Acid phosphatase and leucine aminopeptidase isozymes as biochemical markers of homogeneity in oil seed rape androgenetic lines

Extracts of cotyledons of Brassica napus plants (seed progenies of doubled haploid plants) were separated by electrophoresis on polyacrylamide gels and stained for acid phosphatase (ACP-E.C. 3.1.3.2.) and leucine aminopeptidase (LAP-E.C. 3.4.11.1.) enzymes to investigate the possibility of utilising isozymes as markers of homogeneity (purity) of plant populations. One zone of activity for acid phosphatase and two zones of activity for leucine aminopeptidase were identified on gels, some variation in isozyme patterns occurred in several androgenetic lines. This method is appropriate and consistent for testing the homogeneity of breeding lines-progenies of double haploid (D.H.) plants.     

Association of gene-linked SSR markers to seed glucosinolate content in oilseed rape (Brassica napus ssp. napus)

Breeding of oilseed rape (Brassica napus ssp. napus) has evoked a strong bottleneck selection towards double-low (00) seed quality with zero erucic acid and low seed glucosinolate content. The resulting reduction of genetic variability in elite 00-quality oilseed rape is particularly relevant with regard to the development of genetically diverse heterotic pools for hybrid breeding. In contrast, B. napus genotypes containing high levels of erucic acid and seed glucosinolates (++ quality) represent a comparatively genetically divergent source of germplasm. Seed glucosinolate content is a complex quantitative trait, however, meaning that the introgression of novel germplasm from this gene pool requires recurrent backcrossing to avoid linkage drag for high glucosinolate content. Molecular markers for key low-glucosinolate alleles could potentially improve the selection process. The aim of this study was to identify potentially gene-linked markers for important seed glucosinolate loci via structure-based allele-trait association studies in genetically diverse B. napus genotypes. The analyses included a set of new simple-sequence repeat (SSR) markers whose orthologs in Arabidopsis thaliana are physically closely linked to promising candidate genes for glucosinolate biosynthesis. We found evidence that four genes involved in the biosynthesis of indole, aliphatic and aromatic glucosinolates might be associated with known quantitative trait loci for total seed glucosinolate content in B. napus. Markers linked to homoeologous loci of these genes in the paleopolyploid B. napus genome were found to be associated with a significant effect on the seed glucosinolate content. This example shows the potential of Arabidopsis-Brassica comparative genome analysis for synteny-based identification of gene-linked SSR markers that can potentially be used in marker-assisted selection for an important trait in oilseed rape. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Biochemical markers associated with two Mv chromosomes from Aegilops ventricosa in wheat-Aegilops addition lines

Summary The distribution of three biochemical markers, U-1, CM-4 and Aph_v-a, -b, among wheat-Aegilops addition lines carrying M^v chromosomes from Aegilops ventricosa (genomes D^vM^v) has been investigated. Addition lines which had been previously grouped together on the basis of common non-biochemical characters carried marker U-1, a protein component from the 2M urea extract. The added chromosome, in the appropriate genetic background, seems to confer a high level of resistance to the eyespot disease, caused by the fungus Cercosporella herpotrichoides. The other two markers were concomitantly associated with another similarly formed group of addition lines. Both CM-4, a protein component from the chloroform:methanol extract, and Aph_v-a, -b, alkaline phosphate isozymes, have been previously shown to be associated with homoeologous chromosome group 4, which suggests that the added chromosome in the second group of addition lines is 4M^v.     

Improvement of regeneration ability of androgenetic embryos by early anther transfer in maize

Six media compositions and different transfer times were compared in order to improve the quality of microspore derived structures and their regeneration capacity. The production of regenerated plantlets was increased by a factor of 10 by an early transfer of anthers to medium with low sugar concentration and the use of kinetin-like growth regulators. The best transfer date was 3 weeks after initial plating, onto medium containing 25 gl^–1 sucrose and 1 mg l^–1 kinetin. This study demonstrates that anther transfer at an early developmental stage, before macroscopic appearance of embryo-like structures, improves embryo quality and further regeneration. This production of androgenetic structures showing normal morphological features is the first step to obtain an improvement of androgenetic plant regeneration yield.     

A comparison of androgenetic doubled-haploid,and single seed descent lines in Triticale

Summary Sixty single seed descent (SSD) lines and about 25 anther-derived doubled-haploid (DH) lines were obtained from two triticale crosses. The frequency distributions of 10 quantitative agronomic traits were compared using parametric and non-parametric tests. A multivariate discriminant analysis was subsequently carried out. Gliadin patterns obtained from each line by polyacrylamide gel electrophoresis were used to calculate intra- and inter-population diversities from relative dissimilarity indices. It was found that DH and SSD lines show significant differences in frequency distributions of 1000 grain weight in both crosses, of heading date for one cross, and of lodging susceptibility for the other cross. The results of intra- and inter-population gliadin diversity indicate that although the SSD method theoretically provides more opportunity for recombination to occur than the DH method, it did not produce a greater range of recombinants. Since there is no significant difference between SSD- and DH-line distributions for grain yield, anther culture appears to be an efficient method for producing high yielding homozygous lines from F₁ hybrids of triticale in a relatively short time.     

Developmental specificity and evolution of the acid phosphatase isozymes of Triticum aestivum and its progenitor species

The tissue and developmental specificities of the acid phosphatase (ACPH) isozymes of Triticum aestivum and its progenitor species T. turgidum and T. tauschii have been determined and compared using the zymogram technique. Tissue and/or developmental variation in relative staining intensity, suggestive of variation in the quantity of active enzyme present, was observed for each of the seven major isozymes expressed. Isozymes homologous to each of the major isozymes of the hexaploid were detected in one or the other of the progenitor species. No difference in the pattern of developmental or tissue specificity was observed between the species for any isozyme. However, ACPH-4, encoded by Acph4, a structural gene linked to chromosome 4A, differs in electrophoretic mobility between T. aestivum and T. turgidum, indicating that divergence has occurred between these species at the Acph4 locus since the origin of the hexaploid. The molecular weight of each of five ACPH isozymes of the hexaploid was determined to be approximately 58,000. This finding, plus the results of the developmental study and the earlier demonstration that the structural genes for six isozymes (including four of those whose molecular weight was determined) are linked to homoeologous chromosomes, provides evidence in support of the suggestion that the ACPH structural genes of hexaploid wheat are homoeologously related.Technical article No. 12233 of the Texas Agricultural Experiment Station. Adapted from a dissertation submitted to the Graduate College, Texas A&M University, by M. A. T. in partial fulfillment of the requirements for the Ph.D. in genetics.     

Integration of CAPS markers into the RFLP map generated using recombinant inbred lines of Arabidopsis thaliana

Konieczny and Ausubel have described a technique whereby Arabidopsis thaliana loci can be rapidly mapped to one of the ten chromosome arms using a small number of F2 progeny from crosses between the ecotypes Landsberg erecta and Columbia. The technique involves the use of 18 co-dominant, cleaved amplified polymorphic sequence (CAPS) markers which are evenly distributed throughout the Arabidopsis genome. We have mapped these 18 markers using recombinant inbred (RI) lines generated in our laboratory. These data enable a better integration of loci mapped relative to the CAPS markers into the restriction fragment length polymorphism (RFLP) map generated using Arabidopsis RI lines.     

Evaluation of RFLP and RAPD markers in a comparison of Brassica napus breeding lines

RFLP and RAPD markers were evaluated and compared for their ability to determine genetic relationships in a set of three B. napus breeding lines. Using a total of 50 RFLP and 92 RAPD markers, the relatedness between the lines was determined. In total, the RFLP and the RAPD analysis revealed more than 500 and 400 bands, respectively. The relative frequencies of loci with allele differences were estimated from the band data. The RFLP and RAPD marker sets detected very similar relationships among the three lines, consistent with known pedigree data. Bootstrap analyses showed that the use of approximately 30 probes or primers would have been sufficient to achieve these relationships. This indicates that RAPD markers have the same resolving power as RFLP markers when used on exactly the same set of B. napus genotypes. Since RAPD markers are easier and quicker to use, these markers may be preferred in applications where the relationships between closely-related breeding lines are of interest. The use of RAPD markers in fingerprinting applications may, however, not be warranted, and this is discussed in relation to the reliability of RAPD markers.     

Chromosomal location of isozyme markers in wheat-barley addition lines

Summary The peroxidase (CPX, PER), -amylase (-AMY), acid and alkaline phosphatase (PHE, PHS) and esterase (EST) zymogram phenotypes of Chinese Spring wheat, Betzes barley and a number of presumptive Betzes chromosome additions to Chinese Spring were determined. It was found that five disomic chromosome addition lines could be distinguished from one another and from the other two possible lines on the basis of the zymogram phenotypes of these isozymes. The structural genes Cpxe-H1 and Cpxe-H2 were located in Betzes chromosome 1, the Perl-H5 and Perl-H6 in chromosome 2, the -Amy-H2 and -Amy-H3 in chromosome 7, the Phs-H5 and Phs-H4 in chromosomes 1 and 3 respectively, the Phe-H2, Phe-H3 and Phe-H4 in chromosome 1, the Phe-H1 in chromosome 3, the Ests-H4, Este-H2 and Ests-H6, Este-H8 in chromosomes 1 and 3 respectively and the Estl-H10 and Estl-H2 structural genes were related to chromosomes 3 and 6 respectively. These gene locations provide evidence of homoeology between Betzes chromosomes 1, 2, 3, 6 and 7 and the rye chromosomes 7, 2, 3, 6 and 5, respectively, and also between Betzes chromosomes 1, 2, 3, 6 and 7 and the Chinese Spring homoeologous groups 7, 2, 3, 6 and 5, respectively.     

Detection of androclonal variation in anther-cultured rice lines using rapds

Summary Random amplified polymorphic DNA analysis was used to determine the occurrence and extent of variation in rice (Oryza sativa L.) plants regenerated from anther culture. Androclonal variation in morphologically uniform progenies was detected using 40 10-mer oligonueleotide arbitrary primers. Among 27 plants from nine anther culture-derived lines, variation was detected in three plants from two lines by two primers, namely UBC 160 and UBC 209. Primer UBC 160 amplified a polymorphic band on one of the three progenies from DH-34, while UBC 209 detected polymorphisms on two out of three progenies from line DH-58. Apart from these, the amplification produets were monomorphic across all the regenerants from anther culture-derived plants. Out of 40 tested primers, no difference in the banding pattern was observed in three seed-derived plants. The significance of possible androclonal variation at the DNA level in rice doubled haploid breeding and genetic mapping is discussed.     

Genetic analyses of agronomic and seed quality traits of synthetic oilseedBrassica napus produced from interspecific hybridization ofB. campestris andB. oleracea

The heritability, the number of segregating genes and the type of gene interaction of nine agronomic traits were analysed based on F₂ populations of synthetic oilseedBrassica napus produced from interspecific hybridization ofB. campestris andB. oleracea through ovary culture. The nine traits—plant height, stem width, number of branches, length of main raceme, number of pods per plant, number of seeds per pod, length of pod, seed weight per plant and 1000-seed weight—had heritabilities of 0.927, 0.215, 0.172, 0.381, 0.360, 0.972, 0.952, 0.516 and 0.987 respectively, while the mean numbers of controlling genes for these characters were 7.4, 10.4, 9.9, 12.9, 11.5, 21.7, 20.5, 19.8 and 6.4 respectively. According to estimated coefficients of skewness and kurtosis of the traits tested, no significant gene interaction was found for plant height, stem width, number of branches, length of main raceme, number of seeds per pod and 1000-seed weight. Seed yield per plant is an important target for oilseed production. In partial correlation analysis, number of pods per plant, number of seeds per pod and 1000-seed weight were positively correlated with seed yield per plant. On the other hand, length of pod was negatively correlated (r = -0.69) with seed yield per plant. Other agronomic characters had no significant correlation to seed yield per plant. In this experiment, the linear regressions of seed yield per plant and other agronomic traits were also analysed. The linear regression equation wasy = 0.074x₈ + 1.819x₉ + 6.72x₁₂ -60.78 (R ² = 0.993), wherex ₈, x₉ and x₁₂ represent number of pods per plant, number of seeds per pod and 1000-seed weight respectively. The experiment also showed that erucic acid and oil contents of seeds from F₂ plants were lower than those of their maternal parents. However, glucosinolate content was higher than that of the maternal plants. As for protein content, similar results were found in the F₂ plants and their maternal parents. It was shown that the four quality traits, i.e. erucic acid, glucosinolate, oil content, and protein content, had heritability values of 0.614, 0.405, 0.153 and 0.680 respectively.     

Inheritance of polymorphic markers generated by DNA amplification fingerprinting and their use as genetic markers in soybean

DNA amplification fingerprinting (DAF) using a high primer-to-template ratio and single, very short arbitrary primers, was used to generate amplified fragment length polymorphic markers (AFLP) in soybean (Glycine max (L.) Merr.). The inheritance of AFLPs was studied using a cross between the ancestral Glycine soja PI468.397 and Glycine max (L.) Merr. line nts382, F1 and F2 progeny. The amplification reaction was carried out with soybean genomic DNA and 8 base long oligounucleotide primers. Silver-stained 5% polyacrylamide gels containing 7 M urea detected from 11 to 28 DAF products with primers of varying GC content (ranging from 50 to 100% GC). Depending on their intensity, AFLPs were classified into three classes. DAF profiles were reproducible for different DNA extractions and gels. Forty AFLPs were detected by 26 primers when comparing G. soja and G. max. Most AFLPs were inherited as dominant Mendelian markers in F1 and F2 populations. However, abnormal inheritance occured with about 25% of polymorphisms. One marker was inherited as a maternal marker, presumably originating from organelle DNA while another showed apparent paternal inheritance. To confirm the nuclear origin and utility of dominant Mendelian markers, three DAF polymorphisms were mapped using a F11 mapping population of recombinant inbred lines from soybean cultivars Minsoy × Noir 1. The study showed that DAF-generated polymorphic markers occur frequently and reliably, that they are inherited as Mendelian dominant loci and that they can be used in genome mapping.     

A biochemical genetic study of alcohol dehydrogenase isozymes of the medfly,Ceratitis capitata wied

A concerted effort is under way to analyze, at the genetic, biochemical, and molecular level, theAdh gene system in the medflyCeratitis capitata, an important agricultural pest. The isoelectric focusing (IEF) pattern of alcohol dehydrogenase (ADH) of the medfly demonstrates the presence of two well-differentiated, genetically independent dimeric proteins, called ADH-1 and ADH-2. These proteins do not exhibit interlocus heterodimeric isozymes, and the genes are not controlled coordinately during development,Adh ₁ andAdh ₂ being expressed mainly in muscle or in fat body and ovary, respectively. From the intensity of the IEF isozyme patterns, primary alcohols are judged to be better substrates than secondary alcohols, in contrast withDrosophila melanogaster ADH, and ethanol is probably the most efficient substrate for both sets of isozymes. The isoelectric points of ADH-1 (pI=5.4) and ADH-2 (pI=8.6) are different fromD. melanogaster ADH (pI=7.6), but the medfly ADH-1 has a native molecular weight (approx. 58 kD) close to that ofD. melanogaster. A population survey of samples both from laboratory strains and from wild geographically different populations showed that theAdh ₁ locus is more polymorphic thanAdh ₂. The most variable populations are from Africa, the supposed source area of the species. Further, a case of selection at theAdh ₁ locus under laboratory conditions is reported. The hypothesis ofAdh gene duplication and the degree of similarity between medfly andDrosophila ADH are also discussed. This research was supported mainly by National Research Council of Italy, Special Project RAISA, Sub-project No. 2, Paper No. 342. Grants from the International Atomic Energy Agency, Vienna, Austria, from European Communities Commission, Second R & D Programme, “Science and Technology for Development,” and from the Italian Ministry of University and Scientific Research and Technology (“Funds 40%”) also supported this work. This paper was written when the senior author was on leave of absence at the IMBB, Crete, Greece; he was financially supported by an ECC Senior Fellowship.     

Identification of broccoli and cauliflower cultivars with RAPD markers

Summary RAPD (Random Amplified Polymorphic DNA) markers generated by 4 arbitrary 10-mer primers, discriminated 14 broccoli and 12 cauliflower cultivars (Brassica oleracea L.) by banding profiles. The size of the amplified DNA fragments ranged from 300 to 2600 base pairs. Twenty-eight percent of the markers were fixed in both broccoli and cauliflower, whereas 12.5% were specific to either crop. The rest were polymorphic in either or both crops. The markers generated by two and three primers were sufficient to distinguish each of the broccoli and cauliflower cultivars, respectively. The average difference in markers was 14.5 between broccoli and cauliflower markers, 5.8 between two broccoli cultivars and 7.9 between two cauliflower cultivars. Larger differences for each crop were found between cultivars from different seed companies than within the same company. RAPD markers provide a quick and reliable alternative to identify broccoli and cauliflower cultivars.     

Function of the signal peptide and N- and C-terminal propeptides in the leucine aminopeptidase from Aeromonas proteolytica

The leucine aminopeptidase from Aeromonas proteolytica (also known as Vibrio proteolyticus) (AAP) is a metalloenzyme with broad substrate specificity. The open reading frame (ORF) for AAP encodes a 54 kDa enzyme, however, the extracellular enzyme has a molecular weight of 43 kDa. This form of AAP is further processed to a mature, thermostable 32 kDa form but the exact nature of this process is unknown. Over-expression of different forms of AAP in Escherichia coli (with AAP's native leader sequence, with and without the N- and/or C-terminal propeptides, and as fusion protein) has allowed a model for the processing of wild-type AAP to be proposed. The role of the A. proteolytica signal peptide in protein secretion as well as comparison to other known signal peptides reveals a close resemblance of the A. proteolytica signal peptide to the outer membrane protein (OmpA) signal peptide. Over-expression of the full 54 kDa AAP enzyme provides an enzyme that is significantly less active, due to a cooperative inhibitory interaction between both propeptides. Over-expression of AAP lacking its C-terminal propeptide provided an enzyme with an identical kcat value to wild-type AAP but exhibited a larger Km value, suggesting competitive inhibition of AAP by the N-terminal propeptide (Ki approximately 0.13 nM). The recombinant 32 kDa form of AAP was characterized by kinetic and spectroscopic methods and was shown to be identical to mature, wild-type AAP. Therefore, the ease of purification and processing of rAAP along with the fact that large quantities can be obtained now allow new detailed mechanistic studies to be performed on AAP through site-directed mutagenesis.     

Loss of genetic variation in laboratory colonies of Phormia regina

Genetic variation of laboratory and wild Phormia regina was examined by gel electrophoresis of enzymes. The two old laboratory stocks studied possessed much less genetic variation than wild flies.

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Zusammenfassung Die genetische Variabilität in Kulturen von Laboratoriumstämmen oder wilden Fliegen von Phormia regina wurde mittels Gel Elektrophoresis von 6 Enzymen studiert. Vier verschiedene Stämme wurden untersucht: wilde Fliegen, die F₁ Nachkommen der wilden Fliegen, ein rot-äugiger und ein weiss-äugiger Laboratoriumstamm. Die wilden Fliegen waren genetisch sehr variabel (mittlere Heterozygosität = 0.157). Die geringste Variabilität war in den weiss-äugigen Fliegen (mittlere Heterozygosität = 0.009). Die Laboratoriumstämme sind genetisch dem wilden Fliegenmaterial nicht ähnlich.

     

A full set of monosomic addition lines in Beta vulgaris from Beta webbiana: morphology and isozyme markers

Summary Nine different monosomic additions in Beta vulgaris from Beta webbiana were characterized through morphological characters and isozyme markers. The effect of the alien chromosome on the morphology of the recipient species is chromosome specific, and nine morphotypes could be distinguished. The added chromosome caused a growth reduction in the recipient plants. Eleven isozyme systems were used as marker systems. A 6PGDH band was found as a marker for chromosome 7, which contains a resistance gene for the beet cyst nematode in monosomic additions from Beta procumbens and Beta webbiana. A difference in the 6PGDH zymogram pattern between the two species with respect to this chromosome has been noted.     

Comparison of single seed descent and anther culture-derived lines of three single crosses of rice

Summary Distribution parameters (mean, variance, skewness and kurtosis) of 12 quantitative traits were evaluated for inbred lines generated through single seed descent (SSD) and anther culture (AC) of two japonica x japonica hybrids and one japonica x indica hybrid of rice. For most of the traits the data were normally distributed, and the means and variances were found to be identical for SSD- and AC-derived lines of the given hybrids. However, for some other traits, differences between the two population types were observed, mainly in the lines derived from the intra-japonica crosses. A tentative explanation for these differences is given. Nevertheless, our results suggest that SSD and AC are equally effective breeding techniques for producing agronomically useful lines of rice.     

Comparative analysis of genetic relationship and diagnostic markers of several taxa of Guizotia Cass. (Asteraceae) as revealed by AFLPs and RAPDs

Amplified fragment length polymorphism (AFLP) and random amplified polymorphic DNA (RAPD) were employed to examine the genetic relationship between Guizotia taxa, to suggest the taxonomic status of some of these taxa, and to identify their diagnostic markers. Results from AFLPs and RAPDs share some features in common, both revealing G. scabra ssp. schimperi as the most closely related taxon to G. abyssinica, and indicating that G. arborescens and G. zavattarii are the most divergent taxa. Most of the diagnostic markers revealed in this study were specific to G. arborescens and G. zavattarii. Our analysis suggests that G. scabra ssp. scabra, G. scabra ssp. schimperi, Chelelu and Ketcha are separate species. In this study, AFLP was found to be superior to RAPD in detecting genetic variation, in internal consistency of the data and in the fitness of its clusters to genetic similarity data. AFLPs revealed genetic relationship between Guizotia taxa that is more inline with the cytogenetic and hybridization studies than that revealed by RAPDs.     

Genetics,subcellular localization,and molecular characterization of 6-phosphogluconate dehydrogenase isozymes in tomato

Three independent genes are responsible for 6-phosphogluconate dehydrogenase (6PGDH) activity in tomato. 6Pgdh-2, located on chromosome 12, codes for subunits of an active dimer which is restricted to the plastids. 6Pgdh-1, chromosome 4, and 6Pgdh-3, chromosome 5, code for subunits which form three dimers—two homodimers and an intergenic heterodimer. The latter three isozymes are found in the cytosol. 6Pgdh-1, 6Pgdh-2, and 6Pgdh-3 code for subunits with estimated molecular weights of 49,500, 50,500, and 51,200, respectively. The intergenic heterodimer encoded by 6Pgdh-1 and 6Pgdh-3 is thus composed of subunits that differ in length by approximately 15 amino acid residues. Divergence in the length and primary subunit structure may account for the lower thermal stability of the intergenic heterodimer compared with the corresponding homodimers. A limited survey of other solanaceous plant species suggests that the duplication of cytosolic 6PGDH-coding genes found in tomato may be widespread in the family.This is journal article 1145, Agricultural Experiment Station, New Mexico State University. This research was supported by USDA Grant 82-CRCR-1-1014 to S. D. Tanksley. We acknowledge the technical support of Jaime Iglesias and Fernando Loiaza.