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Barley allele-specific amplicons useful for identifying wheat-barley recombinant chromosomes.

Barley (Hordeum vulgare L.) is potentially a new source of genes for wheat (Triticum aestivum L.) improvement. Wheat-barley chromosome recombinant lines provide a means for introgressing barley genes to wheat genome by chromosome engineering, and since these are expected to occur only rarely in special cytogenetic stocks, an efficient selection skill is necessary to identify them. To convert RFLP markers to barley allele-specific PCR markers useful for effective production of wheat-barley recombinant lines, 91 primer sets derived from RFLP clones which were previously mapped to the barley chromosomes were examined for PCR amplification using 'Chinese Spring' wheat, 'Betzes' barley and the wheat-barley chromosome addition lines. The polymorphisms were detected by an agarose gel electrophoresis of the PCR products without digestion with restriction enzymes. Out of 81 primer sets producing polymorphisms between the wheat and barley genomes, 26 amplified barley chromosome-specific DNAs which were confirmed to be located on the same chromosome as the RFLP markers by using the wheat-barley chromosome addition lines. These amplified DNAs represent barley allele-specific amplicons, which distinguish barley alleles from their wheat homoeologous counterparts. The present investigation revealed a higher probability for obtaining allele-specific amplicons from genomic DNA-derived RFLP markers than from cDNA-derived ones. The barley allele-specific amplicons developed in this study, namely, four for chromosome 2H, two for 3H, seven for 4H, eight for 5H, one for 6H and four for 7H, are suitable for identifying 'Chinese Spring' wheat- 'Betzes' barley recombinant chromosomes. However, one out of eight barley allele-specific amplicons on chromosome 5H did not detect a unique barley band in a 'New Golden' barley chromosome 5H addition line of 'Shinchunaga' wheat, indicating there may be a need to reconstruct allele-specific amplicons with different barley cultivars.

Alleles↗

Genetic diversity of three Elymus species indigenous to Japan and East Asia (E. tsukushiensis, E. humidus and E. dahuricus) detected by AFLP.

With the aim of broadening the gene pool of Triticeae for crop improvement, the genetic diversity of three hexaploid Elymus species indigenous to Japan and East Asia (E. tsukushiensis, E. humidus and E. dahuricus) was evaluated using amplified fragment length polymorphism (AFLP) markers. A total of 34 accessions: five of E. tsukushiensis, 13 of E. humidus, 15 of E. dahuricus, and one tetraploid Elymus species as an outgroup, were analyzed. The AFLP analysis was conducted with 12 selective primer combinations. In all, 329 bands were scored. The average number of bands scored per accession was 135.3. Among the 34 accessions, no polymorphism was detected among 10 accessions of E. humidus and among three accessions of E. tsukushiensis. Each species showed a diagnostic band pattern. One accession of E. humidus showed a band pattern intermediate between those of E. humidus and E. tsukushiensis, suggesting the occurrence of interspecific introgression. The level of intraspecific variation was highest in E. dahuricus (1-psb = 0.17, pi = 0.010), followed by E. tsukushiensis (1-psb = 0.14, pi = 0.008) and E. humidus (1-psb = 0.03, pi = 0.002). The level of interspecific variation was higher than that of intraspecific variation in all the pairs compared. The level of divergence between E. humidus and E. tsukushiensis was relatively low, but even so it was two times higher than that of the intraspecific variation for each species. The phylogenetic tree constructed here showed clear separations of all the species. The results of this study indicate a clear divergence of E. humidus from E. tsukushiensis in spite of the morphological similarity between these two species. Based on the values of nucleotide diversity, the time of divergence between E. humidus and E. tsukushiensis was estimated to be 0.9-1.2 million years ago.

Asia↗

A PCR-based marker for targeting small rye segments in wheat background.

We attempted to develop a PCR-based marker that detects various segments of rye chromosome incorporated into wheat. We designed three sets of PCR primers based on the nucleotide sequence data of a rye repetitive sequence previously reported. One of the primer sets amplified a clear ca. 1.4 kb fragment in a rye cultivar but not in any form of wheat, diploid, tetraploid or hexaploid. We used this critical primer set for PCR of various wild species and cultivars of rye, an array of wheat plants carrying different rye chromosomes or small segments from different regions of rye chromosome 1R, and plants carrying parts of the rye B chromosome. The PCR amplified the 1.4 kb fragment in all the plant materials examined. We believe this PCR primer set will be useful as a universal PCR-based marker for the introgression of rye chromosome segments in the wheat genome.

Chromosomes, Plant↗

Genomic differentiation of Hordeum chilense from H. vulgare as revealed by repetitive and EST sequences.

Hordeum vulgare, cultivated barley, and its wild relative, H. chilense, have several important traits that might be useful for wheat improvement. Here, in situ hybridization and barley expressed sequence tag (EST) markers were used to characterize and compare the chromosomes of H. chilense with those of H. vulgare. FISH with four repetitive DNA sequences, AG, AAG, 5S rDNA and 45S rDNA, was applied to the mitotic chromosomes of H. vulgare, H. chilense and available wheat-H. chilense addition and substitution lines. FISH with the AAG repeat differentiated the individual chromosomes of H. chilense and H. vulgare. The patterns of FISH signals in the two species differed greatly. The 45S rDNA signals were observed on two pairs of chromosomes in both species, while the 5S rDNA signals were observed on four pairs of chromosomes in H. vulgare and on one pair in H. chilense. The AG repeat showed FISH signals at the centromeric regions of all chromosomes of H. vulgare but none of the chromosomes of H. chilense. These results indicate that the chromosomes of the two species are highly differentiated. To study the homoeology between the two species, 209 EST markers of H. vulgare were allocated to individual chromosomes of H. chilense. One hundred and forty of the EST markers were allocated to respective chromosomes of H. chilense using the wheat-H. chilense addition and substitution lines. Twenty-six EST markers on average were allocated to each chromosome except to the chromosome 2H(ch)S, to which only 10 markers were allocated. Ninety percent of the allocated EST markers in H. chilense were placed on H. vulgare chromosomes of the same homo-eologous group, indicating that the expressed sequences of the two species were highly conserved. These EST markers would be useful for detecting chromatin introgressed from these species into the wheat genome.

Chile↗

Genealogy of the "Green Revolution" gene in rice.

During the "Green Revolution" of rice, high-yielding varieties (HYVs) were developed using a semi-dwarf gene (sd1 or OsGA20ox2). The presence or absence of the two mutant alleles (DGWG type in Dee-geo-woo-gen and JKK type in Jikkoku) were surveyed by PCR using 256 accessions of eight wild and two cultivate rice species. The DGWG allele was detected in a landrace (Oryza sativa) and two accessions of wild rice (O. rufipogon), all of which are from China, showing their limited distribution. Genealogical studies of the OsGA20ox2 gene showed that the 62 sequences of O. sativa and O. rufipogon included 20 distinct haplotypes, indicating that the species complex contained OsGA20ox2 genes from two different lineages. The silent site nucleotide diversities (pi and theta(w)) were extremely low in Japonica rice, suggesting a genetic bottleneck. The haplotype network showed that the DGWG and JKK alleles were derived in different lineages. The DGWG carrier (W1944) had unique polymorphisms in the surrounding region of the locus, suggesting that the DGWG allele has been preserved in the wild progenitor, rather than that the DGWG allele has been introgressed from HYVs to W1944. Although a semi-dwarfing plant is a weak competitor under saturated fields, the crossing experiment revealed that the DGWG variant might have been preserved as a hidden variation in the genetic background of wild rice, without expressing a short-stature.

Alleles↗

G4SNVHunter: An R/Bioconductor Package for Evaluating SNV-Induced Disruption of G-Quadruplex Structures Leveraging the G4Hunter Algorithm.

G-quadruplexes (G4s) are nucleic acid secondary structures with important regulatory functions. Single-nucleotide variants (SNVs), one of the most common forms of genetic variation, can potentially impact the formation of G4 structures if they occur within G4 regions. However, there is currently a lack of software tools specifically designed to assess such effects. Here, we present an R/Bioconductor package named G4SNVHunter, which enables rapid detection of variants that may disrupt G4 structures. This tool, based on the core principles of the G4Hunter algorithm, can provide precise quantitative assessment of the propensity for G4 formation within genomic sequences. Specialized experimental methods can then be designed based on the results provided by G4SNVHunter to further verify the specific functions of the affected G4 structures, facilitating deeper insights into the biological impacts of genetic variants from the perspective of G4 structures. To showcase the functionality of the G4SNVHunter package, we analyzed the Neandertal and Denisovan archaic introgressed variants detected by the Sprime software, and identified approximately 5,800 variants located within G4 regions, among which around 230 may impair G4 structure formation propensity. The source code for the G4SNVHunter package has been publicly released under the MIT license at https://github.com/rongxinzh/G4SNVHunter and https://bioconductor.org/packages/devel/bioc/html/G4SNVHunter.html.

G-Quadruplexes↗

Genetic diversity, disease resistance, and environmental adaptation of Arachis duranensis L.: New insights from landscape genomics.

The genetic diversity that exists in natural populations of Arachis duranensis, the wild diploid donor of the A subgenome of cultivated tetraploid peanut, has the potential to improve crop adaptability, resilience to major pests and diseases, and drought tolerance. Despite its potential value for peanut improvement, limited research has been focused on the association between allelic variation, environmental factors, and response to early (ELS) and late leaf spot (LLS) diseases. The present study implemented a landscape genomics approach to gain a better understanding of the genetic variability of A. duranensis represented in the ex-situ peanut germplasm collection maintained at the U.S. Department of Agriculture, which spans the entire geographic range of the species in its center of origin in South America. A set of 2810 single nucleotide polymorphism (SNP) markers allowed a high-resolution genome-wide characterization of natural populations. The analysis of population structure showed a complex pattern of genetic diversity with five putative groups. The incorporation of bioclimatic variables for genotype-environment associations, using the latent factor mixed model (LFMM2) method, provided insights into the genomic signatures of environmental adaptation, and led to the identification of SNP loci whose allele frequencies were correlated with elevation, temperature, and precipitation-related variables (q < 0.05). The LFMM2 analysis for ELS and LLS detected candidate SNPs and genomic regions on chromosomes A02, A03, A04, A06, and A08. These findings highlight the importance of the application of landscape genomics in ex situ collections of peanut and other crop wild relatives to effectively identify favorable alleles and germplasm for incorporation into breeding programs. We report new sources of A. duranensis germplasm harboring adaptive allelic variation, which have the potential to be utilized in introgression breeding for a single or multiple environmental factors, as well as for resistance to leaf spot diseases.

Arachis↗

Lineage allocation during early embryogenesis. Mapping of the neural primordia and application to the analysis of mouse mutants.

The methods outlined in this chapter discuss a range of techniques that have been employed for lineage analysis studies of the neural primordia from the onset of gastrulation and during neurulation. As the mouse has been extensively mapped, lineage analysis during normal morphogenesis is well understood. Attention is now focused on the tissue interactions that are essential for gastrulation and neurulation to proceed normally. The key to understanding these tissue interactions lies in the study of mutant embryos where abnormal development of specific tissue types affects the processes of gastrulation and neurulation. Lineage analysis and tissue potency experiments on particular mutant embryos will provide insight into these essential tissue interactions. As the first step toward undertaking such analysis of the neural derivatives, we have outlined the mutant strains available and detailed a protocol for the introgression of the lacZ transgene onto the mutant background.

Animals↗

Indica rice (Oryza sativa, BR29 and IR64).

Rice is the world's most important food crop. Indica-type rice provides the staple food for more than half of the world population. To satisfy the growing demand of the ever-increasing population, more sustained production of indica-type rice is needed. In addition, because of the high per capita consumption of indica rice, improvement of any traits including its nutritive value may have a significant positive health outcome for the rice-consuming population. Rice yield productivity is greatly affected by different biotic stresses, like diseases and insect pests, and abiotic stresses like drought, cold, and salinity. Attempts to improve resistance in rice to these stresses by conventional breeding through introgression of traits have limited success owing to a lack of resistance germplasm in the wild relatives. Gene transfer technology with genes from other sources can be used to make rice plants resistant or tolerant to insect pests, diseases, and different environmental stresses. For improving the nutritional value of the edible endosperm part of the rice, genes for increasing iron, beta-carotene, or better quality protein can be introduced in rice plants by genetic engineering. Different crops have been transformed using various gene transfer methods, such as protoplast transformation, biolistic, and Agrobacterium-mediated transformation. This chapter describes the Agrobacterium-mediated transformation protocol for indica-type rice. The selectable marker genes used are hygromycin phosphotransferase (hpt), neomycin phosphotransferase (nptII), or phosphomannose isomerase (pmi), and, accordingly, the selection agents are hygromycin, kanamycin (G418), or mannose, respectively.

Adaptation, Physiological↗

Eggplant (Solanum melongena L.).

Eggplant is an economically important vegetable crop in Asia and Africa, and although it is grown in Europe and the United States, it does not account for a significant percentage of agricultural production. It is susceptible to a number of pathogens and insects, with bacterial and fungal wilts being the most devastating. Attempts to improve resistance through introgression of traits from wild relatives have had limited success owing to sexual incompatibilities. Therefore, a crop improvement approach that combines both conventional breeding and biotechnological techniques would be beneficial. This chapter describes an Agrobacterium-mediated transformation protocol for eggplant based on inoculation of seedling explants (cotyledons and hypocotyls) and leaves. We have used this protocol to recover transformants from two different types of eggplant, a Solanum melongena L. breeding line, and S. melongena L. var. Black Eggplant. The selectable marker gene used was neomycin phosphotransferase II (nptII) and the selection agent was kanamycin. In vitro grown transformants acclimated readily to greenhouse conditions.

Agrobacterium tumefaciens↗

Lettuce (Lactuca sativa L.).

Lettuce is a globally important leafy vegetable with the United States being the largest world producers. The crop is susceptible to a number of viruses that are aphid transmitted and also highly vulnerable to post harvest diseases. Although wild species of lettuce are an important source of disease resistance genes, their introgression into commercial lettuce has been limited owing to sexual incompatibilities. Hence, the development of a gene transfer system for lettuce would be extremely valuable both in improving the genetic diversity of the crop and also for the transfer of useful agronomic traits. This chapter describes an Agrobacterium-mediated gene delivery system that is highly adaptable for the production of transgenic plants using a wide range of lettuce germplasms. The system described, commonly referred to as the genotype-independent transformation system, has been used for the transfer of several agriculturally useful traits into commercial varieties of lettuce. In this case, A. tumefaciens strain LBA4404 carrying a binary vector with supervirulent pToK47 was used for infecting excised cotyledonary explants. The plant selectable marker gene neomycin phosphotransferase II (nptII) was used, and transformed plants were selected using kanamycin in the culture medium. The beta-glucuronidase gene with intron (gus-intron) was also used in the gene transfer study to confirm the transgenicity of regenerated plants further.

Agrobacterium tumefaciens↗

SHR.BN-congenic strains for genetic analysis of multifactorially determined traits.

The laboratory rat is an important laboratory animal with multiple well-defined inbred strains, including some of the most widely used animal models of human diseases. Recent advances in the development of rat genetic resources will enable the exploitation of the full potential of rat models of human diseases and, in addition, the rat can provide useful information for comparative genomics of humans and mice. In the current review, we describe the development of congenics strains by introgression of differential chromosome segments from the Brown Norway (BN) rat to the genetic background of the spontaneously hypertensive rat (SHR). These SHR.BN-congenic strains and recombinant sublines derived from them were developed as a model system for genetic analysis of multifactorially determined pathophysiological and morphological conditions.

Animals↗

Metabolic characterization of insulin resistance syndrome feature loci in three brown Norway-derived congenic strains.

Studies on genetic determination of the insulin resistance syndrome in rat models revealed several susceptibility loci for features of this complex phenotype, i.e. dyslipidemia, insulin resistance and obesity. We analysed the influence of introgression of the RNO4, RNO20 segments of SHR origin and RNO8 segment of PD/Cub origin (all previously shown to be involved in (dys)regulation of carbohydrate and lipid metabolism) onto the genetic background of a common progenitor, the Brown Norway (BN/Cub) rat. The differential segments were genetically characterized in the BN.PD-D8Rat39/D8Rat35 (BN-Lx, RNO8 congenic), BN.SHR-Il6/Cd36 (BN.SHR4, RNO4 congenic) and BN.PD-D8Rat39/D8Rat3, SHR-D4Mgh2/Cd36,SHR-D20Wox3/D20Mgh5 (BN-Lx 1K, RNO4, 8, 20 triple congenic) strains and their metabolic profiling was performed. After one week of high-sucrose diet, all congenic strains showed substantially higher levels of serum triglycerides and free fatty acids as well as impaired glucose tolerance in comparison with the BN/Cub progenitor strain. The BN-Lx 1K triple congenic strain displayed the most profound dyslipidemia, glucose intolerance and highest increase of triglyceridemia in response to high-sucrose diet overall, though accompanied with the significantly lowest adiposity index. These results further support the role of genes present within the studied chromosomal regions in observed metabolic disturbances. Furthermore, these findings point to the studied loci within the gene-gene and gene-environment interactions involved in pathogenesis of the insulin resistance syndrome. The set of defined congenic strains provides a possibility of assessing individual features of such a complex phenotype.

Animals↗

Differential linkage of triglyceride and glucose levels on rat chromosome 4 in two segregating rat populations.

The PD/Cub is a recently established model of the IRS. The BN.SHR4 congenic strain was derived by introgression of the chromosome 4 segment of SHR origin (including the defective Cd36/Fat allele) onto the BN/Cub genetic background. We investigated the linkage of metabolic and morphometric phenotypes (total body weight, OGTT, fasting serum levels of TG, FFA) on chromosome 4 in two separate F2 rat populations: the PD/Cub x BN/Cub and PD/Cub x BN.SHR4 (total N = 243). In the PD/Cub x BN.SHR4 F2s, we found significant linkage for fasting TG levels (LOD = 3.26) and suggestive linkage for fasting glycaemia (LOD = 2.80) in the interval Il-6 - D4Bro1, i.e. the part of chromosome 4 of SHR origin in the BN.SHR4 congenic. However, no linkage for fasting TG concentrations, fasting glycaemia or any other followed parameter was found in the second, PD/Cub x BN/Cub F2. The differential linkage of TG and glucose levels to the centromeric part of rat chromosome 4q in the studied F2s points to the importance of this region for the lipid and carbohydrate metabolism at the specific age (10 months) and diet (standard chow) combination. The Cd36/Fat and Il-6 genes are the preliminary positional candidates for the observed effect.

Animals↗

A 14-gene region of rat chromosome 8 in SHR-derived polydactylous congenic substrain affects muscle-specific insulin resistance, dyslipidaemia and visceral adiposity.

The SHR and the PD/Cub are two established rodent models of human metabolic syndrome. Introgression of a ca 30 cM region of rat chromosome 8 from PD/Cub onto the genetic background of SHR was previously shown to influence several of the metabolic syndrome-related traits along with causing the PLS in the SHR-Lx congenic strain. In the process of identification of the causative alleles, we have produced several congenic sublines. The differential segment of SHR-Lx PD5 congenic substrain [SHR.PD(D8Rat42-D8Arb23)/Cub] spans approximately 1.4 Mb encompassing only 14 genes. When comparing the metabolic, morphometric and gene expression profiles of the SHR-Lx PD5 vs. SHR, the polydactyly and several distinct metabolic features observed in the original SHR-Lx congenic were still manifested, suggesting that the responsible genes were "trapped" within the relatively short differential segment of PD/Cub origin in SHR-Lx PD5. Particularly, the SHR-Lx PD5 displayed substantial reduction of insulin sensitivity confined to skeletal muscle. Among the candidate genes, the promyelocytic leukaemia zinc-finger Plzf (Zbtb16) transcription repressor is most likely responsible for the Lx mutation resulting in PLS and could also be involved in the alteration of metabolic pathways. The sequence analysis of the Plzf gene revealed a SNP leading to a threonine to serine substitution in SHR at aminoacid position 208 (T208S). In summary, we have isolated a 1.4 Mb genomic region syntenic to human chromosome 11q23, which, apart from causing polydactyly-luxate syndrome (PLS), affects total body weight, adiposity, lipid profile, insulin sensitivity of skeletal muscle and related gene expression as shown in the SHR-Lx PD5 congenic substrain.

Adipose Tissue↗

Assessing and managing biological risks of plants used for bioremediation, including risks of transgene flow.

The plants used for phytoremediation pose special biological risks, whether transgenic or not, as most of the species: (a) are semi-domesticated; (b) are introduced from other habitats; (c) can become established in the contaminated site; (d) can spread and displace native species, and/or; (e) may introgress transgenes into related species. The addition of transgenes can reduce the risks, e.g. to sterilize or render the species and hybrid offspring hypersensitive to environmental effects (heat, cold), or to a chemical that will cull the species. Various measures can contain transgenes used in phytoremediation species to prevent gene flow, but most containment technologies are both uni-directional (prevent either outflow or influx), and are inherently leaky, even a concept specifically utilizable for phytoremediation--grafting non-transgenic scions on bioremediating transgenic rootstocks. Containment mechanisms should be either stacked with each other or with "mitigator" genes. Transgenic mitigation (TM) has mitigator genes added in tandem to the desired primary transgene (genetically linked) and the mitigator genes confer traits that are positive or neutral to the desired species but are deleterious to hybrids, keeping them at very low frequencies. The concept was demonstrated in tobacco and oilseed rape with a dwarfing mitigator gene that enhanced the reproductive productivity (harvest index) when cultured alone, but eliminated it from mixed populations. Besides the mitigator genes previously proposed for crop species (sterility, no seed shattering, dwarfing, no secondary dormancy) there are genes especially appropriate for phytoremediation, e.g. overexpression of cytokinin oxidase (reduces cytokinin levels) conferring reduced shoot systems (unfitness to compete) with a more extensive root system that is better for extracting toxic wastes as well as no-flowering for vegetatively propagated species. Thus, biotechnology can be harnessed to reduce risks from both non-transgenic and transgenic phytoremediation species.

Biodegradation, Environmental↗

Genetics of hybrid incompatibility between Lycopersicon esculentum and L. hirsutum.

We examined the genetics of hybrid incompatibility between two closely related diploid hermaphroditic plant species. Using a set of near-isogenic lines (NILs) representing 85% of the genome of the wild species Lycopersicon hirsutum (Solanum habrochaites) in the genetic background of the cultivated tomato L. esculentum (S. lycopersicum), we found that hybrid pollen and seed infertility are each based on 5-11 QTL that individually reduce hybrid fitness by 36-90%. Seed infertility QTL act additively or recessively, consistent with findings in other systems where incompatibility loci have largely been recessive. Genetic lengths of introgressed chromosomal segments explain little of the variation for hybrid incompatibility among NILs, arguing against an infinitesimal model of hybrid incompatibility and reinforcing our inference of a limited number of discrete incompatibility factors between these species. In addition, male (pollen) and other (seed) incompatibility factors are roughly comparable in number. The latter two findings contrast strongly with data from Drosophila where hybrid incompatibility can be highly polygenic and complex, and male sterility evolves substantially faster than female sterility or hybrid inviability. The observed differences between Lycopersicon and Drosophila might be due to differences in sex determination system, reproductive and mating biology, and/or the prevalence of sexual interactions such as sexual selection.

Chromosome Mapping↗

High-resolution fine mapping and fluorescence in situ hybridization analysis of sun, a locus controlling tomato fruit shape, reveals a region of the tomato genome prone to DNA rearrangements.

The locus sun on the short arm of tomato chromosome 7 controls morphology of the fruit. Alleles from wild relatives impart a round shape, while alleles from certain cultivated varieties impart an oval shape typical of roma-type tomatoes. We fine mapped the locus in two populations and investigated the genome organization of the region spanning and flanking sun. The first high-resolution genetic map of the sun locus was constructed using a nearly isogenic F(2) population derived from a cross between Lycopersicon pennellii introgression line IL7-4 and L. esculentum cv Sun1642. The mapping combined with results from pachytene FISH experiments demonstrated that the top of chromosome 7 is inverted in L. pennellii accession LA716. sun was located close to the chromosomal breakpoint and within the inversion, thereby precluding map-based cloning of the gene using this population. The fruit-shape locus was subsequently fine mapped in a population derived from a cross between L. esculentum Sun1642 and L. pimpinellifolium LA1589. Chromosome walking using clones identified from several large genomic insert libraries resulted in two noncontiguous contigs flanking sun. Fiber-FISH analysis showed that distance between the two contigs measured 68 kb in L. esculentum Sun1642 and 38 kb in L. pimpinellifolium LA1589, respectively. The sun locus mapped between the two contigs, suggesting that allelic variation at this locus may be due to an insertion/deletion event. The results demonstrate that sun is located in a highly dynamic region of the tomato genome.

Chromosome Mapping↗