Genome-wide association study of the loci and candidate genes associated with agronomic traits in Amomum villosum Lour

Abstract

Amomum villosum Lour. (A. villosum) is a valuable herbaceous plant that produces the famous traditional Chinese medicine Amori Fructus. Identifying molecular markers associated with the growth of A. villosum can facilitate molecular marker-assisted breeding of the plant. This study employed 75 A. villosum accessions as the test material and utilized 71 pairs of polymorphic simple sequence repeat (SSR) molecular markers to genotype the population. The study analyzed the association between SSR markers and phenotypic traits through the linkage imbalance and population structure analysis. Candidate genes associated with the molecular markers were also identified. The results showed that the phenotypic diversity index range of the 12 agronomic traits was 4.081-4.312 and conformed to a normal distribution. Moreover, 293 allelic variations were detected in the 75 accessions, with an average of 5.32 amplified alleles per loci, ranging from 3 to 8. The maximum number of amplified alleles for AVL12 was 8. The population structure and cluster analysis indicated that the accessions could be divided into two subgroups. Using the mixed linear model (MLM) model of population structure (Q)+kinship matrix (K) for association analysis, three SSR molecular markers significantly associated with the agronomic traits were detected. Fluorescence quantification was used to analyze the expression levels of six candidate genes, and it was found that three of the genes were differentially expressed in phenotypically different accessions. This study is the first to use SSR markers for genome-wide association study (GWAS) mapping and identification of the associated agronomic traits in A. villosum. The results of this study provide a basis for identifying genetic markers for growth traits for marker-assisted breeding in A. villosum.

Fig 1. Frequency distribution of the 12 agronomic traits of Amomum villosum Lour.

PH = height; SD = stem diameter; NB = number of blades; NR = number of ramets; LL = leaf Length; LW = leaf width; LEL = ligule length; TLS = total length of stolons; ST = stolon thickness; SIL = stolon internode length; NNS = number of new shoots; TNR = total number of ramets.

Fig 2. Correlation heat map of the 12 agronomic traits of Amomum villosum Lour.

‘*’ indicates correlation significance at the 0.05 level, while ‘**’ represents significance at the 0.01 level and ‘***’ shows significance at the 0.001 level. PH = height; SD = stem diameter; NB = number of blades; NR = number of ramets; LL = leaf Length; LW = leaf width; LEL = ligule length; TLS = total length of stolons; ST = stolon thickness; SIL = stolon internode length; NNS = number of new shoots; TNR = total number of ramets.

Fig 3

Population structure (A) and cluster analysis (B) of the 75 Amomum villosum accessions.

Fig 4. The linkage disequilibrium (LD) distribution among 55 simple sequence repeat (SSR) loci in 75 Amomum villosum accessions.

Fig 5. Genome-wide association analysis of the growth traits of Amomum villosum Lour. using a mixed linear model.

Manhattan plot of (A) ligule length, (B) total number of ramets and (C) number of new shoots. (D) Quantile-quantile (QQ) plot of all the 12 traits.

Fig 6. The expression levels of three candidate genes showing significant differences in different tissues of the phenotypically different accessions (GX16 with the most NNS, YN9 with the least NNS, YN17 with the longest LEL, and YN9 with the shortest LEL).

Data represent the mean of three replicates. Significant differences are indicated as * P < 0.05 and ** P < 0.01. NNS = number of new shoots; LEL = ligule length.