Genetic Diversity Analysis and GWAS of Alloocimene Based on Resequencing of Citron, Lemon Germplasm Resources

To explore the elite germplasm resources of citron and lemon, this study conducted genetic diversity analysis and comprehensive evaluation based on resequencing and phenotypic data. The research aims to elucidate the population genetic structure of citron and lemon germplasm resources, providing foundational materials for lemon germplasm innovation.

In November 2023, 13 fruit traits and 43 volatile functional components in the peel of 158 citron and lemon germplasm resources were measured. In June 2023, the contents of 4 flavonoids in immature fruits were analyzed. Genetic diversity was assessed using the Shannon-Wiener index and coefficient of variation. Combined with correlation analysis, cluster analysis, principal component analysis and other methods, the citron, lemon germplasm resources were comprehensively evaluated. SNP data were used to construct the phylogenetic tree and analyze population genetic structure. Specific accessions were screened based on phenotypic data. Genome-wide association analysis (GWAS) was performed to identify significant variation sites associated with alloocimene. Candidate genes within 10 kb of these sites were annotated and analyzed.

The fruit traits of 158 citron, lemon germplasm resources displayed rich genetic diversity and extensive variation, The coefficient of variation of the 13 phenotypic traits (single fruit weight FW, single fruit peel weight PW, fruit transverse diameter FD, fruit longitudinal diameter FL, fruit shape index FSI, peel thickness PT, number of segments SNPF, seed number per fruit SNF, single seed weight SW, soluble solids content TSS, ascorbic acid content AA, titratable acid content TA, solid acid ratio TSS /TA) ranged from 14.81%-143.47%, and the genetic diversity index ranged from 1.21 to 2.01.The genetic diversity index of 4 flavonoids (rutin, naringin, hesperidin and neohesperidin) in fruits ranged from 0.75 to 1.89, and the genetic diversity index of 43 volatile compounds in peel ranged from 0.25 to 1.93. The principal component analysis on 60 quantitative traits of citron, lemon germplasm resources was carried out, the top 5 principal components explained 66.55% of cumulative variance. A comprehensive evaluation was conducted based on the results of principal component analysis, and comprehensive evaluation score ranged from -0.09 to 2.14, specific germplasms such as Volkamer (Citrus volkameriana Pasquale), Fantastico Bergamot (Citrus bergamia Risso), Castagnaro Bergamot (Citrus bergamia Risso), Amber Lemon (Citrus limon (L.) Burm.f.) and Verna Lemon (Citrus limon (L.) Burm.f.) were identified. Germplasm resources were divided into 7 sub-groups, with high consistency between phenotypic clustering and phylogenetic analysis, except Lime (Citrus aurantifolia Swingle) and Citrus limonia sub-groups showed slight divergence. The elite germplasm clustered predominantly in sub-group 3, indicating shared genetic background. The population genetic structure analysis was consistent with the phylogeny analysis results except that the hybrid varieties (e.g., sweet lemon) formed a distinct sub-group. Significant SNP loci associated with alloocimene content were identified. Two cytochrome P450 family genes (C71D7_SOLCH, C71DB_LOTJA) were proposed as potential regulators of alloocimene metabolism.

Citron and lemon germplasm exhibit extensive genetic diversity in fruit quality traits. Phenotypic clustering analysis was similar to phylogenetic tree analysis and population genetic structure analysis, it showed that gene introgression from mandarin and pomelo likely drove sub-group differentiation. Citron and lemon germplasm resources with higher scores in the comprehensive evaluation were more concentrated in the same sub-group in phylogenetic tree, indicated similar genetic background were presented in these elite and specific germplasms. The candidate genes of C71D7_SOLCH and C71DB_LOTJA may be involved in the metabolism of alloocimene, a volatile substance in the peel.