Whole genome resequencing of four Italian sweet pepper landraces provides insights on sequence variation in genes of agronomic value.

Sweet pepper (Capsicum annuum L.) is a high value crop and one of the most widely grown vegetables belonging to the Solanaceae family. In addition to commercial varieties and F hybrids, a multitude of landraces are grown, whose genetic combination is the result of hundreds of years of random, environmental, and farmer selection. High genetic diversity exists in the landrace gene pool which however has scarcely been studied, thus bounding their cultivation. We re-sequenced four pepper inbred lines, within as many Italian landraces, which representative of as many fruit types: big sized blocky with sunken apex ('Quadrato') and protruding apex or heart shaped ('Cuneo'), elongated ('Corno') and smaller sized sub-spherical ('Tumaticot'). Each genomic sequence was obtained through Illumina platform at coverage ranging from 39 to 44×, and reconstructed at a chromosome scale. About 35.5k genes were predicted in each inbred line, of which 22,017 were shared among them and the reference genome (accession 'CM334'). Distinctive variations in miRNAs, resistance gene analogues (RGAs) and susceptibility genes (S-genes) were detected. A detailed survey of the SNP/Indels occurring in genes affecting fruit size, shape and quality identified the highest frequencies of variation in regulatory regions. Many structural variations were identified as presence/absence variations (PAVs), notably in resistance gene analogues (RGAs) and in the capsanthin/capsorubin synthase (CCS) gene. The large allelic diversity observed in the four inbred lines suggests their potential use as a pre-breeding resource and represents a one-stop resource for C. annuum genomics and a key tool for dissecting the path from sequence variation to phenotype.

Acquadro A ，Barchi L ，Portis E ，Nourdine M ，Carli C ，Monge S ，Valentino D ，Lanteri S ... -  《Scientific Reports》

Whole-genome resequencing reveals genomic footprints of Italian sweet and hot pepper heirlooms giving insight into genes underlying key agronomic and qualitative traits.

Pepper is a major crop species of the Solanaceae family, largely appreciated for its high nutritional and healthy contribution to human diets. In the Mediterranean basin, the favorable pedoclimatic conditions enhanced the selection of several diversified landraces cultivated pepper (Capsicum annuum), for whom Italy can be considered a main pole of diversification. Hence, a survey of traditional C. annuum genetic resources is essential for deep understanding of such diversity and for applications in genomics assisted breeding. Here, we report whole-genome resequencing analyses of two sweet and two pungent genotypes highly diffused in South Italy and representative of the variability for shape, colour and nutritional properties. The four genomes were reconstructed at a chromosomal scale using a reference-guided approach, based on a dataset of 2.6 billion paired-end reads, corresponding to 20× genome coverage and a mapping rate above 99% for a final genomes size of approximately 3 Gb. After five iterations of variant calling, a total of 29,258,818 single nucleotide polymorphisms (SNPs) and 1,879,112 InDels, were identified. Substantial differences were observed among the four genomes based on geographical origin, with chromosomes 9 and 11 showing more polymorphisms in the accessions with higher fruit weight and absence of pungency. Among the identified variants, a small private indel (T - > TA) shared between sweet and big fruits accessions induces a frameshift with the generation of a new stop codon in a gene annotated as extensin, whereas two private SNPs within hot types were identified in 1-aminocyclopropane-1-carboxylate oxidase (ACO), a key gene involved in fruit ripening. The estimation of repetitive elements highlights a preponderant presence of Long Terminal Repeats (LTRs), the majority of which belonged to Gypsy superfamily. By comparing the four genomes with publicly available references including 'CM334' and Zunla-1 highlight the presence of 49,475 shared gene families. The new genomic sequences aim to enrich the whole genome information of pepper local varieties, providing a valuable tool for precision gene mapping, marker discovery, comparative studies. Such knowledge widens the frontiers to understand the selection history of Italian pepper landraces toward the recognition of specificity local agri-food products marks.

Esposito S ，Aiese Cigliano R ，Cardi T ，Tripodi P ... -  《-》

Genome-wide SNP discovery and population structure analysis in pepper (Capsicum annuum) using genotyping by sequencing.

Knowledge on population structure and genetic diversity in vegetable crops is essential for association mapping studies and genomic selection. Genotyping by sequencing (GBS) represents an innovative method for large scale SNP detection and genotyping of genetic resources. Herein we used the GBS approach for the genome-wide identification of SNPs in a collection of Capsicum spp. accessions and for the assessment of the level of genetic diversity in a subset of 222 cultivated pepper (Capsicum annum) genotypes. GBS analysis generated a total of 7,568,894 master tags, of which 43.4% uniquely aligned to the reference genome CM334. A total of 108,591 SNP markers were identified, of which 105,184 were in C. annuum accessions. In order to explore the genetic diversity of C. annuum and to select a minimal core set representing most of the total genetic variation with minimum redundancy, a subset of 222 C. annuum accessions were analysed using 32,950 high quality SNPs. Based on Bayesian and Hierarchical clustering it was possible to divide the collection into three clusters. Cluster I had the majority of varieties and landraces mainly from Southern and Northern Italy, and from Eastern Europe, whereas clusters II and III comprised accessions of different geographical origins. Considering the genome-wide genetic variation among the accessions included in cluster I, a second round of Bayesian (K = 3) and Hierarchical (K = 2) clustering was performed. These analysis showed that genotypes were grouped not only based on geographical origin, but also on fruit-related features. GBS data has proven useful to assess the genetic diversity in a collection of C. annuum accessions. The high number of SNP markers, uniformly distributed on the 12 chromosomes, allowed the accessions to be distinguished according to geographical origin and fruit-related features. SNP markers and information on population structure developed in this study will undoubtedly support genome-wide association mapping studies and marker-assisted selection programs.

Taranto F ，D'Agostino N ，Greco B ，Cardi T ，Tripodi P ... -  《BMC GENOMICS》

Target sequencing reveals genetic diversity, population structure, core-SNP markers, and fruit shape-associated loci in pepper varieties.

The widely cultivated pepper (Capsicum spp.) is one of the most diverse vegetables; however, little research has focused on characterizing the genetic diversity and relatedness of commercial varieties grown in China. In this study, a panel of 92 perfect single-nucleotide polymorphisms (SNPs) was identified using re-sequencing data from 35 different C. annuum lines. Based on this panel, a Target SNP-seq genotyping method was designed, which combined multiplex amplification of perfect SNPs with Illumina sequencing, to detect polymorphisms across 271 commercial pepper varieties. The perfect SNPs panel had a high discriminating capacity due to the average value of polymorphism information content, observed heterozygosity, expected heterozygosity, and minor allele frequency, which were 0.31, 0.28, 0.4, and 0.31, respectively. Notably, the studied pepper varieties were morphologically categorized based on fruit shape as blocky-, long horn-, short horn-, and linear-fruited. The long horn-fruited population exhibited the most genetic diversity followed by the short horn-, linear-, and blocky-fruited populations. A set of 35 core SNPs were then used as kompetitive allele-specific PCR (KASPar) markers, another robust genotyping technique for variety identification. Analysis of genetic relatedness using principal component analysis and phylogenetic tree construction indicated that the four fruit shape populations clustered separately with limited overlaps. Based on STRUCTURE clustering, it was possible to divide the varieties into five subpopulations, which correlated with fruit shape. Further, the subpopulations were statistically different according to a randomization test and F statistics. Nine loci, located on chromosomes 1, 2, 3, 4, 6, and 12, were identified to be significantly associated with the fruit shape index (p < 0.0001). Target SNP-seq developed in this study appears as an efficient power tool to detect the genetic diversity, population relatedness and molecular breeding in pepper. Moreover, this study demonstrates that the genetic structure of Chinese pepper varieties is significantly influenced by breeding programs focused on fruit shape.

Du H ，Yang J ，Chen B ，Zhang X ，Zhang J ，Yang K ，Geng S ，Wen C ... -  《BMC PLANT BIOLOGY》

A further analysis of the relationship between yellow ripe-fruit color and the capsanthin-capsorubin synthase gene in pepper (Capsicum sp.) indicated a new mutant variant in C. annuum and a tandem repeat structure in promoter region.

Mature pepper (Capsicum sp.) fruits come in a variety of colors, including red, orange, yellow, brown, and white. To better understand the genetic and regulatory relationships between the yellow fruit phenotype and the capsanthin-capsorubin synthase gene (Ccs), we examined 156 Capsicum varieties, most of which were collected from Northwest Chinese landraces. A new ccs variant was identified in the yellow fruit cultivar CK7. Cluster analysis revealed that CK7, which belongs to the C. annuum species, has low genetic similarity to other yellow C. annuum varieties. In the coding sequence of this ccs allele, we detected a premature stop codon derived from a C to G change, as well as a downstream frame-shift caused by a 1-bp nucleotide deletion. In addition, the expression of the gene was detected in mature CK7 fruit. Furthermore, the promoter sequences of Ccs from some pepper varieties were examined, and we detected a 176-bp tandem repeat sequence in the promoter region. In all C. annuum varieties examined in this study, the repeat number was three, compared with four in two C. chinense accessions. The sequence similarity ranged from 84.8% to 97.7% among the four types of repeats, and some putative cis-elements were also found in every repeat. This suggests that the transcriptional regulation of Ccs expression is complex. Based on the analysis of the novel C. annuum mutation reported here, along with the studies of three mutation types in yellow C. annuum and C. chinense accessions, we suggest that the mechanism leading to the production of yellow color fruit may be not as complex as that leading to orange fruit production.

Li Z ，Wang S ，Gui XL ，Chang XB ，Gong ZH ... -  《PLoS One》