Genetic determination of ploidy level in Xanthophyllomyces dendrorhous

Xanthophyllomyces dendrorhous (formely Phaffia rhodozyma) is a basidiomycetous yeast-like fungus that produces carotenoids useful for the food industry. Recently, its sexual cycle was reported but little is known about its genetic constitution. To inquire into the ploidy state of X. dendrorhous, biased mutant spectrum, genetic complementation and mitotic recombination analysis were used. A wild-type strain was subjected to N-methyl-N′-nitro-N-nitrosoguanidine mutagenic treatment. Auxotrophic and carotene mutants were forced to revert to the wild-type phenotype. Pigment producing and prototroph revertants behaved as diploid except for adenine less mutants. These results are in agreement with the limited spectrum of auxotrophs obtained in this strain for the ADE1 locus. To analyze the genetic characteristic of the adenine genetic marker of X. dendrorhous, protoplast fusion experiments with several adenine less mutants were performed. The experiments presented in this work suggest that the ATCC 2430 (UDC 67-385) strain of X. dendrorhous is diploid and a heterozygous constitution is proposed for the ADE1 locus. This revised version was published online in June 2006 with corrections to the Cover Date.     

An improvedAgrobacterium-mediated transformation protocol for recalcitrant elite indica rice cultivars

We report here a high-efficiency transformation protocol for recalcitrant indica rice cultivars IR64 and IR72 with the selectable marker genehph and thegusA reporter gene. Factors that favor high-efficiency transformation were found to be use of 2-month-old mature seed-derived embryogenic calli, maltose as a source of carbon, a higher concentration of 2,4-dichlorophenoxyacetic acid, and both phytagel and agar as gelling agents. The putative transgenic (T₀) plants were analyzed for integration of the transgene through polymerase chain reaction and Southern blotting analyses. Various factors thought to be responsible for increased transformation efficiency are discussed.     

Flow cytometric determination of DNA ploidy level in nuclei isolated from paraffin-embedded tissue

Nuclei, isolated from paraffin-embedded tissue, were stained with propidium iodide (PI) and found suitable for DNA analysis by flow cytometry (FCM). DNA-derived fluorescence intensity, however, was always decreased and had a much higher intersample variability as compared to results obtained with fresh material. Using chicken red blood cells (CRBC) as a model system, we found the lower fluorescence intensity to be due to the formalin fixation step in tissue processing. The intersample variability was found to be at least partly caused by variations in the duration of fixation. Overnight trypsinization improved the fluorescence intensity but did not reduce the intersample variability. Under all conditions tested PI binding to CRBC appeared to be saturable. Since fresh diploid or red blood cells could not be used to standardize DNA histograms, an alternative approach was developed in which nuclei from paraffin-embedded normal and tumor tissue of the same specimen were mixed. With this method DNA indices (DI) of 24 colorectal cancers were found to be closely correlated (r = 0.9877, P less than 0.001) with DI obtained with fresh tumor tissue from the same patients. The correlation of the percentages of S-phase nuclei between paraffin-extracted and fresh samples (r = 0.5875, P less than 0.05) was as high as could be expected, taking sampling differences into account. This method is an important tool for the retrospective analysis of FCM-derived DNA parameters in relation to diagnosis and prognosis of neoplasms.     

Nuclear DNA content of Vitis vinifera cultivars and ploidy level analyses of somatic embryo-derived plants obtained from anther culture

Flow cytometry was employed to determine the ploidy level of Vitis vinifera L. somatic embryo-derived plants obtained from anther culture. Only one among the 41 analysed plants (2.4%) presented somaclonal variation (tetraploidy); the other plants were diploid. No significant differences (P≤0.05) were detected between diploid and parental field plants. No haploid or aneuploid plants were observed. The nuclear DNA content of nine V. vinifera cultivars was also estimated using flow cytometry. A non-significant variation was found among the cultivars, with DNA content ranging from 1.17 pg/2C (cv. ‘Tinta Barroca’ and ‘Viosinho’) to 1.26 pg/2C (cv. ‘Cabernet Sauvignon’). These results and previous studies on other Vitis species suggest that Vitis genome is stable with regard to nuclear DNA content.     

Easy determination of ploidy level in Arabidopsis thaliana plants by means of pollen size measurement

Summary Cytogenetic examination of transgenic Arabidopsis thaliana (L.) Heynh. plants obtained by Agrobacterium-mediated gene transfer to cotyledon- and root-explants or by direct gene transfer into protoplasts revealed a high percentage of tetraploid or aneuploid transformants. Depending on the transformation procedure used, 13% (root explant transformation), 33% (cotyledon explant transformation), or 38% (direct gene transfer) of the transformants showed aberrant ploidy levels. A good correlation between the ploidy level of a plant and the size of its pollen grains was observed. This allows quick and simple testing of the ploidy level of transgenic Arabidopsis plants.Abbreviations AM Arabidopsis medium - ANOVA analysis of variance - DAPI 4,6-Diamidino-2-phenylindole - PEG polyethyleneglycol     

Genome-wide association study for soybean cyst nematode resistance in Chinese elite soybean cultivars

Soybean cyst nematode (SCN) (Heterodera glycines Ichinohe) is a highly recalcitrant endoparasite of soybean roots, causing more yield loss than any other pest. To identify quantitative trait loci (QTL) controlling resistance to SCN (HG type 2.5.7, race 1), a genome-wide association study (GWAS) was performed. The association panel, consisting of 120 Chinese soybean cultivars, was genotyped with 7189 single nucleotide polymorphism (SNPs). A total of 6204 SNPs with minor allele frequency >0.05 were used to estimate linkage disequilibrium (LD) and population structure. The mean level of LD measured by r ² declined very rapidly to half its maximum value (0.51) at 220 kb. The overall population structure was approximately coincident with geographic origin. The GWAS results identified 13 SNPs in 7 different genomic regions significantly associated with SCN resistance. Of these, three SNPs were localized in previously mapped QTL intervals, including rhg1 and Rhg4. The GWAS results also detected 10 SNPs in 5 different genomic regions associated with SCN resistance. The identified loci explained an average of 95.5% of the phenotypic variance. The proportion of phenotypic variance was due to additive genetic variance of the validated SNPs. The present study identified multiple new loci and refined chromosomal regions of known loci associated with SCN resistance. The loci and trait-associated SNPs identified in this study can be used for developing soybean cultivars with durable resistance against SCN.     

Characterisation of Chinese elite cultivars and genetic resources of chestnut by AFLP

We selected a informative set of twelve amplified fragment length polymorphism (AFLP) primer pairs suitable for evaluation of Chinese chestnut (Castanea mollissima Blume) genotypes. Cluster analysis based on 198 polymorphic AFLP amplified by these 12 primer pairs clearly divided investigated genotypes according to their place of origin. We showed, that genetic basis of modern genotypes is narrow also in the case of this species.     

Noninvasive determination of genome size and ploidy level in fishes by flow cytometry: detection of triploid Poecilia formosa

BACKGROUND: In order to understand the evolutionary significance of single triploids among the mostly diploid Poecilia formosa we have developed a simple, noninvasive technique for DNA content and ploidy determination. METHODS: From dorsal fin clips of 14 different fish species single cell suspensions were obtained by chopping the material in 2.1% citric acid/0.5% Tween20, passing it through a 0. 6-gauge needle and incubating it for 20 min at room temperature (RT) with gentle agitation. After overnight fixation in 70% ethanol, the cells were treated with 1ml 0.5% pepsin/0.1 M HCl for 15 min at RT before adding DAPI to a final volume of 2 ml. The cells were stained for 1-3 h and then analyzed by flow cytometry. RESULTS: We obtained good measurements with CVs ranging from 1.23% to 3.36%. The poeciliid species measured contain from 1.6 to 2.0 pg/nucleus, Oryzias latipes (Medaka) exhibits a nuclear DNA content of 2.2 pg, Danio rerio (zebrafish) 4.6 pg, Tetraodon fluviatilis (freshwater fugu) 0.70 pg. All values except zebrafish are in good agreement with the literature. CONCLUSIONS: The identification of living specimens of different ploidy for breeding experiments, behavioral studies and tissue transplantations is now made possible. With slight modifications the method can be extended to a field technique, providing therefore a useful tool for a variety of researchers.     

Molecular basis for substrate discrimination by glycine transporters

Glycine is an inhibitory neurotransmitter in the spinal cord and brain stem, where it acts on strychnine-sensitive glycine receptors, and is also an excitatory neurotransmitter throughout the brain and spinal cord, where it acts on the N-methyl-d-aspartate family of receptors. There are two Na(+)/Cl(-)-dependent glycine transporters, GLYT1 and GLYT2, which control extracellular glycine concentrations and these transporters show differences in substrate selectivity and blocker sensitivity. A bacterial Na(+)-dependent leucine transporter (LeuT(Aa)) has recently been crystallized and its structure determined. When the amino acid residues within the leucine binding site of LeuT(Aa) are aligned with residues of the two glycine transporters there are a number of identical residues and also some key differences. In this report, we demonstrate that the LeuT(Aa) structure represents a good working model of the Na(+)/Cl(-)-dependent neurotransmitters and that differences in substrate selectivity can be attributed to a single difference of a glycine residue in transmembrane domain 6 of GLYT1 for a serine residue at the corresponding position of GLYT2.     

High-throughput cell analysis based protocol for ploidy determination in anther-derived rice callus

Plant Cell, Tissue and Organ Culture (PCTOC) - The study provides high-throughput protocol for ploidy determination which may accelerate the production of double haploids in rice. Advancements in...     

NORs and counterstain-enhanced fluorescence studies in Cyprinidae of different ploidy level

The ribosomal RNA gene expression in the genomes of evolutionary diploid (Scardinius erythrophthalmus, Leucaspius delineatus, Tinca tinca) and polyploid species (Cyprinus carpio, Carassius carassius, Carassius auratus gibelio, Carassius auratus auratus) of Cyprinidae has been investigated by means of a silver nitrate technique. The diploid species investigated exhibited only one pair of chromosomes with nucleolus organizers (NOR). Higher numbers of rRNA-expressing chromosomal sites in several evolutionary polyploid species (Carassins) gave evidence against a complete functional diploidization, at least with regard to the NOR bearing chromosomes in these species. The NORs displayed a heterochromatic brilliant chromomycin A₃ fluorescence. No distamycin-A/DAPI-bright heterochromatic blocks were detected in the genomes of the Cyprinidae.     

Correlation between nuclear DNA values and differing optimal ploidy levels inNarcissus,Hyacinthus andTulipa cultivars

4C nuclear DNA amounts were determined in 16 large decorative cultivars ofNarcissus (Amaryllidaceae), 13 ofHyacinthus (Hyacinthaceae) and 12 ofTulipa (Liliaceae) at different levels of ploidy. Within each genus, nuclear DNA amounts and ploidy levels are positively correlated, with no DNA loss in polyploids.Based on wide surveys of chromosome numbers, the maximum numbers of cultivars, interpreted as the optimum levels of selective success or horticultural fitness, were found to be at the tetraploid level inNarcissus (2n=4x=28), the triploid inHyacinthus (2n=3x=24) and the diploid inTulipa (2n=2x=24). All these ploidy optima were shown to correspond to a small range of nuclear DNA amounts (4C=96-139 pg), which could suggest the existence of a single DNA value optimal for the three biologically similar but unrelated genera. In each case the optimum is at an equilibrium reached between enhanced size and other morphological characteristics on one hand and reduced growth rate on the other, both resulting from increase in ploidy and nuclear DNA amounts.     

Correlation between distyly and ploidy level in Damnacanthus (Rubiaceae)

Somatic chromosomes were observed in 661 individuals of 14 taxa, nine species and five varieties, of Damnacanthus (Rubiaceae). Chromosome numbers are reported for the first time for 13 taxa. Diploid (2n = 22) and tetraploid (2n = 44) counts were obtained. Distyly is reported for the first time for four species, D. angustifolius, D. henryi, D. labordei, and D. officinarum. A strong correlation exists between chromosome number and occurrence of distyly. Regardless of taxa in Damnacanthus, distylous populations are diploid, and monomorphic populations are tetraploid. Flowers of the monomorphic populations observed have a long style and short stamens with few exceptions. Polyploidization may have caused the breakdown of distylous to monomorphic flowers. In D. indicus, leaves from the tetraploid populations tend to be larger than those from the diploid populations. Populations of tetraploid D. indicus were distributed in more northern areas than those of the diploid. Three types of sympatric distribution were found for the varieties of D. indicus in Japan: diploid and tetraploid, two diploids, and two tetraploids. Based on the present chromosome number study, the taxonomy of the varieties of D. indicus should be revised.     

Generating and maintaining diversity at the elite level in crop breeding

Most breeding programs develop elite genotypes that are well adapted to the normal range of environmental conditions in the target production region. These elite lines have similar essential alleles for desirable end use characteristics, agronomics, disease resistance, and adaptation in the target region. The genetic makeup of these elite lines is complex. Intermating among the elite lines will often produce new variability through recombination with minimal risk of introducing new undesirable features, and is the source of most new cultivars. Eventually, this variation will be exhausted and new alleles must be introduced into the elite breeding population. Introducing desirable alleles from exotic germplasm may "pollute" the elite gene pool with undesirable alleles. Exotic germplasm may also disrupt essential allele combinations for adaptation, quality, and agronomic performance. New desirable alleles from exotic germplasm can be introgressed into an elite population in a systematic way through limited backcrossing with a minimal disturbance to the finely tuned elite background. Combining recurrent selection within elite germplasm with a systematic introgression from exotic germplasm in the recurrent introgressive population enrichment (RIPE) system has created an open-ended, continually improving, and sustainable elite population breeding system, which is simple, effective, and a regular source of new cultivars.     

Pyramiding transgenic resistance in elite indica rice cultivars against the sheath blight and bacterial blight

Elite indica rice cultivars were cotransformed with genes expressing a rice chitinase (chi11) and a thaumatin-like protein (tlp) conferring resistance to fungal pathogens and a serine-threonine kinase (Xa21) conferring bacterial blight resistance, through particle bombardment, with a view to pyramiding sheath blight and bacterial blight resistance. Molecular analyses of putative transgenic lines by polymerase chain reaction, Southern Blot hybridization, and Western Blotting revealed stable integration and expression of the transgenes in a few independent transgenic lines. Progeny analyses showed the stable inheritance of transgenes to their progeny. Coexpression of chitinase and thaumatin-like protein in the progenies of a transgenic Pusa Basmati1 line revealed an enhanced resistance to the sheath blight pathogen, Rhizoctonia solani, as compared to that in the lines expressing the individual genes. A transgenic Pusa Basmati1 line pyramided with chi11, tlp, and Xa21 showed an enhanced resistance to both sheath blight and bacterial blight. S. Maruthasalam and K. Kalpana have contributed to this article equally.     

A simple method for the calculation of microsatellite genotype distances irrespective of ploidy level

Abstract Microsatellites are powerful molecular markers, used commonly to estimate intraspecific genetic distances. With the exception of band sharing similarity index, available distance measures were developed specifically for diploid organisms and are unsuited for comparisons of polyploids. Here, we present a simple method for calculation of microsatellite genotype distances, which takes into account mutation processes and permits comparison of individuals with different ploidy levels. This method should provide a valuable tool for intraspecific analyses of polyploid organisms, which are widespread among plants and some animal taxa. An illustration is given using data from the planarian flatworm Schmidtea polychroa (Platyhelminthes).