DOI: 10.5061/dryad.k98sf7m72
title: Genetic and fitness measurements of Brighamia accessions for APPS

description: Premise of the study: Living collections maintained for generations are at
 risk of diversity loss, inbreeding, and adaptation to cultivation. To
 address these concerns the zoo community uses pedigrees to track
 individuals and implement crosses that maximize founder contributions and
 minimize inbreeding. Using a pedigree management approach in an
 exceptional plant, we demonstrate how conducting such strategic crosses
 can minimize genetic issues that have arisen under current practices.
 Methods: We performed crosses between these collections and compared the
 fitness of progeny, including plant performance and reproductive health.
 We genotyped the progeny and paternal accessions to measure changes in
 diversity and relatedness within and between accessions. Results: The mean
 relatedness among individuals of an accession, suggests they are full
 siblings. As a result there was high inbreeding and low diversity within
 an accession, although less so among accessions. Progeny from the wider
 crosses had increased genetic diversity, while selfed accessions were
 smaller and less fertile. Discussion: Institutions which hold exceptional
 species should consider how diversity is maintained within their
 collections. Implementing a pedigree-based approach to managing
 reproduction of ex situ plants will slow the inevitable loss of genetic
 diversity and in turn, result in healthier collections.
description: Genomic DNA was extracted from
 parental accessions using the 2× CTAB method (Doyle and Doyle, 1987) and
 the progeny of the aforementioned crosses using DNeasy Plant Mini Kit (Cat
 69104, Qiagen, Germantown, MD, USA). For the parental accession plants, we
 extracted DNA from the maternal accession (CBG), and for
 three of the four paternal accessions (SDZ (Accession 2001-0273-022), NTBG
 (Accession 990833.3) and USBG1 (Accession 2014-0070)), however as the
 fourth accession (USBG2) died before we could extract DNA, we used progeny
 derived (Accession 2019-000) from a self of that plant to estimate
 paternal contribution. All samples were genotyped using markers and
 protocols described in Fant et al. (2019) which had already been tested
 for Hardy-weinberg and presence of null alleles. For this study we used a
 total of 11 primers; eight primers (B05, B08, B43, B44, B46, B47, B51,
 B57) were designed for <i>B. insignis</i> (Fant et al., 2019)
 and three primers (L23, L33, L34) were designed for <i>Lobelia
 villosa</i> (Rock) H. St. John & Hosaka. The eight
 <i>B. insignis</i> primers were visualized using pre-labeled
 forward primers with either WellRed Black (D2), Green (D3) or Blue (D4)
 fluorescent dye (Sigma-Proligo, St. Louis, Missouri, USA), while for
 <i>Lobelia villosa</i> markers, the forward primer was
 modified at the 5’ end (5’-CACGACGTTGTAAAACGAC-3’) so they could be
 labelled separately (Schuelke, 2000). All products were analyzed and
 scored using a CEQ 8000 Genetic Analysis System V9.0 (Beckman Coulter,
 Brea, California, USA). Given that <i>B. insignis</i> is a
 paleotetraploid (Lammers, 1988), four of the primer pairs (B44, B47, B51
 and L23) produced more than two bands. As the peaks were separated from
 other alleles by large range (20-30bp), and segregated independently (Fant
 et al., 2019), they were scored as independent loci.
description: Ready to be used in genalex.