Assignment of genetic linkage maps to diploid Solanum tuberosum pachytene chromosomes by BAC-FISH technology.
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ABSTRACT: A cytogenetic map has been developed for diploid potato (Solanum tuberosum), in which the arms of the 12 potato bivalents can be identified in pachytene complements using multicolor fluorescence in situ hybridization (FISH) with a set of 60 genetically anchored bacterial artificial chromosome (BAC) clones from the RHPOTKEY BAC library. This diagnostic set of selected BACs (five per chromosome) hybridizes to euchromatic regions and corresponds to well-defined loci in the ultradense genetic map, and with these probes a new detailed and reliable pachytene karyotype could be established. Chromosome size has been estimated both from microscopic length measurements and from 4',6-diamidino-2-phenylindole fluorescence-based DNA content measurements. In both approaches, chromosome 1 is the largest (100-115 Mb) and chromosome 11 the smallest (49-53 Mb). Detailed measurements of mega-base-pair to micrometer ratios have been obtained for chromosome 5, with average values of 1.07 Mb/mum for euchromatin and 3.67 Mb/mum for heterochromatin. In addition, our FISH results helped to solve two discrepancies in the potato genetic map related to chromosomes 8 and 12. Finally, we discuss the significance of the potato cytogenetic map for extended FISH studies in potato and related Solanaceae, which will be especially beneficial for the potato genome-sequencing project.
Project description:Integrated cytogenetic pachytene fluorescence in situ hybridization (FISH) maps were developed for chromosomes 1, 3, 4, 5, 6, and 8 of maize using restriction fragment length polymorphism marker-selected Sorghum propinquum bacterial artificial chromosomes (BACs) for 19 core bin markers and 4 additional genetic framework loci. Using transgenomic BAC FISH mapping on maize chromosome addition lines of oats, we found that the relative locus position along the pachytene chromosome did not change as a function of total arm length, indicative of uniform axial contraction along the fibers during mid-prophase for tested loci on chromosomes 4 and 5. Additionally, we cytogenetically FISH mapped six loci from chromosome 9 onto their duplicated syntenic regions on chromosomes 1 and 6, which have varying amounts of sequence divergence, using sorghum BACs homologous to the chromosome 9 loci. We found that successful FISH mapping was possible even when the chromosome 9 selective marker had no counterpart in the syntenic block. In total, these 29 FISH-mapped loci were used to create the most extensive pachytene FISH maps to date for these six maize chromosomes. The FISH-mapped loci were then merged into one composite karyotype for direct comparative analysis with the recombination nodule-predicted cytogenetic, genetic linkage, and genomic physical maps using the relative marker positions of the loci on all the maps. Marker colinearity was observed between all pair-wise map comparisons, although marker distribution patterns varied widely in some cases. As expected, we found that the recombination nodule-based predictions most closely resembled the cytogenetic map positions overall. Cytogenetic and linkage map comparisons agreed with previous studies showing a decrease in marker spacing in the peri-centromeric heterochromatin region on the genetic linkage maps. In fact, there was a general trend with most loci mapping closer towards the telomere on the linkage maps than on the cytogenetic maps, regardless of chromosome number or maize inbred line source, with just some of the telomeric loci exempted. Finally and somewhat surprisingly, we observed considerable variation between the relative arm positions of loci when comparing our cytogenetic FISH map to the B73 genomic physical maps, even where comparisons were to a B73-derived cytogenetic map. This variation is more evident between different chromosome arms, but less so within a given arm, ruling out any type of inbred-line dependent global features of linear deoxyribonucleic acid compared with the meiotic fiber organization. This study provides a means for analyzing the maize genome structure by producing new connections for integrating the cytogenetic, linkage, and physical maps of maize.
Project description:Poplar was one of the first woody species whose individual chromosomes could be identified using chromosome specific painting probes. Nevertheless, high-resolution karyotype construction remains a challenge. Here, we developed a karyotype based on the meiotic pachytene chromosome of Populus simonii which is a Chinese native species with many excellent traits. This karyotype was anchored by oligonucleotide (oligo)-based chromosome specific painting probes, a centromere-specific repeat (Ps34), ribosomal DNA, and telomeric DNA. We updated the known karyotype formula for P. simonii to 2n = 2x = 38 = 26m + 8st + 4t and the karyotype was 2C. The fluorescence in situ hybridization (FISH) results revealed some errors in the current P. simonii genome assembly. The 45S rDNA loci were located at the end of the short arm of chromosomes 8 and 14 by FISH. However, they were assembled on pseudochromosomes 8 and 15. In addition, the Ps34 loci were distributed in every centromere of the P. simonii chromosome in the FISH results, but they were only found to be present in pseudochromosomes 1, 3, 6, 10, 16, 17, 18, and 19. Our results reveal that pachytene chromosomes oligo-FISH is a powerful tool for constructing high-resolution karyotypes and improving the quality of genome assembly.
Project description:The new sequencing technology enables identification of genome-wide sequence-based variants at a population level and a competitively low cost. The sequence variant-based molecular markers have motivated enormous interest in population and quantitative genetic analyses. Generation of the sequence data involves a sophisticated experimental process embedded with rich non-biological variation. Statistically, the sequencing process indeed involves sampling DNA fragments from an individual sequence. Adequate knowledge of sampling variation of the sequence data generation is one of the key statistical properties for any downstream analysis of the data and for implementing statistically appropriate methods. This paper reports a thorough investigation on modeling the sampling variation of the sequence data from the optimized RAD-seq (Restriction sit associated DNA sequencing) experiments with two parents and their offspring of diploid and autotetraploid potato (Solanum tuberosum L.). The analysis shows significant dispersion in sampling variation of the sequence data over that expected under multinomial distribution as widely assumed in the literature and provides statistical methods for modeling the variation and calculating the model parameters, which may be easily implemented in real sequence datasets. The optimized design of RAD-seq experiments enabled effective control of presentation of undesirable chloroplast DNA and RNA genes in the sequence data generated.
Project description:Solanum lycopersicum and Solanum tuberosum are agriculturally important crop species as they are rich sources of starch, protein, antioxidants, lycopene, beta-carotene, vitamin C, and fiber. The genomes of S. lycopersicum and S. tuberosum are currently available. However the linear strings of nucleotides that together comprise a genome sequence are of limited significance by themselves. Computational and bioinformatics approaches can be used to exploit the genomes for fundamental research for improving their varieties. The comparative genome analysis, Pfam analysis of predicted reviewed paralogous proteins was performed. It was found that S. lycopersicum proteins belong to more families, domains and clans in comparison with S. tuberosum. It was also found that mostly intergenic regions are conserved in two genomes followed by exons, intron and UTR. This can be exploited to predict regions between genomes that are similar to each other and to study the evolutionary relationship between two genomes, leading towards the development of disease resistance, stress tolerance and improved varieties of tomato.
Project description:Potato breeding must improve its efficiency by increasing the reliability of selection as well as identifying a promising germplasm for crossing. This study shows the prediction accuracy of genomic-estimated breeding values for several potato (Solanum tuberosum L.) breeding clones and the released cultivars that were evaluated at three locations in northern and southern Sweden for various traits. Three dosages of marker alleles [pseudo-diploid (A), additive tetrasomic polyploidy (B), and additive-non-additive tetrasomic polyploidy (C)] were considered in the genome-based prediction models, for single environments and multiple environments (accounting for the genotype-by-environment interaction or G × E), and for comparing two kernels, the conventional linear, Genomic Best Linear Unbiased Prediction (GBLUP) (GB), and the non-linear Gaussian kernel (GK), when used with the single-kernel genetic matrices of A, B, C, or when employing two-kernel genetic matrices in the model using the kernels from B and C for a single environment (models 1 and 2, respectively), and for multi-environments (models 3 and 4, respectively). Concerning the single site analyses, the trait with the highest prediction accuracy for all sites under A, B, C for model 1, model 2, and for GB and GK methods was tuber starch percentage. Another trait with relatively high prediction accuracy was the total tuber weight. Results show an increase in prediction accuracy of model 2 over model 1. Non-linear Gaussian kernel (GK) did not show any clear advantage over the linear kernel GBLUP (GB). Results from the multi-environments had prediction accuracy estimates (models 3 and 4) higher than those obtained from the single-environment analyses. Model 4 with GB was the best method in combination with the marker structure B for predicting most of the tuber traits. Most of the traits gave relatively high prediction accuracy under this combination of marker structure (A, B, C, and B-C), and methods GB and GK combined with the multi-environment with G × E model.
Project description:Characterization of major resistance (R) genes to late blight (LB) -caused by the oomycete Phytophthora infestans- is very important for potato breeding. The objective of this study was to identify novel genes for resistance to LB from diploid Solanum tuberosum L. Andigenum Group (StAG) cultivar accessions. Using comparative analysis with a edgeR bioconductor package for differential expression analysis of transcriptomes, two of these accessions with contrasting levels of resistance to LB were analyzed using digital gene expression data. As a result, various differentially expressed genes (P ≤ 0.0001, Log2FC ≥ 2, FDR < 0.001) were noted. The combination of transcriptomic analysis provided 303 candidate genes that are overexpressed and underexpressed, thereby giving high resistance to LB. The functional analysis showed differential expression of R genes and their corresponding proteins related to disease resistance, NBS-LRR domain proteins, and specific disease resistance proteins. Comparative analysis of specific tissue transcriptomes in resistant and susceptible genotypes can be used for rapidly identifying candidate R genes, thus adding novel genes from diploid StAG cultivar accessions for host plant resistance to P. infestans in potato.
Project description:BackgroundInvertases are ubiquitous enzymes that irreversibly cleave sucrose into fructose and glucose. Plant invertases play important roles in carbohydrate metabolism, plant development, and biotic and abiotic stress responses. In potato (Solanum tuberosum), invertases are involved in 'cold-induced sweetening' of tubers, an adaptive response to cold stress, which negatively affects the quality of potato chips and French fries. Linkage and association studies have identified quantitative trait loci (QTL) for tuber sugar content and chip quality that colocalize with three independent potato invertase loci, which together encode five invertase genes. The role of natural allelic variation of these genes in controlling the variation of tuber sugar content in different genotypes is unknown.ResultsFor functional studies on natural variants of five potato invertase genes we cloned and sequenced 193 full-length cDNAs from six heterozygous individuals (three tetraploid and three diploid). Eleven, thirteen, ten, twelve and nine different cDNA alleles were obtained for the genes Pain-1, InvGE, InvGF, InvCD141 and InvCD111, respectively. Allelic cDNA sequences differed from each other by 4 to 9%, and most were genotype specific. Additional variation was identified by single nucleotide polymorphism (SNP) analysis in an association-mapping population of 219 tetraploid individuals. Haplotype modeling revealed two to three major haplotypes besides a larger number of minor frequency haplotypes. cDNA alleles associated with chip quality, tuber starch content and starch yield were identified.ConclusionsVery high natural allelic variation was uncovered in a set of five potato invertase genes. This variability is a consequence of the cultivated potato's reproductive biology. Some of the structural variation found might underlie functional variation that influences important agronomic traits such as tuber sugar content. The associations found between specific invertase alleles and chip quality, tuber starch content and starch yield will facilitate the selection of superior potato genotypes in breeding programs.
Project description: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.
Project description:Examining the relationships among DNA sequence, meiotic recombination, and chromosome structure at a genome-wide scale has been difficult because only a few markers connect genetic linkage maps with physical maps. Here, we have positioned 1195 genetically mapped expressed sequence tag (EST) markers onto the 10 pachytene chromosomes of maize by using a newly developed resource, the RN-cM map. The RN-cM map charts the distribution of crossing over in the form of recombination nodules (RNs) along synaptonemal complexes (SCs, pachytene chromosomes) and allows genetic cM distances to be converted into physical micrometer distances on chromosomes. When this conversion is made, most of the EST markers used in the study are located distally on the chromosomes in euchromatin. ESTs are significantly clustered on chromosomes, even when only euchromatic chromosomal segments are considered. Gene density and recombination rate (as measured by EST and RN frequencies, respectively) are strongly correlated. However, crossover frequencies for telomeric intervals are much higher than was expected from their EST frequencies. For pachytene chromosomes, EST density is about fourfold higher in euchromatin compared with heterochromatin, while DNA density is 1.4 times higher in heterochromatin than in euchromatin. Based on DNA density values and the fraction of pachytene chromosome length that is euchromatic, we estimate that approximately 1500 Mbp of the maize genome is in euchromatin. This overview of the organization of the maize genome will be useful in examining genome and chromosome evolution in plants.
Project description:Phloem localization of plant viruses is advantageous for acquisition by sap-sucking vectors but hampers host-virus protein interaction studies. In this study, Potato leafroll virus (PLRV)-host protein complexes were isolated from systemically infected potato, a natural host of the virus. Comparing two different co-immunoprecipitation support matrices coupled to mass spectrometry, we identified 44 potato proteins and one viral protein (P1) specifically associated with virus isolated from infected phloem. An additional 142 proteins interact in complex with virus at varying degrees of confidence. Greater than 80% of these proteins were previously found to form high confidence interactions with PLRV isolated from the model host Nicotiana benthamiana. Bioinformatics revealed that these proteins are enriched for functions related to plasmodesmata, organelle membrane transport, translation and mRNA processing. Our results show that model system proteomics experiments are extremely valuable for understanding protein interactions regulating infection in recalcitrant pathogens such as phloem-limited viruses.