Flax species polymorphism for isozyme and metabolic markers

Genetic diversity of flax isozyme patterns of 6-phosphogluconate dehydrogenase, glutamate-oxaloacetate transaminase, and cytochrome-c-oxidase in leaves, as well as the level and the relative amounts of fatty acids in seed oil were studied in flax species. The isozyme loci examined were found to be polymorphic. It was shown that each of the flax species studied was characterized by strictly defined amounts and proportions of fatty acids, i.e., the fatty acid composition of the seed oil may be used as a species diagnostic trait. Our results suggest that all of the flax species studied have the same ancestor. Linum grandiflorum was shown to be a phylogenetic branch split at an early stage of the flax evolution.     

Phylogenetic relationships among species of Prunus as inferred by isozyme markers

Summary An isozyme survey of 34 species of Prunus representing subgenera Prunus, Amygdalus, Cerasus, and Lithocerasus detected 110 presumptive alleles at 11 isozyme loci. Principal component analysis was conducted on the covariance matrix derived from allelic frequencies calculated for each species. Cluster analysis was performed on the first 30 principal components. Results generally support traditional classification of Prunus at the subgeneric level, except for members of subgenus Lithocerasus and two members of subgenus Amygdalus. Prunus glandulosa Thunb., P. japonica Thunb., and P. tomentosa Thunb. of subgenus Lithocerasus and P. triloba Lindl. of subgenus Amygdalus appear to represent primitive species. P. besseyi Bailey and P. pumila L. of subgenus Lithocerasus and P. andersonii of subgenus Amygdalus should be assigned to subgenus Prunus. Placement of its members indicates that subgenus Lithocerasus is an artificial grouping of species that are very different genetically although similar phenotypically.Paper No. 12529 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC 27695-7643, USA     

Amylase isozyme polymorphism in maize

Summary Amylase isozyme polymorphisms were shown to exist in certain genetic stocks of Zea mays, using the technique, of starch gel electrophoresis. The earliest period of amylase activity was detected during the second day after germination of seeds. The third day after germination gave the best resolution of amylase activity in the form of distinct zones. Tissues examined from adult plants showed no amylase activity. Several hybrid patterns were of interest, but the most unique situation is the F₁ hybrid AA9xAA2. One of the zones in this hybrid appears to be intermediate to the parental types, suggesting the formation of a hybrid enzyme. In the F₁ hybrid AA7xP51 there are two zones of enzyme activity neither of which is present in the parental strains; this is also suggestive of hybrid enzymes with altered electrophoretic mobilities.Supported by the U.S. Atomic Energy Commission under contract No. AT (11-1) 1338. Portion of this work was done in the Department of Genetics, University of Hawaii, under support from the U.S. National Institutes of Health     

A linkage map with RFLP and isozyme markers for almond

Inheritance and linkage studies were conducted with seven isozyme genes and 120 RFLPs in the F₁ progeny of a cross between almond cultivars Ferragnes and Tuono. RFLPs were detected using 57 genomic and 43 cDNA almond clones. Eight of the cDNA probes corresponded to known genes (extensin, prunin (2), -tubulin, endopolygalacturonase, oleosin, actin depolymerizing factor and phosphoglyceromutase). Single-copy clones were found more frequently in the cDNA (65%) than in the genomic libraries (26%). Two maps were elaborated, one with the 93 loci heterozygous in Ferragnes and another with the 69 loci heterozygous in Tuono. Thirty-five loci were heterozygous in both parents and were used as bridges between both maps. Most of the segregations (91%) were of the 11 or 1111 types, and data were analyzed as if they derived from two backcross populations. Eight linkage groups covering 393 cM in Ferragnes and 394 in Tuono were found for each map. None of the loci examined in either map was found to be unlinked. Distorted segregation ratios were mainly concentrated in two linkage groups of the Ferragnes map.     

Fly-pollinated Pleurothallis (Orchidaceae) species have high genetic variability: evidence from isozyme markers

We conducted an isozyme study in 22 populations of five Pleurothallis (Orchidaceae) species (12 loci in nine enzymatic systems). The genetic variability in all populations is surprisingly high (P = 58-83%, A = 2.1-3.8, H(e) = 0.25-0.43) in spite of the fact that the five species are pollinated by small flies whose behavior enables self-pollination. We suggest that self-incompatibility, inbreeding depression, and mechanical barriers that prevent self-pollination in these species are responsible for the maintainance of the high genetic variability. These traits are uncommon in Orchidaceae, but have been observed in these and some other species pollinated by flies or other pollinators with behavior that facilitates self-pollination. The genetic similarity among conspecific populations is also high for species with very short-range flying pollinators. Only one population of P. teres presented values of genetic similarity lower than usually observed in allopatric conspecific populations. Morphology, however, does not support its segregation as a new taxon. All species can be recognized by their enzymatic patterns, and the results agree with recently proposed taxonomic realignments. Conversely, the supposed affinities among these species based on floral morphology are not supported, and we hypothesize that it may be due to convergence in species with similar pollinators.     

A comparative analysis of genetic polymorphism in wild and cultivated barley from Tibet using isozyme and ribosomal DNA markers.

This study was conducted to address some of the issues concerning the possible significance of Tibet in the origin and evolution of cultivated barley. A total of 1757 barley accessions from Tibet, including 1496 entries of Hordeum vulgare ssp. vulgare (HV), 229 entries of the six-rowed wild barley H. vulgare ssp. agriocrithon (HA), and 32 entries of the two-rowed wild barley H. vulgare ssp. spontaneum (HS), were assayed for allozymes at four esterase loci. A subsample of 491 accessions was surveyed for spacer-length polymorphism at two ribosomal DNA loci. Genetic variation is extensive in these barley groups, and the amount of genetic diversity in cultivated barley of this region is comparable with that of cultivated barley worldwide. The level of genetic variation of HA is significantly lower than the other two barley groups, and there is also substantial heterogeneity in the level of polymorphism among different agrigeographical subregions. However, little genetic differentiation was detected among the three barley groups (HV, HA, and HS), as well as among different agrigeographical subregions. Comparison of the results from this and previous studies indicated a strong differentiation between Oriental and Occidental barley, thus favoring the hypothesis of a diphyletic origin of cultivated barley.     

Sweet cherry cultivar identification by leaf isozyme polymorphism

Isozyme polymorphism can assist in the identification of cherry cultivars. Ten isozymes, each showing variation at only one locus, provide 70 unique genotype profiles from leaf extracts of 78 different sweet cherry cultivars. Polymorphism in 6PGD, G6PD, GPI, IDH, PGM, FDP, SKDH and PER is demonstrated for the first time, while observations are extended for the previously described polymorphisms in MDH and GOT. Some cultivars with identical morphological characters and previously treated as one cultivar can be separated on the basis of isozyme genotype. Irradiated mutants and parent material could also be differentiated.     

Development and polymorphism of microsatellite markers for Fagus crenata and the closely related species, F. japonica

 We have developed microsatellite markers (SSRs) applicable to Fagus crenata using the RAHM method and investigated their polymorphisms. We also applied the SSRs in an analysis of a closely related species, F. japonica. Here we describe the isolation and characterization of nine polymorphic microsatellite markers, of which eight are applicable to both species. Among 30 individuals of each of F. crenata and F. japonica we detected a total of 79 and 77 alleles, respectively, with an average of 9.9 and 8.6 alleles per locus. The mean expected heterozygosity (He) was 0.615 (range: 0.216–0.925) in F. crenata and 0.660 in F. japonica (range: 0.259–0.827). The He values were considerably higher than those previously found for isozymes. Paternity exclusion probabilities for multiple loci, calculated over all loci, were extremely high (0.999 and 0.998 in F. crenata and F. japonica, respectively): sufficiently high to study pollen flow in both species. Received: 5 December 1998 / Accepted: 28 December 1998     

Variation of isozyme patterns among Arachis species

The genus Arachis contains a large number of species and undescribed taxa with patterns of genetic variation that are little understood. The objectives of this investigation were to estimate genetic diversity among species of Arachis by utilizing electrophoretic techniques and to establish the potential for use of isozymes as markers for germplasm introgression. One-hundred-and-thirteen accessions representing six of the seven sections of the genus were analyzed for isozyme variation of 17 enzymes. Section Rhizomatosae species were not included because they produce very few seeds. Seeds were macerated and the crude extract was used for starch-gel electrophoretic analyses. Although the cultivated species has few polymorphic isozymes, the diploid species are highly variable and two-to-six bands were observed for each isozyme among accessions. Because of the large number of isozyme differences between A. hypogaea and A. batizocoi (the presumed donor of the B genome), this species can no longer be considered as a progenitor of the cultivated peanut. Seed-to-seed polymorphisms within many accessions were also observed which indicate that germplasm should be maintained as bulk seed lots, representative of many individuals, or as lines from individual plants from original field collections. The area of greatest interspecific genetic diversity was in Mato Grosso, Brazil; however, the probability of finding unique alleles from those observed in A. hypogaea was greatest in north, north-central, south and southeast Brazil. The large number of polymorphic loci should be useful as genetic markers for interspecific hybridization studies.     

Identification of Fusarium species by isozyme analysis

Cellulose-acetate electrophoresis (CAE) was used to investigate isozyme polymorphisms among different isolates of Fusarium cerealis, F. culmorum, F. graminearum and F. pseudograminearum. After initial testing of 18 enzymes in three buffer systems for activity and resolution of bands, 12 proved to be appropriate for analysis of the full sample set. Comparing the different electrophoretic types (ETs), adenylate kinase (AK), NADP dependent glutamate dehydrogenase (NADP GDH), peptidase B (PEP B), peptidase D (PEP D) and phosphoglucomutase (PGM) proved to be diagnostic for at least one species examined. However, only PEP D was useful alone as a marker to distinguish the four taxa studied providing a rapid and simple CAE based diagnostic protocol.     

Genetic diversity of Echinacea species based upon amplified fragment length polymorphism markers.

The taxonomy of Echinacea is based on morphological characters and has varied depending on the monographer. The genus consists of either nine species and four varieties or four species and eight varieties. We have used amplified fragment length polymorphisms (AFLP) to assess genetic diversity and phenetic relationships among nine species and three varieties of Echinacea (sensu McGregor). A total of 1086 fragments, of which approximately 90% were polymorphic among Echinacea taxa, were generated from six primer combinations. Nei and Li's genetic distance coefficient and the neighbor-joining algorithm were employed to construct a phenetic tree. Genetic distance results indicate that all Echinacea species are closely related, and the average pairwise distance between populations was approximately three times the intrapopulation distances. The topology of the neighbor-joining tree strongly supports two major clades, one containing Echinacea purpurea, Echinacea sanguinea, and Echinacea simulata and the other containing the remainder of the Echinacea taxa (sensu McGregor). The species composition within the clades differs between our AFLP data and the morphometric treatment offered by Binns and colleagues. We also discuss the suitability of AFLP in determining phylogenetic relationships.     

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.     

Estimation of outcrossing rates in Duglas-fir using isozyme markers

Summary Seeds produced under open-pollination were collected from eight natural stands and a plus-tree seed orchard of Douglas-fir. These seeds were germinated and both diploid embryos and haploid gametophytes were analyzed by starch-gel electrophoresis. Eleven variable loci were resolved for both kinds of tissue and used as genetic markers for estimating outcrossing rates. Estimates made with single-locus and multilocus methods both indicated that the proportion of viable embryos resulting from outcrossing is about 0.90 for the natural stands, and for the seed orchard. Comparison of single-locus and multilocus estimates of outcrossing rates indicated that little or no inbreeding other than selfing occurred. Estimated outcrossing rates were higher for seeds from the upper portion of the crown than for seeds from the lower crown. It was also found that some trees selfed at a much higher rate than other trees.     

An integrated genetic linkage map for eucalypts using RFLP,RAPD and isozyme markers

An integrated genetic linkage map for E. nitens was constructed in an outbred three-generation pedigree. Analysis of 210 RFLP, 125 RAPD and 4 isozyme loci resulted in 330 markers linked in 12 linkage groups covering 1462 cM (n=11 in eucalypts). The 12th linkage group is comprised of only 5 markers and will probably coalesce with another linkage group when further linked loci are located. Co-dominant RFLP loci segregating in both parents were used to integrate linkages identified in the male and female parents. Differences in recombination frequencies in the two parents were observed for a number of pairs of loci, and duplication of sequences was identified both within and between linkage groups. The markers were distributed randomly across the genome except for the RFLPs in linkage group 10 and for some loci showing segregation distortion, which were clustered into three regions of the map. The use of a large number of co-dominant RFLP loci in this map enables it to be used in other pedigrees of E. nitens and forms a basis for the detection and location of QTL in E. nitens and other eucalypt species.     

Development of PCR-based markers from the tomato glutamate oxaloacetate transaminase isozyme gene family as a means of revitalising old isozyme markers and recruiting new ones

Sequences annotated as aspartate aminotransferases (synonymous with glutamate oxaloacetate transaminases) in the SOL Genomics Network unigene database were used to design 10 pairs of PCR primers for genetic marker development. These primer pairs generated nine CAPS markers, two SCAR markers and one SSR marker, which were bin-mapped using a set of tomato introgression lines (IL) derived from Lycopersicon esculentum cv. M82 and Lycopersicon pennellii LA716. Based on their bin locations, these markers are largely dispersed throughout the tomato genome and appear to have tagged all four of the glutamate oxaloacetate transaminase (Got) isozyme marker genes placed on the classical genetic map of tomato. Orthologous relationships with Arabidopsis aspartate aminotransferase (Asp) genes suggest the existence of at least two additional functional Got genes in tomato that have also been tagged by these markers and likewise an additional functional Asp gene in Arabidopsis. The Got-2 isozyme marker has often been used for the marker-assisted breeding of the I-3 gene for Fusarium wilt resistance introgressed from L. pennellii LA716. The Got-2 CAPS marker that we have developed offers a facile PCR-based alternative to the isozyme marker for the marker-assisted breeding of I-3. However, all of the PCR-based markers we have developed have the potential to assist the breeding of linked traits introgressed from wild relatives of tomato.     

Linkage among isozyme,RFLP and RAPD markers in Vicia faba

Summary Segregating allozyme and DNA polymorphisms were used to construct a preliminary linkage map for faba bean. Two F₂ populations were analyzed, the most informative of which was segregating for 66 markers. Eleven independently assorting linkage groups were identified in this population. One of the groups contained the 45s ribosomal array and could be assigned to the large metacentric chromosome I on which the nucleolar organizer region is located. This linkage group also contained two isozyme loci, Est and Tpi-p, suggesting that it may share some homology with chromosome 4 of garden pea on which three similar markers are syntenic. Additional aspects of the map and the extent of coverage of the total nuclear genome are discussed.     

Genetic control and linkage relations of additional isozyme markers in chick-pea

Summary Allozyme polymorphisms of nine enzymes — aspartate aminotransferase (AAT), diaphorase (DIA), esterase (EST), formate dehydrogenase (FDH), -galactosidase (GAL), -glucosidase (GLU), malate dehydrogenase (MDH), malic enzyme (ME), and peroxidase (PRX) — were described in chick-pea (Cicer L.). Thirteen isozyme loci, Aat-c, Dia-4, Est-2, Est-4, Est-10, Fdh, Gal-2, Gal-3, Gal-4, Glu-3, Mdh-2, Me-2, and Prx-2, were genetically defined. Alleles of each of these isozyme loci expressed codominantly in heterozygotes and exhibited a codominant, single-locus segregation ratio in F₂. The loci Est-2, Mdh-2, and Me-1 were expressed only in flower. Linkage relations were determined for these 13 and several previously defined isozyme loci. The following new genetic linkages were identified: Pgm-p (locus for plastid phosphoglucomutase) — Est-10; Ald-p1 (one of the duplicate loci for plastid aldolase) — Glu-3 — Gal-2 — Est-2,3; Gal-3 — Aco-m (locus for mitochondrial aconitase) — Prx-2,3; Gpi-c (locus for cytosolic glucosephosphate isomerase) — Fdh; and Est-4 — Me-1. This study provides further confirmation on the existence of several conserved linkage groups among Cicer, Pisum, and Lens.     

Investigations on an interspecific hybrid involving three species of the genus Beta,with special reference to isozyme polymorphism

Summary A tetraploid (2n=36) interspecific hybrid was obtained involving three species belonging to three different sections of Beta. The hybrid was highly sterile and did not show apomixis. At meiosis, up to nine bivalents were observed, most probably resulting from autosyndesis of the chromosomes of Beta lomatogona. For nine isozyme systems, individual enzyme expression was investigated in the parental species and in the hybrids. No silencing of genes or genomes was observed. In the case of some polymeric enzymes interspecific heteropolymers could be detected.     

Microsatellite markers for diverse Salix species

Forty‐six microsatellites were isolated from an enriched library of Salix burjatica and tested on 20 individuals (of nine species/hybrids) from the National Willows Collection (IACR‐Long Ashton Research Station, UK). Twenty‐nine were monomorphic, gave multilocus or unscorable patterns, or were duplicates. The remaining 17 microsatellites gave 2–22 alleles/locus. Three microsatellites successfully cross‐amplified in 31 additional Salix species. A further six were tested on panels comprising 6–25 individuals from the 31 species. Cross‐amplification was successful in all cases. These results suggest that the microsatellites isolated here should prove useful for population studies in a wide range of Salix species.