BIGapp: A user-friendly genomic tool kit identified quantitative trait loci for creeping rootedness in alfalfa (Medicago sativa L.)

Abstract

Alfalfa (Medicago sativa L.) is a globally vital forage crop valued for its perennial growth and multiple annual harvests. A breeding effort is underway to improve the crop for productivity and persistence against biotic and abiotic stresses using "creeping rootedness," a trait where plants exhibit horizontal root growth, similar to rhizomes, with increased vegetative ground surface area. In this study, we genotyped a breeding population of 648 alfalfa lines segregating for creeping rootedness using the 3K DArTag marker panel to identify trait-associated genomic loci and evaluate the feasibility of genomic prediction to accelerate breeding cycles. Using genome-wide association studies (GWAS), we identified three quantitative trait loci (QTLs), with one major QTL located on chromosome 6.1 associated with this trait. Genomic prediction showed moderate predictive ability (r = 0.68) for creeping rootedness. A significant advancement in this study was the development and utilization of the Breeding Insight Genomics Application (BIGapp), an R Shiny application designed to streamline the processing of genomic data through an intuitive interface. This tool makes integrating genomics into existing breeding programs accessible, regardless of species ploidy or the researcher's coding proficiency. The identified QTL will be essential in future efforts to develop new alfalfa cultivars with the creeping rootedness trait and accelerate the breeding cycle, with BIGapp playing a pivotal role in these advancements.

FIGURE 1

The creeping rooted phenotype in alfalfa grown at the study site near Logan, UT. (A) Seeded rows of non‐spreading versus spreading alfalfa showing early spring growth. (B) Creeping root structure showing multiple shoots arising from the single creeping root. (C) A 9‐year‐old single alfalfa plant showing spread in comparison to a meter stick. (D) The descriptions for each of the two measured traits used to evaluate creeping rootedness.

FIGURE 2

Summary of two measurements for creeping rootedness across all phenotyped samples. (A) Distribution of the ratings for “Spread 0–10 (5/13/22).” (B) Distribution of the ratings for “Spread 0–10 (5/17/23).” (A–B) The mean value for the spreading parental lines (Entry 3 and Entry 8) and the non‐spreading parental line (CT3‐08) is labeled with vertical dashed lines. (C) Summary metrics for the two creeping root measurements.

FIGURE 3

Principal component analysis of the alfalfa creeping root breeding population (n = 648). (A) Genetic relations plotted for PC1 and PC2 dimensions. (B) Genetic relations plotted for PC1 and PC3 dimensions. Each point on the plot is an individual plant, and the colors/shapes are indicative of their parental cross. The labels denoted with “*” were the parental/check plants. The progeny derives from the following crosses: Orange, White, and Yellow (Entry 8 × CT3‐08); Blue, Pink, and Red (Entry 3 × CT3‐08); and Green (Entry 3 × Entry 8). (A) PC1 versus PC2 and (B) PC1 versus PC3.

FIGURE 4

Manhattan plot for the creeping root trait across all genome‐wide association study (GWAS) models. 2210 single‐nucleotide polymorphisms (SNPs) were evaluated with 417 progeny using GWASpoly. The M.eff significance threshold is the horizontal dashed line. SNPs above that line were significantly associated with the creeping root trait.