DOI: 10.5061/dryad.s4mw6m9d6
title: SNP alleles for pooled DNA samples of Vicia villosa targeting QTL for pod dehiscence and seed dormancy

description: Hairy vetch (Vicia villosa Roth) is a promising legume cover crop, but its
 use is limited by high rates of pod dehiscence and seed
 dormancy.  We used phenotypically contrasting pooled DNA samples
 (n=24 with 29-74 individuals per sample) from an ongoing cover crop
 breeding program across four environments (site-year combinations:
 Maryland 2020, Maryland 2022, Wisconsin 2021, Wisconsin 2022) to find
 genetic associations and genomic prediction accuracies for pod dehiscence
 and seed dormancy. We also combined pooled DNA sample genetic association
 results with the results of a prior genome-wide association study. Genomic
 prediction resulted in positive predictive abilities for both traits
 between environments and with an independent dataset (0.34-0.50), but
 reduced predictive ability for DNA pools with divergent seed dormancy in
 the Maryland environments (0.07-0.15). The pooled DNA samples found six
 significant (false discovery rate q-value&lt;0.01) quantitative trait
 loci (QTL) for seed dormancy and four significant QTL for pod
 dehiscence.  Unfortunately, the minor alleles of the pod
 dehiscence QTL increased the rate of pod dehiscence and are not useful for
 marker-assisted selection. When combined with a prior association study,
 sixteen seed dormancy QTL and zero pod dehiscence QTL were significant.
 Combining the association studies did not increase the detection of useful
 QTL.
description: This study sampled individuals evaluated during ongoing breeding
 efforts of the Cover Crop Breeding Network.  Four environments were
 selected which had sufficient population size and contained meaningful
 variability in pod dehiscence and seed dormancy.  For the remainder of
 this study, I will refer to each environment using an abbreviation and
 final two digits of the harvest year (i.e. Beltsville, MD 2020 and 2022:
 20MD, 22MD, Prairie du Sac, WI 2021 and 2022: 21WI and 22WI). The 20MD
 breeding nursery was planted on Sept 27<sup>th</sup> 2019
 (39°01′50″ N, 76°55′59″ W, Russett–Christiana complex soil) into a tilled
 field which was broadcast seeded with turf red fescue (<em>Festuca
 rubra </em>L.; 340 kg ha<sup>-1</sup>).  On November
 5<sup>th</sup> 2019, 20MD was sprayed with Raptor (ammonium
 salt of imazamox; 0.37 L ha<sup>−1</sup>) to control winter
 weeds. For 20MD, pods were collected from June 23<sup>rd</sup>
 2020 to June 30<sup>th</sup> 2020 (harvest varies with pod
 maturity of individual genotypes, see below).  The 22MD nursery was
 planted at the same location as 20MD on October
 7<sup>th</sup>, 2021 and pods were collected from June
 29<sup>th</sup> to July 5<sup>th </sup>2022.  The
 22MD nursery used black plastic covered raised beds to control weed
 pressure. The 21WI nursery was planted on September
 23<sup>rd</sup> 2020 (43°20′55″ N, 89°45′18″ W, Richwood silt
 loam soil) into landscape fabric.  The 22WI nursery was planted in early
 October on the same location as 21WI into landscape fabric. For 22WI, pods
 were collected between July 20<sup>th</sup> and August
 5<sup>th </sup>2022.  Prior to establishment, all environments
 were supplemented with lime, K and P based on soil test results. 
 Additional details on the breeding program goals and methods can be found
 in prior publications (Kucek et al., 2019; Kissing Kucek et al., 2020b;
 Tilhou et al., 2023).  Breeding site-years consisted of
 direct-seeded spaced individual hairy vetch plants (20MD: n=3696; 21W1:
 n=1200; 22MD: n=3648; 22WI: n=1200) which were visually evaluated for fall
 vigor, spring vigor, and plant maturity (Kalu and Fick, 1981).  Since
 hairy vetch is out-crossing, one round of selection based on vigor occurs
 in late spring prior to cross pollination. Only selected individuals are
 allowed to cross pollinate and are then evaluated for seed production
 characteristics, which includes pod dehiscence and seed dormancy. 
 Selection intensity prior to flowering varied from 3.3 to 47% in each
 environment, allowing the following number of individuals to cross
 pollinate: 20MD: n=124, 21WI: n=560, 22MD: n=155, and 22WI:
 n=363.  Pods were collected for dormancy and dehiscence
 evaluations at ripe seed pod stage according to Kalu and Fick (1981).  Pod
 dehiscence was measured using a visual score with one measurement assigned
 to each pod (targeting a minimum of 50 pods per individual; Kissing Kucek
 et al., 2020b).  During 2020, visual scores were on a 0-3 scale (described
 in Kissing Kucek et al., 2020b) while 2021 and 2022 used a 0 or 1 scale
 (0: pod is closed enough that a seed could not fall out, 1: pod is open
 enough that a seed could fall out).  Green, flat, or immature pods were
 discarded prior to scoring.  Seed dormancy was measured as a proportion of
 25 seeds which imbibed water after 7 d per individual plant, with three
 replicates per individual (detailed methods in Kissing Kucek et al.,
 2020a).  Seeds which did not imbibe water after 7 d were scarified and
 observed after an additional 7 d to determine seed viability for
 environments collected in 2020. For 2021 and 2022 environments, hard seeds
 were determined to be viable and not scarified.  Dead seeds were not
 included in the dormant seed proportion. Subsequent
 field selection for disease resistance and seed production resulted in
 smaller population sizes available for pod dehiscence ratings, seed
 dormancy ratings, and DNA pool construction (20MD: n=115; 20W1: n=206;
 22MD: n=109; 22WI: n=287).  Hairy vetch tissue from these individuals were
 collected for sequencing during active vegetative growth in mid-summer.
 Each leaf sample was placed into a labeled coin envelope and immediately
 placed on ice before transport to a laboratory freezer (-20°C) and stored
 until freeze drying. For each location, trait-based
 pooled DNA samples were constructed from stored leaf samples using the
 best 25% and worst 25% performance for pod dehiscence and seed dormancy. 
 In addition, one random pooled DNA sample was constructed for each
 environment from a random sample of 25% of the population size. 
 Individuals included in a trait-based pooled DNA sample were not included
 in the random sample.  Pool construction was achieved  by combining equal
 sized leaf tissue from each individual prior to pulverizing (Craig et al.,
 2009).  A subsample of homogenized samples was then used to extract DNA. 
 To help validate this method, four technical replicates were created from
 four randomly selected tissue samples. 
 <em>Sequencing and SNP filtering</em>
 In total, 24 DNA pools were submitted for sequencing (five pools
 [high pod dehiscence, low pod dehiscence, random, high seed dormancy, and
 low seed dormancy] by four environments [21MD, 22MD, 21WI, and 22 WI] and
 four technical replicates.  The University of Wisconsin Biotechnology
 Center prepared libraries for genotype-by-sequencing using
 an <em>NsiI-BfaI </em>double digestion restriction enzyme
 digestion. Fragments were then ligated to barcoded adaptors prior to
 polymerase chain reaction amplification and sequencing on an Illumina
 sequencer (NovaSeq 6000) targeting a 20 million reads per sample (mean of
 approximately 15x coverage). Bioinformatics processing
 was completed at the University of Wisconsin Biotechnology Center using
 the TASSEL analysis platform (Glaubitz et al., 2014) in parallel with
 re-calling single nucleotide polymorphisms (SNPs) from the Tilhou et al.
 2023 GWAS panel which are hairy vetch individuals evaluated in Oregon and
 Texas in 2019 (n=869). Barcoded sequence read outputs were collapsed into
 a set of unique sequence tags with counts. These tags were aligned to the
 reference genome (<em>V. villosa</em> v1.1; Fuller et al.,
 2023). Each tag was assigned to a position with the best unique alignment,
 and the occupancies of tags for each sample were observed from barcode
 data. For pooled DNA samples, allele states were analyzed as two-times
 alternate allele frequencies (continuous 0-2) and individual DNA samples
 were analyzed as alternate allele dosages (0, 1, or 2).  Overall,
 2,877,384 SNPs were present prior to filtering.  Of these, only 2,105,338
 SNPs were mapped to the seven main chromosome fragments.  Within these,
 165,057 of the remainder had &gt;30 read depths within the pooled DNA
 samples.  This cut-off was based on the accuracy among technical
 replicates at varying read depths.  At this point, the SNPs had 14.8% of
 missing values which were imputed to the mean values for the marker. Last,
 SNPs were removed with &lt;0.025 minor allele frequency, resulting in
 122,801 SNPs.
description: # SNP alleles for pooled DNA samples of Vicia villosa targeting QTL for
 pod dehiscence and seed dormancy
 ## Description of the data
 and file structure VetchPoolDNA_MarkerInformation.csv contains columns
 indicating the reference and alternate alleles for each SNP. It also
 indicates the percent individuals with missing date in the individual
 sequencing dataset for that SNP. Similarly, there is a column with the
 mean read depth within the sequencing of pooled DNA.
 VetchPoolDNA_Dosage.csv contains allele dosages with columns representing
 sites and rows representing individuals or DNA pools (ID column). 99
 indicates missing data. Pool allele frequencies are the mean allele read
 frequency! Therefore, these are on different scales (individuals 0-2 and
 DNA pools 0-1). VetchPoolDNA_Performance.csv contains the pod dehiscence
 rates, seed dormancy rates, and number of individuals with phenotypic
 information included in each pool. ## Sharing/Access information
 Individuals in this file are identical to those measured in the prior
 published GWAS. DOI: 10.5061/dryad.z34tmpgm1 ##