Whole-Genome Resequencing of Near-Isogenic Lines Reveals a Genomic Region Associated with High Trans-Lycopene Contents in Watermelon

Trans-lycopene is a functional phytochemical abundant in red-fleshed watermelons, and its contents vary among cultivars. In this study, the genetic basis of high trans-lycopene contents in scarlet red flesh was evaluated. Three near-isogenic lines (NILs) with high trans-lycopene contents were derived from the scarlet red-fleshed donor parent DRD and three coral red-fleshed (low trans-lycopene contents) recurrent parents. The lycopene contents of DRD (589.4 ± 71.8 µg/g) were two times higher than that of the recurrent parents, and values for NILs were intermediate between those of the parents. Coral red-fleshed lines and F1 cultivars showed low trans-lycopene contents (135.7 ± 18.0 µg/g to 213.7 ± 39.5 µg/g). Whole-genome resequencing of two NILs and their parents and an analysis of genome-wide single-nucleotide polymorphisms revealed three common introgressed regions (CIRs) on chromosomes 6, 9, and 10. Twenty-eight gene-based cleaved amplified polymorphic sequence (CAPS) markers were developed from the CIRs. The CAPS markers derived from CIR6 on chromosome 6, spanning approximately 1 Mb, were associated (R2 = 0.45–0.72) with the trans-lycopene contents, particularly CIR6-M1 and CIR6-M4. Our results imply that CIR6 is a major genomic region associated with variation in the trans-lycopene contents in red-fleshed watermelon, and CIR6-M1 and CIR6-M4 may be useful for marker-assisted selection.

Identification of starch candidate genes using SLAF-seq and BSA strategies and development of related SNP-CAPS markers in tetraploid potato

Potato starch is an essential nutrient for humans and is widely used worldwide. Locating relevant genomic regions, mining stable genes and developing candidate gene markers can promote the breeding of new high-starch potato varieties. A total of 106 F1 individuals and their parents (YSP-4 × MIN-021) were used as test materials, from which 20 plants with high starch content and 20 with low starch content were selected to construct DNA pools for site-specific amplified fragment sequencing (SLAF-seq) and bulked segregation analysis (BSA). A genomic region related to the starch traits was first identified in the 0–5.62 Mb of chromosome 2 in tetraploid potato. In this section, a total of 41 non-synonymous genes, which were considered as candidate genes related to the starch trait, were annotated through a basic local alignment search tool (BLAST) search of multiple databases. Six candidate genes for starch (PGSC0003DMG400017793, PGSC0003DMG400035245, PGSC0003DMG400036713, PGSC0003DMG400040452, PGSC0003DMG400006636 and PGSC0003DMG400044547) were further explored. In addition, cleaved amplified polymorphic sequence (CAPS) markers were developed based on single nucleotide polymorphism (SNP) sites associated with the starch candidate genes. SNP-CAPS markers chr2-CAPS6 and chr2-CAPS21 were successfully developed and validated with the F2 population and 24 tetraploid potato varieties (lines). Functional analysis and cloning of the candidate genes associated with potato starch will be performed in further research, and the SNP-CAPS markers chr2-CAPS6 and chr2-CAPS21 can be further used in marker-assisted selection breeding of tetraploid potato varieties with high starch content.

Identification of Fruit Firmness QTL ff2.1 by SLAF-BSA and QTL Mapping in Melon

Fruit firmness is an important target of melon breeding, as it is associated with shelf life and economic value; however, the precise mechanism determining fruit firmness during fruit ripening remains elusive. In the present study, one hundred forty-four F2 plants and F2-3 families derived from the high-firmness melon line M2-10 and the low-firmness melon line ZT091 were used to identify major quantitative trait loci (QTLs) by specific-locus amplified fragment (SLAF) sequencing with bulked segregant analysis (BSA). Simple sequence repeat (SSR) and cleaved amplified polymorphic sequence (Caps) markers based on the resequencing of parental lines were also used to narrow the associated region to identify candidate genes. Two regions associated with fruit firmness were investigated, including a 4.87 Mb region on chr. 2 and a 28.7 Mb region on chr. 5 of the melon genome. SSR and Caps markers were used to construct a genetic map of the associated regions: QTL ff5.1 was located between CmSSR13509 and CmSSR13423 and explained 38.44% of the observed variation, with an LOD threshold of 17.44; ff2.1 was located between CmSSR07709 and SNP22228 and explained 28.14% of the variation, with an LOD threshold of 3.8, and this region included 106 Kb and 10 candidate genes. Quantitative Real-time PCR (qRT-PCR) was used to investigated the investigate candidate gene expression at 15, 20 and 25 days after pollination (DAP) in the parental lines, and significant expression levels were detected for most of the genes, including four genes of unknown function and MELO3C017519, MELO3C017520, MELO3C017522, MELO3C029506, and MELO3C029520. These results revealed a new QTL, ff2.1, for melon fruit firmness-related gene identification.

VCF2CAPS–A high-throughput CAPS marker design from VCF files and its test-use on a genotyping-by-sequencing (GBS) dataset

Next-generation sequencing (NGS) is a powerful tool for massive detection of DNA sequence variants such as single nucleotide polymorphisms (SNPs), multi-nucleotide polymorphisms (MNPs) and insertions/deletions (indels). For routine screening of numerous samples, these variants are often converted into cleaved amplified polymorphic sequence (CAPS) markers which are based on the presence versus absence of restriction sites within PCR products. Current computational tools for SNP to CAPS conversion are limited and usually infeasible to use for large datasets as those generated with NGS. Moreover, there is no available tool for massive conversion of MNPs and indels into CAPS markers. Here, we present VCF2CAPS–a new software for identification of restriction endonucleases that recognize SNP/MNP/indel-containing sequences from NGS experiments. Additionally, the program contains filtration utilities not available in other SNP to CAPS converters–selection of markers with a single polymorphic cut site within a user-specified sequence length, and selection of markers that differentiate up to three user-defined groups of individuals from the analyzed population. Performance of VCF2CAPS was tested on a thoroughly analyzed dataset from a genotyping-by-sequencing (GBS) experiment. A selection of CAPS markers picked by the program was subjected to experimental verification. CAPS markers, also referred to as PCR-RFLPs, belong to basic tools exploited in plant, animal and human genetics. Our new software–VCF2CAPS–fills the gap in the current inventory of genetic software by high-throughput CAPS marker design from next-generation sequencing (NGS) data. The program should be of interest to geneticists involved in molecular diagnostics. In this paper we show a successful exemplary application of VCF2CAPS and we believe that its usefulness is guaranteed by the growing availability of NGS services.

Development of CAPS Markers for Evaluation of Genetic Diversity and Population Structure in the Germplasm of Button Mushroom (Agaricus bisporus)

Agaricus bisporus is a globally cultivated mushroom with high economic value. Despite its widespread cultivation, commercial button mushroom strains have little genetic diversity and discrimination of strains for identification and breeding purposes is challenging. Molecular markers suitable for diversity analyses of germplasms with similar genotypes and discrimination between accessions are needed to support the development of new varieties. To develop cleaved amplified polymorphic sequences (CAPs) markers, single nucleotide polymorphism (SNP) mining was performed based on the A. bisporus genome and resequencing data. A total of 70 sets of CAPs markers were developed and applied to 41 A. bisporus accessions for diversity, multivariate, and population structure analyses. Of the 70 SNPs, 62.85% (44/70) were transitions (G/A or C/T) and 37.15% (26/70) were transversions (A/C, A/T, C/G, or G/T). The number of alleles per locus was 1 or 2 (average = 1.9), and expected heterozygosity and gene diversity were 0.0–0.499 (mean = 0.265) and 0.0–0.9367 (mean = 0.3599), respectively. Multivariate and cluster analyses of accessions produced similar groups, with F-statistic values of 0.134 and 0.153 for distance-based and model-based groups, respectively. A minimum set of 10 markers optimized for accession identification were selected based on high index of genetic diversity (GD, range 0.299–0.499) and major allele frequency (MAF, range 0.524–0.817). The CAPS markers can be used to evaluate genetic diversity and population structure and will facilitate the management of emerging genetic resources.

Selection of F2 homozygous plants over the QTL9 related to rice panicle structure by using CAPS markers

QTL9 is a new potential quantitative trait locus related to rice panicle structure, identified through a GWAS analysis of Vietnamese local rice panel. To validate the QTL9, a biparental population was developed using the low branching (G6) and the high branching (G189) accessions from two haplotypes characterised by a contrasting phenotype for the secondary branch number and spikelet number traits. In parallel, using Gene capture technology combined with new generation sequencing identified 1,002 genetic variants in the QTL9 region between two parents, of which 12 SNPs were found in the cleavage site of 5 restriction enzyme. In this study, 12 primer pairs were tested to amplify the CAPS markers, based on the 12 SNPs, in order to be applied to select homozygous F2 lines over the QTL9 region for both haplotypes. 4 CAPS markers were selected distributing over the QTL9 region and displaying polymorphism between two parents. 275 F2plants were genotyped using 3 CAPS markers leading to the selection of 74 homozygotes plants over the QTL9region for haplotype 1, 66 homozygotes plants over the QTL9 region for haplotype 2, and 153 heterozygous plants. Homozygous lines will be used to develop the F3population, phenotyping of two F3 haplotypes will lead to discovering the role of QTL9 related to rice panicle structure.

THE ESTIMATION OF HEAT AND DROUGHT TOLERANCE MAIZE LINES USING DNA MARKERS

The main factor which causes to decrease maize grain yield is drought. In most regions where maize is grown, the water stress during the growing period is caused by both lack of soil moisture and high air temperature. The purposes of our study were the estimation and selection of maize lines for drought and heat tolerance based on DNA markers and determination of the correlation between CAPS markers and plant ability to resist the water stress. As the result of study, the significant differences were found between leaf temperature of maize lines which contained favorable alleles by both CAPS markers in 2018 and 2019 (35.72 and 34.41ºC respectively), LSD0.05=1.27. The leaf temperature of maize lines which had SNP (A) by dhn С397 (36.95ºC) differenced significantly with lines contained favorable allele by rspC1090 or lines with no favorable alleles in 2018 (33.68 and 34.35ºC respectively). Based on analysis by seeds germinating in sucrose solution the significant differences were observed between the amount of sprouted seeds in lines contained SNP(G) by rspC1090 and lines without any favorable allele (4% and 2.25% respectively), LSD0.05= 1.70. As the result of correlation analysis, the positive correlation was determined between SNP(A) by dhnС397 marker in maize lines and leaf temperature in 2018-2019 (r=0.16). The positive correlation was observed between SNP(G)byrspC1090 and the percent of sprouted seeds in sucrose solution (r=0.31). Thus, for complex estimation and maize line selection for drought and heat tolerance it could be recommended to use two CAPS markers dhnC397 and rspC1090.