Manuscript Click here to access/download;Manuscript;Siniscalchi et al primer note_final_final_final_resub_edits.docx Click here to view linked References 1 2 3 4 Using genomic data to develop SSR markers for species of Chresta (Vernonieae, Asteraceae) 5 6 7 from the Caatinga 8 9 10 11 Carolina M. Siniscalchi12*, Benoit Loeuille3, José R. Pirani2, Jennifer R. Mandel1 12 13 14 15 16 17 18 1 19 Department of Biological Sciences, University of Memphis, Memphis, TN 38152, USA 20 21 2 Departamento de Botânica, Instituto de Biociências, Universidade de São Paulo, Rua do Matão, 22 23 24 277, 05508-090, São Paulo, SP, Brazil 25 26 3 Universidade Federal de Pernambuco, Departamento de Botânica - CCB, Av. Prof. Moraes Rego, 27 28 1235, 50670-901, Recife, PE, Brazil. 29 30 * 31 Author for correspondence: [email protected] 32 33 34 35 36 ORCID ID: 37 38 Carolina M. Siniscalchi: 0000-0003-3349-5081 39 40 41 Benoit Loeuille: 0000-0001-6898-7858 42 43 José Rubens Pirani: 0000-0001-7984-4457 44 45 Jennifer R. Mandel: 0000-0003-3539-2991 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 1 64 65 1 2 3 4 Abstract 5 6 Chresta is a genus mostly endemic to Brazil that presents several rupiculous species with naturally 7 8 9 fragmented distributions. Aiming to facilitate studies about genetic diversity and structure in these 10 11 species, we developed a set of 22 nuclear and 6 plastid microsatellite markers that are transferable 12 13 14 among different species of the genus. We used previously obtained genomic data from target 15 16 capture and Illumina sequencing to identify putative repeat regions, designed and synthetized 17 18 19 primers, and genotyped individuals from different populations of three species. All loci were 20 21 successfully amplified in all three species and were overall variable, except for the plastid 22 23 24 markers, which were monomorphic in two species. These newly developed microsatellites will be 25 26 useful in studies focusing on the population genetics of Chresta. 27 28 29 30 31 Key Words: Chrestinae, microsatellite, rock outcrops, target capture. 32 33 34 35 36 1. Introduction 37 38 Microsatellites, or Simple Sequence Repeats (SSRs), still are some of the most frequently used 39 40 41 markers for population and conservation genetics studies. They are prevalent among different 42 43 organisms, abundant in the genome, often highly polymorphic, and relatively inexpensive (Hodel 44 45 et al. 2016). The traditional method to develop microsatellite markers from a genomic library 46 47 48 enriched for repeat motifs is work-intensive and usually results in markers that are hard to 49 50 successfully transfer among different species (Squirrell et al 2003). With Next Generation 51 52 53 Sequencing methods becoming more widespread and less expensive, and with the increased 54 55 availability of genomic data in public databases, developing microsatellites from in silico mining 56 57 58 of sequences has become more common (Zalapa et al. 2012). Another advantage is the ability to 59 60 61 62 63 2 64 65 1 2 3 4 generate non-anonymous markers that can be more easily transferred across species, especially 5 6 when they are located in conserved genomic regions, such as within genes and expressed regions 7 8 9 of the DNA (Ellis and Burke 2007). 10 11 The recent development of target capture probes specific to the Asteraceae, targeting ca. 1000 12 13 14 nuclear orthologous regions (Mandel et al. 2014), has considerably advanced our understanding of 15 16 the phylogeny of the family (Mandel et al. 2015; 2017; 2019), and provided an abundance of 17 18 19 genomic data that have yet to be explored in other ways. Marker development is one of the 20 21 potential uses, and has already been tested with the development of microsatellites for genus 22 23 24 Antennaria (Thapa et al. 2019). 25 26 Asteraceae is the third most diverse plant family in Brazil, with more than 2000 species occurring 27 28 in the country, being among the top five more numerous families in five of the six tradionally 29 30 31 recognized phytogeographic domains (BFG 2015). Chresta Vell. ex DC. has 18 species mostly 32 33 endemic to Brazil (Siniscalchi 2019a), which are distributed mainly in the Cerrado, the savanna- 34 35 36 like environment in central Brazil, and in the Caatinga, a diverse phytogeographic domain 37 38 composed mainly of Seasonally Dry Tropical Forests in a semi-arid region in Northeastern Brazil. 39 40 41 The group of species that occurs in the Caatinga is composed exclusively of rupiculous plants, 42 43 usually represented by populations that are isolated from one another due to rock outcrops being 44 45 surrounded by a dry forest matrix (Siniscalchi et al. 2018). Some species, like Chresta harleyi 46 47 48 H.Rob., C. hatschbachii H.Rob. and C. subverticillata Siniscalchi and Loeuille are restricted to 49 50 small areas that are relatively close to one another, without showing overlap or signs of 51 52 53 hybridization, while others, like C. martii (DC.) H.Rob, have a wider distribution, showing 54 55 morphological differentiation between populations on the extremes of the distribution (Siniscalchi 56 57 58 et al. 2019b). Although these seven species share many morphological similarities and 59 60 61 62 63 3 64 65 1 2 3 4 environmental preferences, they do not form a monophyletic group, with C. martii actually being 5 6 the sister taxa to the rest of the genus. The other six Caatinga species form a clade which is further 7 8 9 subdivided into two clades (Siniscalchi et al. 2019c). 10 11 The natural isolation of the populations of Chresta in the Caatinga and the presence of 12 13 14 morphological variability throughout a geographical gradient raise interesting questions about the 15 16 evolutionary processes that act on these species, as the gene flow between different populations is 17 18 19 likely limited. Aiming to facilitate future studies of the genetic variation in Chresta species, we 20 21 developed microsatellite markers using previously obtained genomic data from a phylogenetic 22 23 24 study of Vernonieae (Siniscalchi et al. 2019c), comprising both nuclear and chloroplast sequences. 25 26 27 28 2. Material and Methods 29 30 31 DNA extraction—Total DNA was extracted from silica-gel dried leaves using the E.Z.N.A.® SQ 32 33 Plant DNA Kit from Omega Bio-Tek (Norcross, GA, USA), with the addition of PVP and 34 35 36 Ascorbic Acid to the first extraction buffer (10 mL SQ1 buffer, 100 mg PVP, 90 mg ascorbic 37 38 acid). One extra step was added for Chresta martii extractions, consisting of two washes with 1 39 40 41 mL of STE buffer (0.25 M sucrose, 0.03 M Tris, 0.05 M EDTA), followed by 10 minutes of 42 43 centrifugation at 2,000 g, in order to remove mucilage (adapted from Shepherd and McLay 2011). 44 45 Target capture and genomic data assembly—Total DNA extracted from 17 Chresta species 46 47 48 (Table 1) was quantified using fluorometry (Qubit 3.0, ThermoFisher Scientific), then sheared to 49 50 300 bp fragments using a sonicator (Covaris S series or QSonica Q500). Illumina libraries were 51 52 53 prepared with the NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs 54 55 Inc., Ipswich, MA, USA), according to the manufacturer’s instructions, using 15 cycles on the last 56 57 58 amplification step. Final library concentrations and sizes were checked using Qubit and libraries 59 60 61 62 63 4 64 65 1 2 3 4 were then pooled in groups of four. Target capture was carried out using the myBaits COS 5 6 Compositae/Asteraceae 1Kv1 kit (Mandel et al. 2014) (Arbor Biosciences, Ann Arbor, MI, USA), 7 8 9 according to the manufacturer’s instructions and using a 36-hour incubation step. Sequencing was 10 11 performed at Macrogen Inc. (Seoul, South Korea), on an Illumina HiSeq2500 device, in paired- 12 13 14 end, high-throughput mode. 15 16 Reads were trimmed for quality using Trimmomatic (Bolger et al. 2014) and assembled into 17 18 19 contigs using SPAdes (Bankevich et al. 2012), with kmer lengths of 21, 33, 55, 77, 99 and 127. 20 21 The sequences were matched back to the original probes using the phyluce pipeline (Faircloth 22 23 24 2016), resulting in approximately 700 individual alignments corresponding to the recovered 25 26 targeted loci, containing all taxa for which that given locus was recovered. 27 28 29 30 31 Repeat identification and primer design—The identification of putative microsatellites was 32 33 carried out in the individual matrices obtained for each locus, using the plugin Phobos on 34 35 36 Geneious (Mayer 2006–2010), searching for di- to pentanucleotides motifs, with a minimal length 37 38 of 15 base pairs and allowing for imperfect motifs. All loci identified as having repeats were then 39 40 41 inspected by eye, and those showing the longest repeats or that presented variation among the 42 43 sequences in the matrix were selected. Loci that were found in more than one taxon present in the 44 45 matrix were preferred, in order to ensure transferability. The consensus for each alignment was 46 47 48 subjected to a BLAST search on GenBank, using BLASTn, to identify in which part of the gene 49 50 the repeat was. We gave preference to repeats located in introns or less conserved regions (Table 51 52 53 2).