Genome-wide association analysis of 101 accessions dissects the genetic basis of shell thickness for genetic improvement in Persian walnut (Juglans regia L.)

Abstract

Background: Understanding the underlying genetic mechanisms that drive phenotypic variations is essential for enhancing the efficacy of crop improvement. Persian walnut (Juglans regia L.), which is grown extensively worldwide, is an important economic tree fruit due to its horticultural, medicinal, and material value. The quality of the walnut fruit is related to the selection of traits such as thinner shells, larger filling rates, and better taste, which is very important for breeding in China. The complex quantitative fruit-related traits are influenced by a variety of physiological and environmental factors, which can vary widely between walnut genotypes.

Results: For this study, a set of 101 Persian walnut accessions were re-sequenced, which generated a total of 906.2 Gb of Illumina sequence data with an average read depth of 13.8× for each accession. We performed the genome-wide association study (GWAS) using 10.9 Mb of high-quality single-nucleotide polymorphisms (SNPs) and 10 agronomic traits to explore the underlying genetic basis of the walnut fruit. Several candidate genes are proposed to be involved in walnut characteristics, including JrPXC1, JrWAKL8, JrGAMYB, and JrFRK1. Specifically, the JrPXC1 gene was confirmed to participate in the regulation of secondary wall cellulose thickening in the walnut shell.

Conclusion: In addition to providing considerable available genetic resources for walnut trees, this study revealed the underlying genetic basis involved in important walnut agronomic traits, particularly shell thickness, as well as providing clues for the improvement of genetic breeding and domestication in other perennial economic crops.

Keywords: Fruit-related traits; GWAS; Juglans regia; Shell thickness.

Fig. 1

Principal component analysis (PCA) biplot of ten phenotypic traits within 101 walnut individuals

Fig. 2

Population structure and linkage disequilibrium (LD). A Neighbor-joining tree and population structure of 101 accessions; B Principal component analysis (PCA); C Linkage disequilibrium (LD) analysis

Fig. 3

GWAS for shell thickness and in-depth analysis of candidate genes. A Manhattan plot shows that SNPs were significantly correlated with walnut shell thickness characteristics. Grey dashed lines represent significance threshold of -log10 (p-value); B Haplotype map and LDBlock of the JrFKR1 gene, from 43.83 Mb to 43.90 Mb of chromosome 1; C Phylogenetic relationships and conserved domains of FRK1 genes in 11 species; D Phenotypic differences between the two JrFRK1 gene haplotypes

Fig. 4

GWAS for shell thickness and in-depth analysis of candidate genes. A Manhattan plot shows SNPs significantly associated with walnut shell thickness characteristics. Grey dashed lines represent significance threshold of -log10 (p-value); B Haplotype map and LDBlock of the JrGAMYB gene, from 23.62 Mb to 23.64 Mb of chromosome 13; C Phylogenetic relationships and conserved domains of GAMYB genes in 11 species; D Phenotypic differences between the three JrGAMYB gene haplotypes

Fig. 5

GWAS for shell thickness and candidate gene analysis. A Manhattan and B Q-Q plots show that SNPs were significantly correlated with walnut shell thickness characteristics. Grey dashed lines represent the significance threshold of -log10 (p-value) = 5, black arrows indicate JrPXC1 (C). Haplotype map and LDBlock of the JrPXC1 gene, from 334.89 kb to 354.60 kb of chromosome 10; D Phenotypic differences between the two JrPXC1 gene haplotypes

Fig. 6

Phylogeny and expression profiles of JrPXC1. A Phylogenetic relationships and conserved domains of 11 species of PXC1 genes. B Protein sequences of PXC1 genes. C Expression of JrPXC1 gene in 17 different tissues. X-axis represents the different tissues of the walnut: CE5 (callus exterior); CK3 (catkins); EM8 (embryo); FL3 (pistillate flower); FL6 (pistillate flower); HC2 (hull cortex); HL6 (hull immature); HP3 (hull peel); HU3 (hull immature); IF6 (fruit immature); LE5 (leaves); LY2 (leaf young); LY7 (leaf young); RT6 (root); SE7 (somatic embryo) and VB5 (vegetative bud). D Expression of JrPXC1 gene in the husk and leaves at four different stages of fruit development. GL indicates walnut green leaves and HU indicates walnut husks. Each of the three biological replicates represents a development period, for a total of four periods. E Results of QRT-PCR for JrPXC1 at three developmental stages of the shell and husk. Samples are from walnut fruit of different periods, * indicates p < 0.05, ** represents p < 0.01