, 2010;González-Megías and Menéndez, 2012), as well as the study of plant reproductive ecology (e.g., Gómez, 1996).
Moricandia moricandioides belongs to the small but economically relevant genus Moricandia DC., with nine recognized species distributed in North Africa, the Middle East, Central Asia, and Southern Europe (Perfectti et al., 2017). Other members of the genus are narrow endemics (e.g., M. foetida Bourg.) restricted to limited areas in southeastern Spain, as well as widespread weeds (e.g., M. arvensis (L.) DC.) that likely colonized the Iberian Peninsula and southern Italy from northwestern Africa. In addition, it has recently been observed that M. moricandioides individuals may discriminate kin, allowing them to adjust their floral display depending on the genetic configuration of their neighborhood (Torices et al., 2018), thus enabling potential cooperation with kin. We here present and characterize microsatellite markers for M. moricandioides and two related species. The development of these markers will allow the exploration of the finespatial genetic structure within M. moricandioides populations, which is essential for testing hypotheses concerning kin selection and altruism in plants.

Plant collection and DNA purification
We collected leaves from individuals of M. moricandioides cultivated in a greenhouse from seeds sampled in three different natural populations (Appendix 1). To test cross-species amplification, two related species were also collected (Appendix 1): M. arvensis (seeds sampled in June 2016) and M. foetida (leaves collected from a natural population in January 2017). All leaves were conserved in silica gel before DNA purification. We extracted genomic DNA using a BioSprint 96 DNA Plant Kit (QIAGEN, Hilden, Germany) following the manufacturer's instructions and eluted in 100 μL of nuclease-free water.

Development of microsatellite primers
Microsatellite markers were developed using an enrichment protocol at AllGenetics & Biology (A Coruña, Spain). The library, prepared using the Nextera XT DNA kit (Illumina, San Diego, California, USA), was enriched in the following motifs: AT, AAG, ACG, ATCT, and ACAT. The enriched library was then sequenced on an Illumina MiSeq platform (PE300). Assembly of the reads was performed in Geneious 8.1.8 (Biomatters Ltd., Auckland, New Zealand) after quality checking and removal of duplicate sequences. Primers were then designed with default parameters in Primer3 (Koressaar and Remm, 2007;Untergasser et al., 2012) using sequences with at least 80 nucleotides, and 500 primer pairs were developed that hybridized in the microsatellite flanking regions. We first tested and checked for polymorphism in 72 primer pairs using 11 M. moricandioides samples from the three populations. Of these 72 primer pairs, 15 polymorphic microsatellite markers were selected (with three or more alleles per locus) and tested using 77 samples from the three populations. The markers were organized into five multiplexes according to the primer properties and size ranges (Table 1).

Data analysis
Genetic diversity parameters, pairwise linkage disequilibrium (LD), and Hardy-Weinberg equilibrium (HWE) were calculated using GENEPOP 4.7.0 (Rousset, 2008) for each of the three populations of M. moricandioides. We adjusted both tests for multiple comparisons using Benjamini and Hochberg's (1995)   The number of alleles per locus ranged from one to 11 ( Table 2). The observed heterozygosity ranged from 0.000 to 1.000, and the expected heterozygosity ranged from 0.153 to 0.865 (Table 2). Loci 185 and 335 showed significant deviation from HWE in all three populations. Loci 335 showed two alleles fixed in heterozygosity. Loci 170,176,235,393,406,439,and 465 showed deviation from HWE in one or two populations. Loci 170 and 393 were monomorphic in population B. Loci 031, 096, 212, 262, 319, and 402 showed no deviation from HWE. There was no significant LD between any pairs of loci. There was no evidence of null alleles for any locus in population B. Loci 170, 176, 235, and 465 showed evidence of null alleles in population A, as did loci 170 and 439 in population C. Loci 031 and 096 did not amplify consistently in all individuals of populations A and C ( Table 2). The significant deviation from HWE and the presence of null alleles might be a consequence of the high genetic relationship between the individuals sampled, some of which were half-siblings. All sequences, along with primers, were submitted to the European Nucleotide Archive (Table 1; http://www.ebi. ac.uk/ena/data/view/LS483218-LS483232).

Cross-amplification in related species
Tests of amplification of the 15 loci were performed on two related species, M. arvensis and M. foetida (Table 3). Loci 031,170,185,212,235,319,393,406,and 439 amplified in both species;loci 176,402,and 465 amplified only in M. foetida; and loci 096, 262, and 335 did not amplify in either species. The number of alleles per locus ranged between one and five. Loci 406 and 439 were monomorphic in M. arvensis, whereas loci 185, 393, and 402 were monomorphic in M. foetida.

CONCLUSIONS
We developed and successfully amplified 15 polymorphic markers in M. moricandioides and in its close relatives M. arvensis and M. foetida. These polymorphic loci will be valuable for future studies in M. moricandioides, primarily those related to fine-scale population genetic structure and patterns of mating. In addition, the successful cross-amplification in M. arvensis and M. foetida suggests that these loci may be suitable to study gene flow, population structure, and potential introgression of the widespread M. arvensis into the other more narrowly distributed species of the genus Moricandia.

SUPPORTING INFORMATION
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APPENDIX S1. Primer mix setup for multiplex reactions and final PCR concentration.