Abstract: 【Objective】Tangelo is a type of citrus hybrid that has mandarin (Citrus reticulata Blanco) and pummelo (C. maxima (Burm.) Merr.) pedigrees. Owing to the characteristic blended flavors of mandarin and pummelo, tangelo is increasingly favoured by consumers, and receiving attentions from citrus breeders. Besides the conventional tangelos such as Sweet Spring and Haruka, previously we have developed two elite tangelo varieties called Yanggaung No. 1 and Yanggaung No. 2 derived from a crossing of Ehime Kashi No. 28 and Haruka. At present, the cultivated area of Yanggaung No. 1 is expanding. Precise and efficient variety identification is crucial for enforcing plant variety rights. Molecular markers, for example SNP, SSR and InDel, have been broadly applied for genotyping, variety identification, genetic diversity analysis and breeding in various crops. This study aims to discover whole-genome InDels by comparison of whole-genome sequencing data of Yangguang No. 1, Yangguang No. 2 and their parents, and developed agarose gel-resolved InDel markers for genotyping and discriminating different tangelo varieties. Furthermore, the application of the selected InDel markers for genetic analysis of a wider range of citrus germplasm resources is investigated. 【Methods】The plant materials for whole genome re-sequencing were the healthy leaves of Yangguang No. 1 and Yangguang No. 2 tangelos, and their seed parent Ehime Kashi No. 28 and pollen parent Haruka. Ten tangelo varieties were tested for the genotyping, discrimination and identification with the select InDel markers. Altogether, 20 mandarins, 10 tangelos, 2 pummelos, 5 sweet oranges (C. sinensis (L.) Osbeck), 2 citrons (C. medica L.), 3 lemons (C. limon (L.) Burm. f.), 2 yuzu (C. junos Siebold. ex Tanaka), 2 grapefruits (C. paradisi Macf.), 2 kumquats (Fortunella spp.), 3 trifoliate oranges (Poncirus trifoliata Raf.) were used for genetic diversity analysis. The whole-genome re-sequencing was carried out with NovaSeq 6000 Sequencer (Illumina Inc., San Diego, USA) in Biomarker Technologies (Beijing, China). The clean reads were mapped against the genome of clementine mandarin (https://phytozome.jgi.doe.gov/pz/portal.html) by using BWA; GATK was used to call and filter InDels; and SnpEff was applied for variant annotation. Primers for detecting the InDels were designed with BatchPrimer3 according to the flanking sequences. Population genetic parameters including major allele frequency (MAF) number of alleles (Na), number of genotypes (Ng), effective number of alleles (Ne), expected heterozygosity (He), observed heterozygosity (Ho) and polymorphic information contents (PIC) were calculated by using PowerMarker V3.25. PCoA was conducted by using “ade4” package of R program, and then the scatter plot was performed by using “ggplot2” package. 【Results】The four varieties each generated ＞ 10 million clean paired-end reads with ＞ 3 billion base, Q20 and Q30 of ＞ 96% and 92%, properly mapped ratio of ＞ 85%, 1× and 5× coverage ratio of ＞ 89.9% and 67.95% and average depth of ＞ 7×. A total of 285757, 338510, 320991 and 296648 InDels in 281458, 330079, 314081 and 289508 loci were called in the genomes of Ehime Kashi No.28, Haruka, Yangguang No. 1 and Yangguang No. 2, respectively. Clearly, InDels and InDel loci in the genome of Haruka was more than that of Ehime Kashi No. 28. Indeed, it is reasonable to deduce that Ehime Kashi No. 28 (Nanko × Amakusa), whose seed parent Nanko has half clementine mandarin pedigree, should show more identity to clementine mandarin than the other three. Moreover, it was not surprising that their hybrid offspring, Yangguang No. 1 and Yangguang No. 2 had the intermediate amounts of InDels and InDel loci compared to their parents. After removing the common InDels among the different varieties, we identified 607376 InDel loci (657414 variants) with an average density of 1.08 InDel/locus. Most (92.5%, 561914) of the loci had one InDel, and 41112, 4127, 220 and 3 loci had two, three, four and five InDels, respectively. The majority (488369, 80.4%) of InDel loci were intergenic, whereas only 19.6% (119007) were intragenic. Most of the InDel were mononucleotide, followed by dinucleotide. Notably, we observed that 5315 (2.1%), 6402 (2.2%), 6226 (2.2%) and 5766 (2.3%) InDels were ＞ 30 or ＜ -30 bp in length. They could be potentially used as InDel markers that can be detected through simple agarose gel electrophoresis. With the genomic DNA of the 10 tangelos and Ehime Kashi No. 28 as templates, PCR products corresponding to the 48 InDels were successfully amplified, and the bands could be separated by 2.5% agarose gel electrophoresis. The genotypes of the tested tangelos and Ehime Kashi No. 28 could be unambiguously and easily discriminative with these InDel markers. Moreover, a total of 51 citrus germplasm resources were further genotyped with these InDel markers. The PICs of these 48 markers in the total 51 citrus germplasms were ranged from 0.055 to 0.450 with an average of 0.281; the MAFs ranged from 0.510 to 0.971 with an average of 0.739; the Ho and He were in the range of 0.039 to 0.647 and 0.059 to 0.546 with averages of 0.306 and 0.350, respectively. Some markers with higher He, PIC and lower MAFs, for example I3_744, I7_170, I8_516, I8_533 and I9_669, showing high genetic diversity, were potentially important for the genetic diversity analysis for citrus crops. In addition, we built a PCoA plot to illustrate the genetic distances among all the 51 varieties. The different varieties in same types generally tend to cluster close, fitting the conventional citrus taxonomic system, although the mandarin and kumquat groups were relatively scattering, which could be explained by interspecific or intergenetic hybrids. However, the precise genetic relations among these citrus germplasms could not be inferred from the PCoA.【Conclusions】The genome-wide InDels from two novel tangelos and their parents are characterised. Forty-eight InDels (＞ 30 or ＜ -30 bp) are useful for the effective discrimination and identification of various tangelos through low-cost agarose gel electrophoresis. They are also applicable for genetic diversity and classification of true citrus fruit tree group.
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