Use of electrophoresis for variety testing in carrot

Electrophoretic markers were used to describe varieties of carrot and tested for their use in distinguishing and identifying them. Seed proteins and ten enzyme systems (PGI, PGM, PGD, ACP, ADH, GOT, IDH, MDH, MR and SkDH) were studied in a set of varieties and selection lines. Using the proteins from bulk samples of more than 25 seeds per variety, consistent results were obtained which enabled the 38 varieties studied to be divided into 14 groups. Among the enzymes studied, PGI, PGM and PGD were found to be convenient for distinction purposes. They demonstrated the heterogeneity of the varieties and their combined use enabled most of them to be distinguished from one another. The use of electrophoretic markers for distinguishing varieties is discussed.     

Use of isozyme patterns in characterization of potato mutant lines selected for agronomic quantitative traits

Summary Seven varieties and 57 spontaneous or induced in vitro mutant lines (20 macromutant and 37 micromutant events) of potato were tested by starch gel electrophoresis for ADH, GOT, PGI, PGM, ACO, IDH, MDH and 6PGDH isozymes in tuber extracts. The data showed that in contrast to variety comparisons, the isozyme patterns rarely differentiate mutant lines which have altered morphological traits. But trying to identify isozyme differences in mutants can still be useful for a chimeric structure for GOT-2 alleles in a mutant from Atlantic and a new tuber specific locus for 6PGDH in mutants from Russet Burbank were found.Abbreviations ACO aconitase - ADH alcohol dehydrogenase - GOT glutamate oxaloacetate transaminase - IDH isocitric acid dehydrogenase - MDH malate dehydrogenase - 6PGDH 6-phosphogluconate dehydrogenase - PGI phosphoglucoisomerase - PGM phosphoglucomutase - SGE starch gel electrophoresis - EMS ethyl metanesulfonate     

Characterization of isozymes in Salvia columbariae

Summary Salvia columbariae is a herbaceous annual species which has potential for domestication as a new source of industrial oil. Isozyme markers provide a mean by which this process may be facilitated. Isozyme survey of field grown Salvia columbariae plants showed variation for malate dehydrogenase (MDH), phosphoglucose isomerase (PGI), and phosphogllucomutase (PGM). Selfed seed was obtained from the field and was grown in the greenhouse for segregation analyses. Electrophoretic results indicated that MDH was variable at zone 1, showing presence or absence of a band. The observed segregation ratio was not significantly different from expected ratio for Pgi-4 and Pgm-3 isozymes, indicating monogenic control of the two loci. Pgi-4 locus was heterozygous for a null allele. Cross dimerization between the allozyme Pgi-3 and Pgi-4 loci resulted in an intergenic band for this isozyme system.Abbreviations ACO aconitase - ADH alcohol dehydrogenase - DTT-DL dithiothreitol - FDH formate dehydrogenase - GOT glutamate-oxalacetate transaminase - IDH isocitric dehydrogenase - MDH malate dehydrogenase - MNR menadione reductase - 6PGD 6-phosphogluconate dehydrogenase - PGI phosphoglucose isomerase - PGM phosphoglucomutase - PVP-40 polyvinylpyrrolidone - SKDH shikimate dehydrogenase - TPI triosephosphate isomerase     

Inheritance of isozyme variants and their linkage relationships in Chinese fir (Cunninghamia lanceolata Hook.)

Summary 49 single-clone seed samples of Chinese fir (Cunninghamia lanceolata) were used to investigate genetic variation for nine enzyme systems. LAP, PGM, and SOD were invariant, whereas GDH, GOT, IDH, MDH, 6PDH, and SKDH were highly variable. Inheritance patterns of the variable enzyme systems were mostly in accordance with Mendelian expectations. Linkage was studied by analysing 43 two-locus combinations with the following two-locus combinations significantly linked in at least 50% of the respective double-heterozygous clones: Gdh1: Got3; Gdh1: Idh1; Got2: 6Pdh2; Idh2: Skdh1; Mdh1: 6Pdh1. Estimates of recombination frequencies for each locus pair varied considerably among clones.Supported by the Carl Duisberg Gesellschaft.     

Phenotypic polymorphism of six enzymes in the grasspea (Lathyrus sativus L.)

Summary The phenotype variation in six enzymes, 6-phosphogluconate dehydrogenase (6-PGD), malate dehydrogenase (MDH), peroxidase (PRX), isocitrate dehydrogenase (IDH), galactose dehydrogenase (GD) and glutamate oxaloacetate transaminase (GOT), was investigated using horizontal starch gel electrophoresis in 52 accessions of the grasspea, Lathyrus sativus. Phenotypic polymorphism was observed for all six enzymes. High phenotypic polymorphism (Pj) was observed for PRX and 6-PGD, while there was little polymorphism for GOT, with only two accessions showing variation. There was no correlation between phenotypic polymorphism and region of origin, or groupings of accessions made on the basis of flower colour. Tentative genetic interpretations of banding patterns are given for five of the enzyme systems. The level of apparent heterozygosity was higher than expected in this predominantly autogamous species. The level of variation in the grasspea is discussed in terms of its potential for exploitation through plant breeding.     

Genetic and linkage analysis of isozyme loci in Daucus carota L.

Summary Electrophoretic polymorphisms of eight enzyme systems were studied in leaves of Daucus carota ssp. sativus in order to identify additional isozyme loci and generate first linkage groups of genetic markers. The genetic analysis of aconitase (ACO), leucin aminopeptidase (LAP), menadione reductase (MDR), phosphoglucomutase (PGM), 6-phosphogluconic dehydrogenase (6-PGD), shikimate dehydrogenase (SKD), and triose phosphate isomerase (TPI) zymograms resulted in the identification of 8 isozyme marker loci, designated as Aco-1, Lap-1, Pgm-1, Pgm-3, 6-Pgd-2, Skd-1, Tpi-1, and Tpi-2. All loci segregated with codominant alleles and encoded for monomers (ACO, LAP, PGM, SKD), and dimers (6-PGD, TPI), respectively. MDR enzymes of the variable region MDR-2 appeared to be identical with Dia-2 isozymes. Tests of joint segregation for pairwise comparisons of all 14 isozyme marker loci now available in carrots indicate that 12 loci are linked in 4 linkage groups (marked K1 to K4) in the following order: Aco-1, Pgi-1, and Dia-3 (K1), Tpi-2, Got-2, and Lap-1 (K2), Got-3 and Tpi-1 (K3) and Pgm-1, Pgm-3, 6-Pgd-2 and Skd-1 (K4). Dia-2 and Got-1 remained unlinked. The possible duplication of a PGM locus and a 6-PGD locus is discussed.     

Genetic control of isozymes in eggplant and its wild relatives

Summary This study was conducted to elucidate the inheritance and linkage relationships of isozymes in aspartate aminotransferase (AAT, EC 2.6.1.1), alcohol dehydrogenase (ADH, EC 1.1.1.1), phosphogluconate dehydrogenase (PGD, EC 1.1.1.43), phosphoglucomutase (PGM, EC 2.7.5.1) and shikimate dehydrogenase (SKDH, EC 1.1.1.25) in eggplant and its wild relatives. Segregating populations were generated by backcrossing of hybrids among the species. Evidence of Mendelian inheritance was obtained for seven loci: Aat-1, Adh-1, Adh-2, Pgd-1, Pgm-1, Pgm-2 and Skdh-1. Twenty-one pairs of loci were tested for independent assortment, suggesting three linked pairs, Aat-2 with Pgd-2 (R=0.35±0.07), Adh-2 with Pgm-1 (R=0.33±0.07) and Pgd-2 with Pgm-2 (R=0.32±0.06).Abbreviations AAT aspartate aminotransferase - ADH alcohol dehydrogenase - PGD phosphogluconate dehydrogenase - PGM phosphoglucomutase - SKDH shikimate dehydrogenase     

Geographical distribution of allozyme patterns in shallot (Allium cepa var. ascalonicum Backer) and wakegi onion (A. × wakegi Araki)

Summary Shallot and wakegi were collected in the main islands of Indonesia, and in Japan, Korea, Taiwan, Malaysia, Thailand and Bangladesh. Five isozyme resolutions, phosphoglucomutase (PGM), glutamate oxaloacetate (GOT), glutamate dehydrogenase (GDH), esterase (EST) and peroxidase (POX) were employed for demonstrating inter-and intraspecific differences. A dendrogram separated 189 collected accessions into 25 types of wakegi onion and 18 types of shallot. All accessions of Japan, Korea and Taiwan were determined to be wakegi onion, whereas those of Bangladesh, Malaysia and Thailand were shallot. Twenty-six out of 165 Indonesian accessions indicated wakegi onion distribution in Sumatra, West Java province and in Sulawesi Island. This confirmed that there is mixed-cultivation of the two Allium species with no distinction made between them. Japan and Indonesia had respectively 12 and eight unique types of wakegi onion, while Korea had only one type. West Java showed the most various type of wakegi onion, whereas East Java had many types of shallot. Shallots collected from Bangladesh were distinetly different from those of South East Asian types.     

Identification of triploid Citrus by isozyme analysis

Summary Seedlessness is a desirable horticultural attribute in Citrus and is positively associated with triploidy. The conventional cytological method for triploid identification is a laborious technique involving the preparation of root tips for chromosomal analysis. Digital densitometry of isozymes, however, offers the possibility of distinguishing triploid Citrus from large populations of seedlings both quickly and cheaply. Where there are no gene dosage regulation effects, greater band density should be evident in the allozyme contributed by the diploid gamete for a heterozygous locus. The isozymes of 4 enzymes; malate dehydrogenase, 6-phosphogluconate dehydrogenase, shikimate dehydrogenase, and phosphoglucose isomerase, were investigated with polyacrylamide gel electrophoresis. Band densities of these isozymes for triploid Citrus, their diploid siblings and diploid progenitors were measured using a digital densitometer. Of the 4 enzymes investigated only allozymes for shikimate dehydrogenase exhibited consistent differences over a wide range of Citrus cultivars. Greater band density was evident in the allozyme contributed by the diploid gamete. The band density ratio between allozymes for triploid Citrus was close to 0.5, while for diploid Citrus band density ratios were close to 1.0. This effect is due to the extra protein coded by the additional gene dose and was not observed in diploids. Shikimate dehydrogenase proved to be an accurate molecular marker for distinguishing between diploid and triploid Citrus for heterozygous progeny.Abbreviations PAGE Polyacrylamide gel electrophoresis - MDH malate dehydrogenase - 6PGD 6-phosphogluconate dehydrogenase - SkDH shikimate dehydrogenase - PGI phosphoglucose isomerase     

Inheritance of isozymes in peach x Prunus kansuensis and peach x Prunus davidiana hybrids

Summary Isozyme variation and inheritance were investigated with starch gel electrophoresis in peach (Prunus persica L. Batsch) x P. kansuensis Rehd. and peach x P. davidiana (Carr.) Franch. interspecific hybrids. Of five enzyme systems surveyed for polymorphism, four systems were identified as polymorphic [isocitrate dehydrogenase (IDH, EC 1.1.1.41), phosphoglucomutase (PGM, EC 2.7.5.1), aspartate aminotransferase (AAT, EC 2.6.1.1), and 6 phosphogluconate dehydrogenase (PGD, EC 1.1.1.44)] and may be useful as genetic markers in future cultivar and rootstock development. Analysis of progenies segregating for pairs of loci suggests a possible linkage between the loci coding for Aat-1 and Pgd-2. Independent assortment was observed for isozyme loci Idh/Pgm-2, Idh/Aat-1, Idh/Pgd-2, Pgm-2/Aat-1, Pgm-2/Pgd-2, and Aat-2/Aat-1. The red leaf locus, Gr, assorted independently of the isozyme loci: Idh, Pgm-2, Aat-1, and Pgd-2.     

Cytogenetic and isozymic characterization of anther-panicle culture derived tall fescue aneuploids

Summary Chromosome associations of 44 tall fescue (Festuca arundinacea Schreb., 2n=6x=42) plants derived from anther-panicle culture of Kentucky 31 were evaluated to determine if new genetic stocks could be identified. Seventeen of the plants were euploids (21 II), two were monosomic (20II+1I), 22 were double monosomic (19II+2I) and three were triple monosomic (18II+3I). Zymograms were obtained for phosphoglucoisomerase (PGI), glutamate oxaloacetate transaminase (GOT), acid phosphatase (ACPH), malate dehydrogenase (MDH) and 6-phosphogluconate dehydrogenase (6-PGD). The zymograms were identical for GOT, ACPH, and 6-PGD but different patterns were found for MDH and PGI in some of the double monosomics and euploids from a different piece of callus than the majority of the plants.Contribution of the USDA-ARS in cooperation with the Kentucky Agricultural Experiment Station. Scientific Journal Series No. 86-3-61.     

Identification of tomato cultivars (Lycopersicon esculentum) by polyacrylamide isoelectric focusing

Summary Isozyme analyses have been used for the definitive identification of many plant cultivars, but not for cultivated tomatoes. Six isozyme systems, namely alcohol dehydrogenase, acid phosphatase, phosphoglucomutase, esterase, phosphoglucoisomerase and 6-phosphogluconate dehydrogenase of tomato seed extracts were resolved by isoelectric focusing on polyacrylamide gels with a narrow pH gradient. Nine alcohol dehydrogenase phenotypes were distinguished which, with three acid phosphatase phenotypes, identified twelve of the seventeen cultivars. Fewer differences were found for the other isozymes. Since this method could differentiate between breeding parents and their progeny it is concluded that further investigations are warranted.Abbreviations APS acid phosphatase - ADH alcohol dehydrogenase - EST esterase - IEF isoelectric focusing - PGI phosphoglucoisomerase - PGM phosphoglucomutase - 6-PGDH 6-phosphogluconate dehydrogenase - RFLP restriction fragment length polymorphism - VOPRI Vegetable and Ornamental Plant Research Institute     

Isozyme variation in taro (Colocasia esculenta (L.) Schott) from Asia and Oceania

Summary Isozyme variation was studied in 1,417 cultivars and wild forms of taro collected in Asia and Oceania. Seven polymorphic enzyme systems (MDH, IDH, PGI, 6-PGD, ME, SkDH, and ADH) revealed 143 isozyme phenotypes, or zymotypes, each uniquely characterized by the presence or absence of 56 electromorphs. Results showed greater isozyme variation in Asia than in Oceania, with Indonesia being the area of greatest diversity. No correlations were found between zymotypes and morphotypes or ploidy levels (as described by other investigators). Multivariate analyses of the isozyme data indicated that the majority of the Indonesian cultivars were different from the Philippine and Oceanian taro cultivars. Oceanian cultivars constituted a continuum of clusters and are thought to have originated from a narrow genetic base introduced from Indonesia. If taro breeding is to have any future in Oceania, it is important to exchange genotypes to broaden the base of existing breeding programmes.     

Genetic analysis and nomenclature for seven isozyme systems in Brassica nigra, B. oleracea and B. campestris

General criteria for the assignment of names to enzyme systems, regions of activity, isozyme loci and allozymes have been lacking in crucifer species. This paper proposes a standard nomenclature for seven isozyme systems in the three diploid species of U's triangle: Brassica nigra, B. oleracea and B. campestris. Gel/electrode buffers, which provided the best resolution for seven isozyme systems, acid phosphatase (APS), aconitase (ACO), leucine aminopeptidase (LAP), 6-phosphogluconate dehydrogenase (6 PGD), phosphoglucoisomerase (PGI), phosphoglucomutase (PGM), and triosephosphate isomerase (TPI), were proposed as standards. Isozyme genetic analysis was determined for B. oleracea and B. campestris from previous studies and by segregation of selfed progenies of heterozygous B. nigra plants. Several populations were studied and 148 allozymes at the 18 loci observed were described for the three species. Their relative mobility was studied using a pure line of oilseed rape as reference. The comparison of the different alleles within and between the species is discussed.     

Enzyme polymorphism in faba bean (Vicia faba L. minor) accessions. Genetic interpretation and value for classification

Three isozyme sytems glutamate oxaloacetate transaminase (GOT), superoxide dismutase (SOD), and malic enzyme (ME) have been analysed electrophoretically to study the diversity within and between 33 faba bean (V. faba L. type minor) accessions. For comparison, two cultivars (Vesuvio and Talo) were included in the study. Further objective of this study was to classify populations into genetic similar groups. SOD and GOT showed large variation, while ME presented minor change. Principal components, hierarchical cluster, and canonical discriminant analyses produced five groups with significant differences among them. The alleles with low frequency played a significant role in the discrimination. It was concluded that more detailed investigations regarding genetic aspects of enzyme diversity are needed to support the data here reported. This revised version was published online in July 2006 with corrections to the Cover Date.     

Segregation of isozymes in selfed progenies of a synthetic amphidiploid between Solanum integrifolium and S. melongena

Isozyme and cytogenetic analyses were performed on selfed progenies of a synthetic amphidiploid between scarlet eggplant, Solanum integrifolium (= S. aethiopicum),and eggplant, Solanum melongena `DMP', for estimating genetic uniformity. Isozymes in the 379 examined seedlings segregated into five genotypes (phenotypes) each at the four loci examined, Pgd-2 of phosphogluconate dehydrogenase (E.C.1.1.1.43), Idh-2 of isocitrate dehydrogenase (E.C.1.1.1.41), Pgm-2 of phosphoglucomutase (E.C.2.7.5.1)and Skdh-1 of shikimate dehydrogenase (E.C.1.1.1.25), indicating that the selfed seedlings were not genetically uniform. Most of the examined 15 selfed seedlings exhibited a somatic chromosome number of 48, that is the same number of the synthetic amphidiploid, whereas isozyme genotypes among them were variable. It is suggested that the segregation of isozymes was not caused by variation of chromosome number but by genetic segregation of isozyme genes. The genome of the synthetic amphidiploid was indicated to be unstable. This revised version was published online in July 2006 with corrections to the Cover Date.     

Inheritance of isoenzymes and their linkage relationships in two interspecific cherry progenies

Two interspecific cherry progenies, Prunus avium 'Napoleon' × P. incisa E621 and × P. nipponica F1292, were analysed by polyacrylamide gel electrophoresis for 14 enzyme systems: aconitase (ACO), acid phosphatase (ACP), alcohol dehydrogenase (ADH), amylase (AMY), glutamate oxaloacetate transaminase (GOT), glucose–6-phosphate isomerase (GPI), isocitrate dehydrogenase (IDH), leucine aminopeptidase (LAP), malate dehydrogenase (MDH), malic enzyme (ME), phosphogluconate dehydrogenase (PGD), phosphoglucomutase (PGM), shikimate dehydrogenase (SKD) and superoxide dismutase (SOD). Thirty-one loci were deduced from segregating banding patterns, Aco–2, Acp–1 to –5, Acp–8, –9, Adh–1 to –6, Amy–2, –3, Got–1 to –3, Gpi–2, Idh–1 to –4, Lap–1, Me–1, –2, Mdh–2, Pgd–1, –2 and Sod–2. Only ten of these had previously been established. Seven putative loci were polymorphic but did not segregate in the progenies. Analysis of cosegregations and calculation of recombination fractions revealed that 15 loci could be grouped into four linkage groups: Acp–1/–2/–3–-Acp–5; Gpi–2–- Got–2–-Got–1–-Lap–1; Adh–4/–6– -Amy–2; and Adh–1–-Adh–5–-Adh–2–- Me–2. These consolidate two previously reported linkage groups and establish three new groups. The previously reported linkage of Lap–1 with Me–1 was not confirmed. Fourteen cultivars of P. avium were analysed for the same 14 enzyme systems and showed polymorphism for just 17 of the established loci and for none of the putative loci, indicating far less scope for linkage analysis in intraspecific progenies from crosses among these cultivars.     

Genetic diversity of natural populations of Atriplex halimus L. in Morocco: An isoenzyme-based overview

Based on polyacrylamide gel electrophoresis, nine natural populations of Atriplex halimus L., a perennial shrub, collected in different regions of Morocco, were studied for their genetic variation using isoenzyme polymorphism of the highly active enzyme systems: esterases (EST), acid phosphatases (ACP) and glutamate oxaloacetate transaminase (GOT). Different allozyme frequencies from 7 different loci were obtained for all populations of this halophyte species. High levels of genetic diversity were revealed. The mean number of alleles per locus (A = 1.9–2.0), the percentage of polymorphic loci (p = 71.4–85.7) and the mean expected heterozygosity (H_e = 0.339–0.385) showed an important variability in all populations. Gene diversity was essentially explained by the within population component. The between populations differentiation accounted for 8% of the whole diversity (F_ST, averaged over all loci, is 0.08). The relationships among the 9 populations were inferred from the Nei’s genetic distances. Four major groups were formed. The northern population ‘Tanger’, forming a unique group, was highly divergent from the other groups. It appeared that the genetic distance between all groups was related to the geographic distance that separates them. This revised version was published online in August 2006 with corrections to the Cover Date.     

Correlation of isoenzyme polymorphism and Bayoud-disease resistance in date palm cultivars and progeny

Summary Seedlings of date palm (Phoenix dactylifera L.), obtained from seven cultivars crossed with two males, were analyzed by polyacrylamide gel electrophoresis for esterase (EST), glutamate oxaloacetate transaminase (GOT), endopeptidase (ENP) and alcohol dehydrogenase (ADH) polymorphisms. Eleven, eight, five and two phenotypes were revealed for the enzymes tested, respectively. Seedlings of F₁ populations derived from Bayoud (Fusarium)-resistant and low fruit quality cultivars were characterized by a high electrophoretic polymorphism, when compared with progenies of Bayoud-susceptible and high fruit quality cultivars. In almost all cases, the most frequent electrophoretic phenotypes scored for each enzyme in different F₁ populations, were similar to those of the corresponding parent cultivars. Heterozygous phenotypes have been found for, at least, 3 loci; Got-2, Est-1 and Enp, indicating that such loci could be used to screen for hybrid seedlings. The expected Mendelian segregation of allozymes has been observed for these 3 loci, in many F₁ populations. It seems that progenies of Bayoud-resistant cultivars are characterized by a high level of electrophoretic polymorphism. The estimation of this index and the search for genetic linkage with segregating allozymes, may be biochemical criteria useful as an aid in distinguishing date palm seedling populations derived from Bayoud-resistant cultivars and suitable for breeding programs.     

Genetics of isozyme variants in Populus tremula,P. tremuloides and their hybrids

Summary Eight Populus tremula and six P. tremuloides clones as well as 49 full-sib families were studied in GOT, LAP, 6-PGDH, and SKDH by horizontal starch gel electrophoresis. For GOT one polymorphic zone was found and segregation of seven full-sib families suggests Mendelian inheritance. For LAP and 6-PGDH two zones each were clearly scored. For LAP two polymorphic loci were proposed based on the phenotypic segregation of isozyme variants in six and 34 full-sib families, respectively. In 24 full-sibs families the presence of null alleles was inferred for both loci. The genetic control of the upper zone of 6-PGDH was demonstrated by a segregation analysis of 17 full-sib families. SKDH also demonstrated a Mendelian inheritance pattern in 12 of the full-sib families analysed. The electrophoretic patterns of pollen were similar to those of buds, but migration rates of the supposed corresponding isozymes were slightly modified (Lap-B, Skdh, 6Pgdh-A). Lap-A was not present in pollen extracts and hybrid bands were not found when gels were stained for dimeric enzymes (6-PGDH, GOT).