To reveal the phylogeny, population genetic structure and diversity level of pomelo (Citrus grandis (L.) Osbeck) germplasm, and to efficiently utilize them to explore genes related to important fruit quality traits, this research provided an insight into the population genetic structure and phylogeny of pomelo germplasm and facilitated the pomelo varieties innovation.

A total of 282 pomelo accessions including landraces from different geographical regions and hybrid offspring of kiyomi tangor and pomelo were contained in this study. GBS library was constructed with genomic DNAs digested by EcoR I restriction endonuclease and sequenced on Illumina HiSeq PE150 platform, the clean short reads were then mapped to pomelo reference genome by BWA, and SNPs were called out with SAMTOOLS pipeline. Based on 121 726 SNPs genotyping data, principal component analysis (PCA) and population genetic structure analysis were carried out and phylogenetic trees were constructed with Neighbor-joining method. Furthermore, two sub-populations containing high-acid accessions (32) and low-acid accessions (23) were used to identify candidate genes related to fruit acidity by Fst and XP-CLR selective sweeping analysis. Meanwhile, the genotype data of 282 pomelo accessions and the phenotypic data of titratable acid content in fruit were used for GWAS.

A total of 201.66 Gb original reads were generated from 282 pomelo germplasm by GBS approach, in average each sample produced 0.72 Gb reads. After the screening conditions of sequencing depth of dp5, the miss less than 0.2 and minor alleles frequency (MAF)>0.01, and a total of 121 726 SNPs were selected out for subsequent analysis. The PCA, structure and phylogenetic analysis all supported that the 282 pomelo germplasm could be divided into 6 subgroups, among which pomelo and kiyomi hybrid population, grapefruits and other pomelo hybrid populations could be obviously different from true-to-type pomelo populations, pomelos originated from different geographical region displayed unique genetic feature. The pomelo germplasm from Thailand and Vietnam formed a relatively unique group different from other domestic groups, such as ShaTian pomelo, Wen Dan pomelo, and Dian Jiang pomelo in China. The genetic introgression from Vietnam pomelos were exhibited in most pomelo germplasm in southern China, suggested that Vietnam was the origin center for pomelo. In addition, some pomelo germplasm with unknown origin have been identified accurately by GBS technology. This study showed that different geographical distribution and artificial selection pressure had great effect on the genomic composition of pomelo. Besides, Fst, XP-CLR selective sweeping analysis revealed a strong selection signal region on chromosome 7 contained genes annotated as dihydrolipoyl transacetylase (DLT-E2) of pyruvate dehydrogenase complex (PDC) and aluminum-activated malate transporter (ALMT9), which involved in the synthesis and transportation of citric acid. In additional GWAS genome-wide association analysis identified another region on chromosome 2, which was also highly associated with fruit acidity.

GBS technology provided reliable and efficient method for studying the phylogeny and evolution of pomelo. The study showed that artificial cross breeding, long-term artificial selection, geography isolation and domestication were the major driving forces for the formation of different types of pomelo germplasm. In addition, it clearly showed that Southeast Asian was primary center for pomelo origin and China mainland was secondary evolutionary center. Several candidate genes related to citric acid content in pomelo fruits were identified by Fst, XP-CLR selective sweeping and GWAS. This study provided important gene resources for the further genetic improvement and breeding of pomelo fruits.

Bud sports mutation is a DNA mutation occurred in somatic meristem, and it often displays visible morphological and other characteristic changes different from its mother plants in branches, leaves, flowers and fruits. However, the discriminating bud sports mutation from the epigenetic variation caused by environmental conditions and cultivation measures etc. external factors was still mostly depended on the molecular fingerprint detection.

This study objective was to identify citrus bud sports mutant through Target-SSR sequencing technology.

Firstly, the genome of clementine mandarin (Citrus reticulata Blanco) and satsuma (Citrus unshiu Macf.), as well as GSS and EST sequences of satsuma were used to scan SSRs loci with GMATA, and the highly polymorphic SSRs loci were screened out to design primers. The multiplex PCR with optimized primers were ampllified on 60 citrus bud sports mutants to construct high-thoughout sequencing library, and the amplification products were then sequenced on illumina Minseq platform. The clean sequencing short reads were mapped to reference target sequences to find differentiated SSRs loci presented in citrus bud sports mutants.

A total of 77 pairs of SSR primers were designed from highly polymorphic SSRs loci. The primer pair combinations were optimized and 18 multiplex PCR amplification products were sequenced. The target SSR-seq analysis showed that the genotyping data of SSRs could divided 60 citrus accessions into two groups corresponding to sweet orange and mandarin, and the mandarin group could be further subdivided into different citrus cultivars, such as Orah, ponkan etc. 11 SSR loci containing mostly ATT motif were found in 7 Tarroco blood orange mutants, 8 SSR loci containing mostly TAA motif were found in 2 ‘Wu Yue Hong’ mutants and 5 navel orange, 16 SSR loci containing mostly GA motif were found in 9 Bing Tang Cheng mutants, 9 SSR loci containing mostly AAT motif were found in 2 Sha Tang Ju mutants, and 15 SSR loci with mostly AAT motif were found in 4 satsuma mutants. This study showed that Target-SSR sequencing technology provided an excellent resolving approach to discriminate citrus bud mutations.

In this study, an effective method for citrus bud mutant identification by using Target SSR-seq technology were established, and 60 citrus germplasm accessions could be discriminated. The precision and reliability of SSR genotyping information could be utilized in citrus germplasm resources management and variety intellectual property protection.

By using specific locus amplified fragment sequencing (SLAF-seq) sequencing method and combined with fixation indices analysis on different sweet orange populations, the candidate genes related with some excellent traits in sweet orange were mined, so as to provide some candidate genes for the further molecular breeding in sweet orange.

SLAF-seq technology was used to genotype 240 sweet orange germplasm resources, which represented extensive genetic diversity and from different geographical origins accessions preserved in the National Citrus Germplasm Repository in Beibei of Chongqing. The genomic SNP genotype data were obtained using the sweet orange genome as the reference, and the Fst and XP-CLR values between different subpopulations were calculated.

A total of 497.82 Mb_- reads were obtained by sequencing 240 sweet oranges accessions with SLAF-seq technology, after the sequencing reads were mapped on to the reference genome by BWA software, and a total of 1 467 968 SNPs were identified by GATK and samtools. The Fst and XP-CLR indices were used to screen out candidate genes related to three traits, including fruit navel occurrence, fruit weight, and titratable acid content. Those genes adjacent to the selected SNPs were called. The result showed that orange1.1G044639m and orange1.1g023641m which annotated auxin efflux vector were related to the formation of navel in sweet orange. SNP in orange1.1G023641m which encoded E3 ubiquitin ligase had the highest Fst score between two different single-fruit weight population, and orange1.1G046891m might affect fruit weight by regulating carbohydrate formation; orange1.1G011684m encoding dihydrolipoamide transacetylase of PDH, and the SNP caused a nonsynonymous mutation in its CDS region, which might determine the citric acid content in fruit; orange1.1G034502m encodes Serine/threonine protein kinase, annotated as phosphorylation dephosphorylation function also related with the citric acid content in fruit.

In this study, the Fst indices were used to screen six candidate genes associated with three fruit traits in sweet orange by using SNPs from SLAF-seq data. Finally, the relevant genes were screened out for three horticultural traits, including navel occurrence, fruit weight and titratable acid content, and the identified candidate genes should be giving further investigation to explore their regulation mechanism in these traits.

The flesh color is an important appearance and quality trait of pomelo varieties. The aim of this study was to excavate variation loci and genes significantly related to the flesh color of pomelo varieties, so as to lay a foundation for further understanding the flesh color development mechanism and marker-assisted breeding of pomelo varieties.

100 pomelo accessions were used to identify their flesh color with a color difference meter, and classified based on their phenotype results. The simplified genome sequencing of 100 pomelo accessions was carried out using GBS (genotyping-by-sequencing) technology. The eigenvalues and eigenvectors of the genotype data obtained from sequencing were calculated by GCTA software to analyze the population structure, and the genetic differentiation index (Fst) of two different flesh color populations were calculated using Plink 2.0 software. The GLM model in GEMMA software was adopted to perform genome-wide association analysis, and the variation loci significantly associated with color phenotype were identified for further allelic variation analysis. According to the citrus linkage disequilibrium (LD) distance, the candidate genes located in the 25 kb adjacent region around the variation loci that might be related to the forming of flesh color were screened out, four white and four red pomelo accessions were randomly selected to perform qPCR to obtain the candidate genes expression at different fruit development stages.

According to the phenotype of flesh color, 100 accessions were divided into two categories corresponding to white and red pomelo accessions, including 58 white flesh pomelos and 42 red flesh pomelos. Six SNP sites with Fst index greater than 0.4 and -log₁₀ (P)>9 were screened out after Fst genetic differentiation analysis and GWAS genome-wide association analysis. The genotypes of six variation SNPs sites were extracted from 100 pomelos accessions and the genotypes of these SNPs could be used to predict the flesh color of pomelos. A total of 14 genes that function annotated as isoprene synthesis, plastid development, abscisic acid signal regulation, ethylene response related genes and transcription factors were screened and selected. Genes expression analysis showed that Cg7g013760 (mitogen-activated protein kinase kinase MAPKKK17), Cg7g013840 (chloroplast porphyrinogen deaminase), Cg7g014020 (transcription factor TCP7), Cg7g014120 (chloroplast superoxide dismutase SOD3), Cg7g014190 (FAD synthetase 2) were related to the forming of flesh color in pomelos.

A total of 6 SNP sites significantly associated with flesh color in pomelos accessions were identified by GWAS and Fst analysis. A total of 14 genes within the 25 kb regions around the mutation SNPs site were screened and annotated. The gene expression analysis showed that five genes might be involved in the forming of flesh color in pomelo accessions.

The study objective was to explore candidate genes associated with resistance to Alternaria brown spot disease in citrus mandarin, so as to provide the basis for developing suitable molecular markers for further citrus resistance breeding work.

In summer and autumn of 2022, the young leaves of 136 citrus mandarin accessions were picked and inoculated with the fungus mycelium of Alternaria alternata in the laboratory. The combined results of two experiments were used to obtain the reliable results of 121 citrus mandarins responding to Alternaria brown spot. The phenotypes of 121 citrus mandarins were selected to verify the effectiveness of the CAPS marker developed in a previous study. Then, in order to obtain the candidate SNPs related to Alternaria brown spot resistance, the phenotypes of 121 citrus mandarins and corresponding SNP genotype data from GBS sequencing were analyzed with PCA, GWAS, and F_st methods, respectively. Candidate genes had been selected from the flanking region of 25 kb sequences surrounding the candidate SNPs site, and then they were screened out according to phytozome annotations. The expressions of candidate genes were analyzed after inoculating with the pathogen fungus on leave of Chengtuo hongju and Clementina (Algeria) for 24, 48, and 72 h.

Among 121 citrus mandarins, 67 varieties such as Clementine and Satsuma were resistant to Alternaria brown spot disease, whereas 54 varieties such as Hong Ju and Ponkan were susceptible. Some varieties resistance to Alternaria brown spot disease could discriminated by CAPS, but its accuracy only accounted for 76.86% in the study. GWAS analysis identified six significant SNPs highly associated with disease resistance, among which SNP1 located in Ciclev10021676 at 24 838 146 bp of chromosome 3 could be used to predicate the resistance of varieties, and their genotype showed a strong correlation with phenotype. Eight significant SNPs highly associated with disease resistance selected by F_st analysis. Finally, five genes of Ciclev10018604, Ciclev10023485, Ciclev10023486, Ciclev10024586 and Ciclev10019874 were screened out. The expression levels of these five genes in Chengtuo Hongju were up-regulated extensively after inoculating leaves with the pathogen fungus, and their expression levels reached the highest 48 hours later.

Through GWAS, the SNP at 24 838 146 bp on chromosome 3 was found to be the most significant one with high resistance correlation to Alternaria brown spot disease, and the genotype at this location could be effectively used to distinguish the resistant varieties. The candidate genes responding to Alternaria brown spot disease in mandarin were discussed, i.e., Ciclev10019874, Ciclev10018604, Ciclev10023485, Ciclev10024586, and Ciclev10023486.

In this study, the whole-genome resequencing data and phenotypic identification information were used to reveal the genetic variation and population structure of mandarin germplasm resources and to explore elite citrus germplasms with these information, the aim was to promote citrus variety improvement in the direction of high fruit quality and diversification.

In 2021 and 2022, 25 phenotypic traits of fruits and flowers of 473 mandarin accessions were identified in two consecutive years, and the young leaves were collected for DNA extraction, library construction and sequencing. Diversity analysis of phenotypic information was carried out using the variation coefficient and Shannon-Wiener index. To screen out excellent mandarin germplasm, a comprehensive evaluation on mandarin germplasm resources was conducted using correlation analysis, cluster analysis, and principal component analysis. Phylogenetic tree analysis was performed based on resequencing data to analyze the characteristics of the group to which the excellent germplasm belongs.

There are extensive variations and rich genetic diversity in 473 mandarin accessions. The variation coefficient of 21 quantitative traits ranges from 9.36% to 76.98%, with an average of 34.95%. and the genetic diversity index from 1.71 to 2.08, with an average of 1.93. The diversity index of the four qualitative traits ranged from 0.35 to 0.98, which was significantly lower than the quantitative traits. Among them, the variation coefficient of segment membrane and juice sac wall weight is the largest, and peel color have the largest genetic diversity index. Principal component analysis was performed on 17 fruit phenotypes, and the top five principal components have the cumulative contribution rate more than 70%. Factor analysis was used to conduct a comprehensive evaluation, it was used to select elite mandarin accessions with the comprehensive evaluation score which ranged from -1.23 to 1.59, the cultivar with the highest score was LP31 (Nishinokaori×Jin Kui), followed by LP45 (Nishinokaori×Jin Kui), LP18 (Benimadona×American Tang Ju), LP3 (Tarocco Blood Orange×Jin Kui). Both the phenotypic cluster analysis results and the phylogenetic tree analysis divided the tested mandarin germplasm resources into 7 groups, different groups can reflect the differences in the origin, variety type and hybrid combination derived from different genetic backgrounds. The germplasms with high comprehensive evaluation scores mostly are concentrated in Group Ⅳ, which showed that these excellent germplasms have similar genetic component.

The 473 mandarin accessions used in this study has extensive phenotypic variation and rich genetic diversity. The phenotypic cluster analysis results and phylogenetic tree gave similar classification results, and the excellent germplasm selected based on comprehensive evaluation score have high genetic similarity.