Genetics Analyses

Measuring Population Diversity

Students/personnel at MSU conducted the DNA laboratory work and screening for amplification and variation of the fresh material. Genetic diversity was assessed using chloroplast sequence (cpDNA) variation and highly variable nuclear microsatellites. Tissue collected from the study populations was homogenized using a Retsch MM 301 Ball Mill (Retsch, Newtown, PA) and whole genomic DNA (both cpDNA and nuclear DNA) were isolated using a Maxwell® 16 MDx Research Instrument and Maxwell 16 and Tissue DNA Purification Kits (Promega, Mannheim, Germany). Three cpDNA regions were sequenced using universal PCR primers (Taberlet et al. 1991). While the chloroplast genome is notoriously invariant, these regions have previously proven valuable for investigating genetic structure in this species (Blum et al. 2007). The microsatellites to be used are also known to be highly variable in populations located on the Gulf Coast (Blum et al. 2004; Sloop et al. 2005). There are 35 microsatellites characterized for this species. We screened these loci for variation, and selected 15 that are highly variable and exhibit robust disomic genotypes. The cpDNA marker should only spread through populations via seed because chloroplasts are typically maternally inherited (Reboud and Zeyl 1994). Contrasting chloroplast variation with nuclear variation will allow us to assess the relative roles of seed and pollen in dispersing genes within and among populations (McCauley 1997). Degree of genetic structure will be estimated and tested for significance using AMOVA for both classes of molecular markers (Weir and Cockerham 1984; Excoffier et al. 1992). High levels of genetic structure would indicate the potential for greater local adaptation and would suggest the use of more geographically proximate accessions for use during restoration. More importantly, these genetic data will allow us to identify populations that exhibit ideal reproductive strategies for use in restoration. Each ramet will be assigned to a genet by matching multilocus genotypes using GENELEX v. 6.5 (Peakall and Smouse 2012). This will allow us to infer the degree to which a population propagates clonally and sexually using established statistical methods (Yakimowski and Barrett 2014).

Nuclear DNA was extracted from all plants, a necessary first step for conducting the complete molecular analysis. We screened the nuclear DNA microsatellite loci for variability. This was the most time consuming step. The genotyping was essentially done for S. alterniflora in summer 2017. However, we had some issues isolating DNA with J. roemerianus. We used a different approach that included an additional mechanical breakdown step that seemed to have improved matters. While DNA concentrations were low, it turns out that wasn’t the main problem. The microsatellites we were trying to score were originally characterized for J. effusus. It appears that J. roemerianus is different enough that those markers aren’t tractable in that species. Fortunately, microsatellite markers for J. roemerianus have recently been developed.

At end of 2018, all 10 populations (500 plants) had been completed for nuclear DNA extraction, microsatellite loci were screened for variability, and preliminary data processing for both S. alterniflora (Table 2) and J. roemerianus (Table 3) at MSU. Genetic diversity was generally higher (allelic richness – Na and heterozygosity – Ho, He) for S. alterniflora populations collected within MS, than for those populations collected from outside of MS, whereas the opposite was generally true for J. roemerianus. The effective population (Ne) sizes for both species is really small. That means that rates of sexual reproduction are really low. S. alterniflora and J. roemerianus correlation indices (rbarD) were illustrated as heat maps (Figure 7) to reveal high and low levels of Linkage Disequilibrium (LD). LD serves as an indicator of inbreeding (self-crossing) which is known to lead to low genetic diversity. Both species are known to rely primarily on asexual vegetative propagation and clonal expansion, and use sexual seed-based reproduction primarily to colonize disturbed areas or to expand their range. It is not yet known how long seeds may remain viable during water-borne transport, or how this might affect dispersal distance.

We have some preliminary cpDNA sequence data to analyze as well. J. roemerianus and S. alterniflora cpDNA were sequenced and analyzed at the rbcL (Rubisco Large subunit) DNA region. Sequences were viewed and edited using Sequencher software. Resulting haplotypes were illustrated for each species via haplotype network (Figures 5 and 6). Our first early glimpse at that data suggests there is little cpDNA variation in S. alterniflora.

Tables 2 and 3: Results from the genetics analysis. Sites in grey shaded rows are MS sites. Data for Fort Pike, LA and Popps Ferry bridge, MS sites are still being summarized. Na = number of alleles (a measure of genetic diversity within the population), Ho = observed heterozygosity, He = expected heterozygosity (heterozygosity is a measure of genetic variation within the sample plants). SE is standard error of the mean for a sample size of 20 plants per site.

Figure 5 and 6. Left panel - Network analysis of Spartina cpDNA variation (RBCL). Eight unique haplotypes were revealed amongst the 10 populations analyzed. Right panel - Network analysis of the 1352 base pair region (rbcL) within Juncus cpDNA. Four unique haplotypes were revealed amongst the 10 populations analyzed.