HT-B and S-RNase CRISPR-Cas9 double knockouts show enhanced self-fertility in diploid Solanum tuberosum.

The Gametophytic Self-Incompatibility (GSI) system in diploid potato (Solanum tuberosum L.) poses a substantial barrier in diploid potato breeding by hindering the generation of inbred lines. One solution is gene editing to generate self-compatible diploid potatoes which will allow for the generation of elite inbred lines with fixed favorable alleles and heterotic potential. The S-RNase and HT genes have been shown previously to contribute to GSI in the Solanaceae family and self-compatible S. tuberosum lines have been generated by knocking out S-RNase gene with CRISPR-Cas9 gene editing. This study employed CRISPR-Cas9 to knockout HT-B either individually or in concert with S-RNase in the diploid self-incompatible S. tuberosum clone DRH-195. Using mature seed formation from self-pollinated fruit as the defining characteristic of self-compatibility, HT-B-only knockouts produced little or no seed. In contrast, double knockout lines of HT-B and S-RNase displayed levels of seed production that were up to three times higher than observed in the S-RNase-only knockout, indicating a synergistic effect between HT-B and S-RNase in self-compatibility in diploid potato. This contrasts with compatible cross-pollinations, where S-RNase and HT-B did not have a significant effect on seed set. Contradictory to the traditional GSI model, self-incompatible lines displayed pollen tube growth reaching the ovary, yet ovules failed to develop into seeds indicating a potential late-acting self-incompatibility in DRH-195. Germplasm generated from this study will serve as a valuable resource for diploid potato breeding.

Genomic selection for morphological and yield-related traits using genome-wide SNPs in oil palm.

Oil palm is the most important oil crop worldwide. Colombia is the fourth largest producer, primarily relying on production from interspecific hybrids, derived from crosses between Elaeis oleifera and Elaeis guineensis (OxG). However, conventional breeding can take up to 20 years to generate a new variety. Therefore, reducing the breeding cycle while improving the genetic gain for complex traits is desirable. Genomic selection (GS) is an approach with the potential to achieve this goal. In this study, we evaluated 431 F1 interspecific hybrids (OxG) and 444 backcrosses (BC1) for morphological and yield-related traits. Genomic predictions were performed with the G-BLUP model using three different population datasets for training the model: the same population (TRN1), the other population (TRN2), and both populations (TRN1+2). Higher multi-family prediction accuracies were obtained for foliar area (0.3 in OxG) and trunk height (0.47 in BC1) when the model was trained with TRN1. Single-family prediction accuracies were lower in the OxG compared to BC1 families for traits such as trunk diameter, trunk height, bunch number, and yield using TRN1. Conversely, lower prediction accuracies were obtained for most traits when the model was trained using TRN2 (< 0.1). Multi-trait models showed a substantial increase of the predictions for traits such as yield (0.22 for OxG and 0.44 for BC1), because of the genetic correlations between traits. The results herein highlighted the potential of GS for parental selection in OxG and BC1 populations, but further studies are required to improve the models to select individuals by their genetic value. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-022-01341-5.

Combining transcriptome analysis and GWAS for identification and validation of marker genes in the Physalis peruviana-Fusarium oxysporum pathosystem.

Vascular wilt, caused by the pathogen Fusarium oxysporum f. sp. physali (Foph), is a major disease of cape gooseberry (Physalis peruviana L.) in Andean countries. Despite the economic losses caused by this disease, there are few studies related to molecular mechanisms in the P. peruviana-Foph pathosystem as a useful tool for crop improvement. This study evaluates eight candidate genes associated with this pathosystem, using real-time quantitative PCR (RT-qPCR). The genes were identified and selected from 1,653 differentially expressed genes (DEGs) derived from RNA-Seq analysis and from a previous genome-wide association study (GWAS) of this plant-pathogen interaction. Based on the RT-qPCR analysis, the tubuline (TUB) reference gene was selected for its highly stable expression in cape gooseberry. The RT-qPCR validation of the candidate genes revealed the biological variation in their expression according to their known biological function. Three genes related to the first line of resistance/defense responses were highly expressed earlier during infection in a susceptible genotype, while three others were overexpressed later, mostly in the tolerant genotype. These genes are mainly involved in signaling pathways after pathogen recognition, mediated by hormones such as ethylene and salicylic acid. This study provided the first insight to uncover the molecular mechanism from the P. peruviana-Foph pathosystem. The genes validated here have important implications in the disease progress and allow a better understanding of the defense response in cape gooseberry at the molecular level. Derived molecular markers from these genes could facilitate the identification of tolerant/susceptible genotypes for use in breeding schemes.

Optimization of the genotyping-by-sequencing SNP calling for diversity analysis in cape gooseberry (Physalis peruviana L.) and related taxa.

A robust Genotyping-By-Sequencing (GBS) pipeline platform was examined to provide accurate discovery of Single Nucleotide Polymorphisms (SNPs) in a cape gooseberry (Physalis peruviana L.) and related taxa germplasm collection. A total of 176 accessions representing, wild, weedy, and commercial cultivars as well as related taxa from the Colombian germplasm bank and other world repositories were screened using GBS. The pipeline parameters mnLCov of 0.5 and a mnScov of 0.7, tomato and potato genomes, and cape gooseberry transcriptome for read alignments, were selected to better assess diversity and population structure in cape gooseberry and related taxa. A total of 7,425 SNPs, derived from P. peruviana common tags (unique 64 bp sequences shared between selected species), were used. Within P. peruviana, five subpopulations with a high genetic diversity and allele fixation (HE: 0.35 to 0.36 and FIS: -0.11 to -0.01, respectively) were detected. Conversely, low genetic differentiation (FST: 0.01 to 0.05) was also observed, indicating a high gene flow among subpopulations. These results contribute to the establishment of adequate conservation and breeding strategies for Cape gooseberry and closely related Physalis species.

Genome-wide association study (GWAS) for morphological and yield-related traits in an oil palm hybrid (Elaeis oleifera x Elaeis guineensis) population.

BACKGROUND: The genus Elaeis has two species of economic importance for the oil palm agroindustry: Elaeis oleifera (O), native to the Americas, and Elaeis guineensis (G), native to Africa. This work provides to our knowledge, the first association mapping study in an interspecific OxG oil palm population, which shows tolerance to pests and diseases, high oil quality, and acceptable fruit bunch production. RESULTS: Using genotyping-by-sequencing (GBS), we identified a total of 3776 single nucleotide polymorphisms (SNPs) that were used to perform a genome-wide association analysis (GWAS) in 378 OxG hybrid population for 10 agronomic traits. Twelve genomic regions (SNPs) were located near candidate genes implicated in multiple functional categories, such as tissue growth, cellular trafficking, and physiological processes. CONCLUSIONS: We provide new insights on genomic regions that mapped on candidate genes involved in plant architecture and yield. These potential candidate genes need to be confirmed for future targeted functional analyses. Associated markers to the traits of interest may be valuable resources for the development of marker-assisted selection in oil palm breeding.

Association analysis for disease resistance to Fusarium oxysporum in cape gooseberry (Physalis peruviana L).

BACKGROUND: Vascular wilt caused by Fusarium oxysporum is the most important disease in cape gooseberry (Physalis peruviana L.) in Colombia. The development of resistant cultivars is considered one of the most cost-effective means to reduce the impact of this disease. In order to do so, it is necessary to provide breeders with molecular markers and promising germplasm for introgression of different resistance loci as part of breeding schemes. Here we described an association mapping study in cape gooseberry with the goal to: (i) select promising materials for use in plant breeding and (ii) identify SNPs associated with the cape gooseberry resistance response to the F. oxysporum pathogen under greenhouse conditions, as potential markers for cape gooseberry breeding. RESULTS: We found a total of 21 accessions with different resistance responses within a diversity panel of 100 cape gooseberry accessions. A total of 60,663 SNPs were also identified within the same panel by means of GBS (Genotyping By Sequencing). Model-based population structure and neighbor-joining analyses showed three populations comprising the cape gooseberry panel. After correction for population structure and kinship, we identified SNPs markers associated with the resistance response against F. oxysporum. The identification of markers was based on common tags using the reference genomes of tomato and potato as well as the root/stem transcriptome of cape gooseberry. By comparing their location with the tomato genome, 16 SNPs were found in genes involved in defense/resistance response to pathogens, likewise when compared with the genome of potato, 12 markers were related. CONCLUSIONS: The work presented herein provides the first association mapping study in cape gooseberry showing both the identification of promising accessions with resistance response phenotypes and the identification of a set of SNP markers mapped to defense/resistance response genes of reference genomes. Thus, the work also provides new knowledge on candidate genes involved in the P. peruviana - F. oxysporum pathosystem as a foundation for further validation in marker-assisted selection. The results have important implications for conservation and breeding strategies in cape gooseberry.

Genetic diversity and population structure in Physalis peruviana and related taxa based on InDels and SNPs derived from COSII and IRG markers.

The genus Physalis is common in the Americas and includes several economically important species, among them Physalis peruviana that produces appetizing edible fruits. We studied the genetic diversity and population structure of P. peruviana and characterized 47 accessions of this species along with 13 accessions of related taxa consisting of 222 individuals from the Colombian Corporation of Agricultural Research (CORPOICA) germplasm collection, using Conserved Orthologous Sequences (COSII) and Immunity Related Genes (IRGs). In addition, 642 Single Nucleotide Polymorphism (SNPs) markers were identified and used for the genetic diversity analysis. A total of 121 alleles were detected in 24 InDels loci ranging from 2 to 9 alleles per locus, with an average of 5.04 alleles per locus. The average number of alleles in the SNP markers was two. The observed heterozygosity for P. peruviana with InDel and SNP markers was higher (0.48 and 0.59) than the expected heterozygosity (0.30 and 0.41). Interestingly, the observed heterozygosity in related taxa (0.4 and 0.12) was lower than the expected heterozygosity (0.59 and 0.25). The coefficient of population differentiation FST was 0.143 (InDels) and 0.038 (SNPs), showing a relatively low level of genetic differentiation among P. peruviana and related taxa. Higher levels of genetic variation were instead observed within populations based on the AMOVA analysis. Population structure analysis supported the presence of two main groups and PCA analysis based on SNP markers revealed two distinct clusters in the P. peruviana accessions corresponding to their state of cultivation. In this study, we identified molecular markers useful to detect genetic variation in Physalis germplasm for assisting conservation and crossbreeding strategies.

Identification of immunity related genes to study the Physalis peruviana--Fusarium oxysporum pathosystem.

The Cape gooseberry (Physalisperuviana L) is an Andean exotic fruit with high nutritional value and appealing medicinal properties. However, its cultivation faces important phytosanitary problems mainly due to pathogens like Fusarium oxysporum, Cercosporaphysalidis and Alternaria spp. Here we used the Cape gooseberry foliar transcriptome to search for proteins that encode conserved domains related to plant immunity including: NBS (Nucleotide Binding Site), CC (Coiled-Coil), TIR (Toll/Interleukin-1 Receptor). We identified 74 immunity related gene candidates in P. peruviana which have the typical resistance gene (R-gene) architecture, 17 Receptor like kinase (RLKs) candidates related to PAMP-Triggered Immunity (PTI), eight (TIR-NBS-LRR, or TNL) and nine (CC-NBS-LRR, or CNL) candidates related to Effector-Triggered Immunity (ETI) genes among others. These candidate genes were categorized by molecular function (98%), biological process (85%) and cellular component (79%) using gene ontology. Some of the most interesting predicted roles were those associated with binding and transferase activity. We designed 94 primers pairs from the 74 immunity-related genes (IRGs) to amplify the corresponding genomic regions on six genotypes that included resistant and susceptible materials. From these, we selected 17 single band amplicons and sequenced them in 14 F. oxysporum resistant and susceptible genotypes. Sequence polymorphisms were analyzed through preliminary candidate gene association, which allowed the detection of one SNP at the PpIRG-63 marker revealing a nonsynonymous mutation in the predicted LRR domain suggesting functional roles for resistance.