Project Number: 2080-21000-019-045-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Jul 1, 2022
End Date: Jun 30, 2023
Our research goal is to provide alfalfa seed growers knowledge on the genetic diversity and migration rates of managed alkali bee populations in the Touchet-Gardena-Lowden (TGL) area. This knowledge is important to growers in making effective breeding and translocation decisions across bee beds. We will accomplish our goal by pursuing the following specific objectives: 1) Identify microsatellite markers using the published alkali bee genome (Kapheim et al., 2019). We will survey the alkali bee genome for 50 microsatellite markers using bioinformatic analysis. These novel microsatellite markers will be rigorously evaluated for utility with alkali bee DNA using PCR, gel imaging, and fragment analysis. 2) Determine population structure (degree of genetic isolation), and rates of inbreeding and population bottlenecks (loss of genetic diversity). In collaboration with TGL area alfalfa seed growers, we aim to sample 450 bees across 15 bee beds in the TGL area. Genetic diversity will be estimated with markers identified in Objective 1. 3) Estimate migration rates among beds. Migration rates will be estimated by examining gene flow across bee beds using the same microsatellite marker dataset as in Objective 2. We will test for effects of geographic distance, bee bed size, and nest density.
Objective 1. Building off previous APRI funded genome research (Kapheim et al., 2019), we will identify 50 novel microsatellite loci for amplification and genotyping that will allow us to characterize the genetic diversity and population dynamics of sampled bee beds. Our preliminary analysis of the alkali bee genome identified >21,039 microsatellite loci ranging from 100-250 nucleotides (nts) in length. Once candidate microsatellite loci are identified, we aim to test consistent amplification with PCR and gel imaging with unlabeled microsatellite primers. Analysis. To achieve this goal, we will extract DNA from n = 96 specimens using the methods described in Koch et al. (2017) from either museum or contemporary specimens collected in Objective 2. The 50 markers will be tested in two replicates of n = 3 specimens across a random sample of the DNA extracts, culminating to a total of 300 PCR reactions. These reactions will be visualized on an agarose gel with a molecular ladder to confirm amplification. Once a microsatellite marker demonstrates consistent PCR amplification, we will proceed to purchase dye-labeled primers and PCR amplify the extracted alkali bee DNA in a four-marker multiplex using protocols described in Koch et al. (2017). Objective 2. In partnership with TGL area alfalfa seed growers, we will survey for alkali bees during the end of alfalfa seed pollination in 2022 across 15 bee beds. We aim to collect 30 female bees at each bee bed. Female alkali bees will be placed into 70% EtOH. Mid-leg tissue will be used for molecular work. Additionally, we aim to sample two unmanaged populations of alkali bees in Idaho (locations provided by J. Cane). Sampling will be done as above and these unmanaged populations will provide a comparison for measurements of genetic diversity, inbreeding, and relatedness estimates. Analysis. DNA extraction and 4-marker PCR multiplexes will be performed following the methods described in Obj 1. We will test for differences in genetic diversity across populations (across managed bee beds and compared to unmanaged populations) using an ANOVA. Next, we will do an analysis of molecular variance to test for differences in genetic structure across populations. Evidence for inbreeding and genetic bottlenecks will be examined with a sign test and M-ratio analysis in Bottleneck 1.2.02 (Cristescu et al., 2010). We will identify full-siblings across bee beds by assigning individuals to families with Colony v2.0 (Jones & Wang, 2010). Obj 3. Some bee beds are geographically isolated from other beds (>2 miles) in the TGL area. This may limit migration and genetic mixing among beds, though we do not have a good understanding of dispersal in this species. Analysis. Microsatellite genotypes of the female alkali bees generated in Obj. 2 will be used to estimate migration rates. We aim to estimate gene flow patterns across bee beds with both a coalescence maximum likelihood method with Migrate-n 3.2.6 (Beerli & Palczewski, 2010) as well as with relatedness measures following the methods described in Lopez-Uribe et al (2015).