Double Haploid Development and Assessment of Androgenic Competence of Balkan Pepper Core Collection in Bulgaria

This study was designed to assess the androgenic potential of 180 pepper accessions and 11 progenies (four F1 and seven BC) possessing PMMoV resistance in order to complement an ongoing pepper breeding program. The experiment was carried out in 10 replications with 20 anthers for each accession in two different induction mediums from 2017 to 2019. The highest androgenic response was observed in culture medium 17-2 but differences between two mediums were nonsignificant. From a total of 191 genotypes, 102 genotypes expressed a potential for direct embryogenesis. Embryo induction was seen to be genotype-dependent and decreased in the following order: Pumpkin > Conical > Bell or blocky > Round > Elongate as the most responsive genotypes with over 10% reacted anthers being observed in CAPS-23, CAPS-29, CAPS-127, CAPS-157, CAPS-169, F1 and BC 887 derived from CAPS-23. The number of regenerated plants was higher in the conical group and least in the round varietal group. Regenerated plants were examined visually and by flow cytometry for identification of spontaneous doubled haploids (DH) and haploids. Those originating from F1 and BC progenies were additionally evaluated by a CAPS marker targeting L4 allele for resistance against PMMoV. Obtained results revealed two groups consisting of homozygous susceptible and resistant plants. Therefore, use of anther culture in ongoing breeding will greatly facilitate the pepper genetic improvement.

Marker-Assisted Evaluation of Two Powdery Mildew Resistance Candidate Genes in Korean Cucumber Inbred Lines

Two genes, CsLRR-RPK2 (CsGy5G015660) and CsaMLO8 (Csa5G623470), have been considered as powdery mildew (PM) resistance genes in cucumbers. In this study, we evaluated the involvement of the alleles of these two genes in PM resistance in 100 commercial Korean cucumber inbred lines. To achieve this, we developed cleaved amplified polymorphic sequences (CAPS) and InDel markers from CsLRR-RPK2 and CsaMLO8. Genotyping analysis indicated that the CsLRR-RPK2-CAPS marker showed a stronger correlation with the PM-resistant phenotype, with an 84% consistency compared to the CsaMLO8-InDel marker. The use of the CsaMLO8-InDel marker showed a 70% consistency between phenotype and genotype results. It was proposed that the CsLRR-RPK2-CAPS marker successfully eliminated PM-susceptible inbred lines, since both genotype and phenotype results were 100% identical. Furthermore, the present study revealed that the introduction of one of these alleles is probably enough to confer PM resistance in cucumbers. However, seven PM-resistant inbred lines harbored either CsaMLO8 or CsLRR-RPK2 alleles, indicating that there is another PM-resistant resource(s) besides CsaMLO8- and CsLRR-RPK2–originated resistance in the commercial Korean inbred lines. Our results provide reliable evidence confirming two PM-resistant candidate genes for the detection of PM resistance resources in cucumber inbred lines.

Marker-Assisted Pyramiding of Multiple Disease Resistance Genes in Coffee Genotypes (Coffea arabica)

The use of resistant cultivars is the most effective strategy for controlling coffee leaf rust caused by the fungus Hemileia vastatrix. To assist the development of such cultivars, amplified fragment-length polymorphism (AFLP) markers linked to two loci of coffee resistance to races I and II as well as pathotype 001 of H. vastatrix were converted to sequence-characterized amplified region (SCAR) and cleaved amplified polymorphic site (CAPS) markers. In total, 2 SCAR markers and 1 CAPS marker were validated in resistant and susceptible parents as well as in 247 individuals from the F2 population. The efficiency of these markers for marker-assisted selection (MAS) was evaluated in F2:3 and backcross (BCrs2) populations genotyped with the developed markers and phenotyped with race II of H. vastatrix. The markers showed 90% efficiency in MAS. Therefore, the developed markers, together with molecular markers associated with other rust resistance genes, were used for F3:4 and BCrs3 coffee selection. The selected plants were analyzed using two markers associated with coffee berry disease (CBD) resistance, aiming for preventive breeding. MAS of F3:4 and BCrs3 individuals with all resistance loci was feasible. Our phenotypic and genotypic approaches are useful for the development of coffee genotypes with multiple genes conferring resistance to coffee leaf rust and CBD.

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 Marker Linked to H2 Locus of Fusarium Wilt Resistance in Chickpea

Background: Fusarium wilt is one of the widely distributed biotic stress of chickpea limiting its productivity worldwide. The major problem limiting the resistance breeding is screening of germplasm and breeding lines for disease resistance. To address such problems, identification of molecular marker closely linked to resistance locus is an effective strategy.Methods: The RAPD marker A07C417 closely linked H2 locus of Fusarium wilt resistance locus has been converted into SCAR and the loss of initial polymorphism was recaptured by alignment of consensus sequences from SCAR amplified locus for the identification of common motifs. Further, the linkage association of this marker with Fusarium wilt resistance locus has been reaffirmed using Fusarium wilt response of RILs phenotyped for wilt reaction using wilt sick pots and also wilt sick plots.Result: Identification of single nucleotide polymorphism at the consensus SCAR locus between susceptible and resistant cultivar, enabled development of CAPS marker through suitable restriction enzyme Aci I making it an effective codominant marker system for resistance deployment via marker assisted selection.

Involvement of purple acid phosphatase gene into nitrogen uptake of oil palm (Elaeis guineensis)

Maryanto SD, Tanjung ZA, Roberdi, Sudania WM, Pujianto, Hairinsyah, Utomo C, Liwang T. 2021. Involvement of purple acid phosphatase gene into nitrogen uptake of oil palm (Elaeis guineensis). Biodiversitas 22: 1385-1390. Nitrogen is the most important nutrient element in terms of plant growth. Plant purple acid phosphatases (PAPs) are known to participate in the phosphate (Pi) acquisition and utilization. Moreover, PAP gene plays an important role in nitrogen fixation. A single nucleotide polymorphism (SNP) was previously detected in the exon 7 of EgPAP3, based on SNP mining analysis of oil palm (Elaeis guineensis Jacq.). genome database. This study was aimed to obtain a Cleaved Amplified Polymorphic Sequences (CAPS) marker based on SNP within EgPAP associated with efficient nitrogen uptake oil palm progenies. Primer pairs were designed and used for PCR amplification of 3 oil palm progenies that showed low N-content, 3 progenies with moderate N-content, and 3 progenies with high N-content. The amplicon was purified prior to single-pass DNA sequencing analysis. Based on Pearson’s chi-square and odds ratio statistical analysis, the SNP has strong positive correlation with the phenotype. The SNP is located at chromosome 13 with a distance of 17.7771 cM from start codon. The sequencing analysis revealed that three progenies with high N-content samples had GG allele motif, while moderate N-content progenies had GA allele and low N-content progenies had AA allele motifs respectively. In addition, a restriction site of NIaIV was found to be adjacent to the SNP location, thus the PCR products of all samples were digested with NIaIV restriction enzyme. NIaIV was able to distinguish between high, medium and low efficient DNA samples. Whole high N-content progenies with GG allele motifs were undigested indicating a single band size of 670 bp identical to the untreated PCR product (control). Moderate N-content progenies produced a 670 bp, 550 bp, and 120 bp bands because of digested by NIaIV. Low N-content progenies also resulted in double bands of 550 bp and 120 bp due to digested by NIaIV. Furthermore, NIaIV restriction enzyme was applied to digest other 54 oil palms DNA samples with unknown genotypes. Whole GG samples were consistently shown to have single band, GA and AA samples were also consistent in producing two bands with different lengths. Based on this result, CAPS marker based on SNP in EgPAP3 was successfully developed to screen between high and low efficient N-uptake of oil palm progenies.

Genetic mapping and candidate gene analysis for melon resistance to Phytophthora capsici

Phytophthora blight is one of the most serious diseases affecting melon (Cucumis melo) production. Due to the lack of highly resistant germplasms, the progress on disease-resistant research is slow. To understand the genetics of melon resistance to Phytophthora capsici, an F2 population containing 498 individuals was developed by crossing susceptible line E31 to highly resistant line ZQK9. Genetic analysis indicated that the resistance in ZQK9 was controlled by a dominant gene, tentatively named MePhyto. Through bulked-segregant analysis (BSA-Seq) and chromosome walking techniques, the MePhyto gene was mapped to a 52.44 kb interval on chromosome 12. In this region, there were eight genes and their expression patterns were validated by qRT-PCR. Among them, one wall-associated receptor kinase (WAK) gene MELO3C002430 was significantly induced in ZQK9 after P. capsici inoculation, but not in E31. Based on the non-synonymous mutation site in MELO3C002430, a cleaved amplified polymorphic sequence (CAPS) marker, CAPS2430, was developed and this maker was co-segregated with MePhyto in both F2 population and a collection of 36 melon accessions. Thus MELO3C002430 was considered as the candidate gene and CAPS2430 was a promising marker for marker-assisted selection (MAS) in breeding. These results lay a foundation for revealing the resistance mechanism of melon to P. capsici.

Cleaved amplified polymorphic sequences (CAPS) marker for identification of two mutant alleles of the rapeseed BnaA.FAD2 gene

Two mutants of winter rapeseed (Brassica napus L. var. oleifera) with an increased amount of oleic acid in seeds were created by chemical mutagenesis (HOR3-M10453 and HOR4-M10464). The overall performance of the mutated plants was much lower than that of wild-type cultivars. Multiple rounds of crossing with high-yielding double-low (“00”) cultivars and breeding lines having valuable agronomic traits, followed by selection of high oleic acid genotypes is then needed to obtain new “00” varieties of rapeseed having high oleic acid content in seeds. To perform such selection, the specific codominant cleaved amplified polymorphic sequences (CAPS) marker was used. This marker was designed to detect the presence of two relevant point mutations in the desaturase gene BnaA.FAD2, and it was previously described and patented. The specific polymerase chain reaction product (732 bp) was digested using FspBI restriction enzyme that recognizes the 5′-C↓TAG-3′ sequence which is common to both mutated alleles, thereby yielding band patterns specific for those alleles. The method proposed in the patent was redesigned, adjusted to specific laboratory conditions, and thoroughly tested. Different DNA extraction protocols were tested to optimize the procedure. Two variants of the CAPS method (with and without purification of amplified product) were considered to choose the best option. In addition, the ability of the studied marker to detect heterozygosity in the BnaA.FAD2 locus was also tested. Finally, we also presented some examples for the use of the new CAPS marker in the marker-assisted selection (MAS) during our breeding programs. The standard CTAB method of DNA extraction and the simplified, two-step (amplification/digestion) procedure for the CAPS marker are recommended. The marker was found to be useful for the detection of two mutated alleles of the studied BnaA.FAD2 desaturase gene and can potentially assure the breeders of the purity of their HOLL lines. However, it was also shown that it could not detect any other alleles or genes that were revealed to play a role in the regulation of oleic acid level.

PCR-Based InDel Marker Associated with Powdery Mildew-Resistant MR-1

Powdery mildew (PM) is a fungal disease occurring in both field and greenhouse conditions worldwide. It infects many plant species and reduces both the productivity and quality of crops. Melon (Cucumis melo L.) is an economically important crop. In order to develop a molecular marker that can be used more conveniently in the development of PM-resistant melon using MR-1 melon resources, the previously reported cleaved amplified polymorphic sequence (CAPS) marker was improved with a length polymorphism PCR marker. Two cleaved CAPS markers—BSA12-LI3ECORI and BSA12-LI4HINFI—associated with BPm12.1, a major quantitative trait locus (QTL) corresponding to the PM resistance of MR-1, have been reported. In this study, we found that in the BSA12-LI3ECORI CAPS marker specifically, a 41 bp deletion was present in the PCR DNA region of the MR-1 melon genome. A new marker capable of distinguishing polymerase chain reaction (PCR) length polymorphism was produced using insertion-deletion (InDel) information in this region. This PCR-based InDel marker distinguished the genotypes of PM-resistant MR-1, PM-susceptible Top Mark, and their F1 progeny. These results suggest that this InDel marker could be used to develop PM-resistant melon varieties based on MR-1.