Advanced High-Throughput Root Phenotyping and GWAS Identifies Key Genomic Regions in Cowpea During Vegetative Growth Stage

Abstract

Improving crop production in changing environments can be achieved through selective breeding; however, limited advanced root phenotyping and genotyping in early growth stages hinder assessing root architecture variation and diversity, despite its importance. Therefore, this study utilized advanced image phenotyping on a diverse set of 222 cowpea accessions, revealing significant variations in key phenotypic traits and the genomic regions influencing them. Our study revealed a total of 55 genes linked to major root traits. Among eight root traits-total root length (TRL), surface area (SA), average diameter (AD), root volume (RV), tip number (TN), fork number (FN), primary root length (PRL), and lateral root length (LRL), analyzed, seven significant single nucleotide polymorphisms (SNPs) demonstrated particularly strong associations with three key traits, including surface area (SA), tip number (TN), and fork number (FN). SA emerged as a significant trait, exhibiting considerable variation across the studied accessions. The mean SA was 59.59 cm², with some genotypes surpassing 140.72 cm². Further analysis identified two SNPs that showed significant association with SA, located on two distinct chromosomes: 3 and 11. Similarly, two significant SNPs associated with TN were found on chromosome 3, while three SNPs associated with FN were identified on chromosomes 2, 3, and 8. These findings significantly advance our understanding of the genetic foundations underlying important phenotypic traits in cowpeas, offering a robust framework for future genetic improvement initiatives. The results strongly suggest that implementing breeding programs focused on selecting root phenotypes could significantly enhance cowpea productivity across various environments.

Keywords: Vigna unguiculata; advance phenotyping; genotyping; root architecture; single nucleotide polymorphisms.

FIGURE 1

Workflow for root phenotype collection: (a) Seedlings at the V2 growth stage grown in PVC pipes within the greenhouse. (b) Cowpea plants with roots cleaned to remove soil particles. (c) Roots, after cleaned with tap water, were placed in a container and scanned using an EPSON scanner. (d) 2‐D images of the scanned roots were analysed using WinRHIZO Pro software.

FIGURE 2

Variation among cowpea root genotypes analyzed during the present study. The scale bar represents 10 cm and applies uniformly to all root images, which were consistently resized to ensure a standard size.

FIGURE 3

Correlation of root traits among different cowpea genotypes, ***p < 0.001 shows significance.

FIGURE 4

PCA Biplot of Root Traits in Diverse Cowpea Accessions. The biplot shows the variation in root traits among the cowpea accessions, with PC1 and PC2 accounting for 58.87% and 14.07% of the total variance, respectively.

FIGURE 5

Manhattan plots of (a) Surface area, (b) Tip number, (c) Fork number. Each Manhattan plot is highlighted with red dots pointed out by blue arrows and labelled with marker names representing the significant SNPs identified by GWAS.

FIGURE 6

Quantile‐quantile (QQ) plots of (a) Surface Area, (b) Tip number, and (c) Fork number, traits falling above the threshold linear line showing significant marker‐trait associations.

FIGURE 7

The results of the GWAS for three cowpea root traits: (a) SA‐VU03, (b) SA‐VU11, (c) TN (position 2_11370), (d) TN (position 2_51446), (e) FN‐VU02, and (f) FN‐VU08. Each trait is depicted with a Manhattan plot, where significant loci are highlighted as red dots above the green threshold lines. The genes that directly influence the root traits are indicated in bold. The box plots illustrate that different alleles at the significant SNP loci have distinct effects on these traits. The LD blocks demonstrate that the significant SNPs are within haplotype blocks. Moreover, the LD blocks show coefficients ranging from 0 to 1, with a color gradient from blue to orange representing the R² values.

FIGURE 8

This figure illustrates the diversity in root system architecture among some selected cowpea genotypes, highlighting differences in SA, TN, and root depth. (A) IT343993 and IT195385 are examples of genotypes with low SA, low TN, and shallow root systems. (B) IT162768 and IT180838 are genotypes exhibiting high SA and high TN but with shallow root systems. (C) IT104192 and IT236285 are genotypes demonstrating high surface area, high density, high tip number, and deep root systems.