STABLE DIPLOIDS FROM INTRAGROUP ZYGOSPORES OF CLOSTERIUM EHRENBERGII MENEGH. (CONJUGATOPHYCEAE)1

Two pairs of stable diploid clones were obtained as aberrant forms among F₁ progeny of an intragroup (intraspecific) cross between R-11-4 (mating type +) and M-16-4b (mating type -) of Group A of Closterium ehrenbergii Menegh. Each pair was derived from the two germination products of a single zygospore, and both clones were mating type minus. The cell size range of these four diploid minus clones was considerably above that of normal (haploid) Group A clones. Chromosome counts at the second meiotic metaphase indicated that these clones were diploid with approximately 200 chromosomes, which was double the number for normal Group A clones. Diploid minus clones conjugated normally with any haploid Group A plus clones, and yielded many triploid zygospores. Triploid zygospores germinated normally as did intragroup diploid zygospores. In metaphase I preparations, only bivalents were observed except on a few occasions where some uni- and multivalents were also detected. Viability of F₁ progeny from triploid zygospores (55–74%) was somewhat lower than from diploid zygospores of Japanese Group A populations (65–90%), but higher than intergroup (interspecific) hybrid zygospores from Groups A, B and H (0–12%). In addition to lower viability, some F₁ progeny from triploid zygospores exhibited slow vegetative growth. Almost all pairs of F₁ clones from single triploid zygospores were of opposite mating type, similar to normal diploid zygospores of the intragroup cross. Morphological variability of F₁ progeny of triploid zygospores was great. The apparently normal meiosis of triploid zygospores and the high viability of F₁ progeny suggested that the genome of Group A contains several sets of chromosome complements with mechanisms by which bivalents are regularly formed in the first meiotic division.     

Biosystematic studies of theClosterium peracerosum-strigosum-littorale complex IV. Hybrid breakdown between two closely related groups,group II-A and group II-B

Since a pre-zygotic isolating mechanism has been shown to be functioning completely between Group II-B plus and Group II-A minus (Watanabe and Ichimura, 1978b), the reciprocal cross was investigated in order to clarify the presence of a postzygotic isolating mechanism between the two sympatric closely related groups of theClosterium peracerosum-strigosum-littorale complex. Viabilities and fertilities of F₁, F₂ and backcross progenies of crosses within and between the two groups were studied using two representative pairs, IB-4-2 and IB-4-9 of Group II-A and KAS-4-30 and KAS-4-29 of Group II-B. Viability was estimated by % survival of isolated gones. Viability of F₁ progeny was 31.7% in the intergroup cross, while it was 70.0 and 46.0% in the intragroup cross of Group II-A and that of Group II-B, respectively. Viabilities of intragroup F₂ and backcross progenies were shown to be in the range of 32.0–76.0%. In contrast with this, those of F₂ and backcross progenies of the hybrids obtained in the intergroup cross were shown to be markedly reduced to the range of 0.0–2.5%. Viable clones obtained in these intra-and intergroup crosses were almost all fertile, but one sterile clone was fonnd among F₁ progeny of Group II-B. It was concluded that the so-called hybrid breakdown is also at work as, an isolating mechanism between the two groups of this complex. This study was supported by the Grants in Aid. Nos. 00554220 and 56122019, from the Scientific Research Found of the Ministry of Education, Science and Culture, Japan.     

Using SSR markers for hybrid identification and resource management in Vietnamese <Emphasis Type="Italic">Acacia</Emphasis> breeding programs

We used a set of 16 SSR markers to check the identity of pure-species and hybrid clones in Vietnam’s Acacia auriculiformis, Acacia mangium, and acacia hybrid (A. mangium × A. auriculiformis) breeding programs. The statistics package HIest, applied to a large synthesized population, enabled accurate allocation of genotypes to the two pure species, F₁ and F₂ inter-specific hybrids and backcrosses, based on estimates of hybridity and heterozygosity. The hybridity status of putatively pure A. mangium and A. auriculiformis clones in adjacent clonal seed orchards was checked. Four out of 100 clones selected as A. mangium were found to be backcrosses (A. mangium × F₁ inter-specific hybrid) while out of 96 clones selected as A. auriculiformis, two were F₁ hybrids and two were backcrosses (A. auriculiformis × F₁ hybrid). The markers were then applied to check the hybridity status of 160 putative acacia F₁ hybrid genotypes that had been selected on morphological criteria from open-pollinated progenies collected from A. auriculiformis and A. mangium parents. Many selections based on morphology were found to be mistaken. Only thirteen of 63 clones originating from A. auriculiformis mothers were F₁ hybrids, four were backcrosses, and the remaining 46 were pure A. auriculiformis. Fewer mistakes were evident for clones selected from A. mangium mothers, with 82 out of 89 clones confirmed as F₁ hybrids, three as backcrosses, and four as pure A. mangium. The occurrence of F₁ hybrids and backcrosses in pure-species seed orchards and their progeny shows that inter-species contamination is an issue requiring management in both pure-species and in hybrid breeding of these species in Vietnam. Examination of genetic distances among verified clones showed patterns of relatedness that were consistent with pedigree records. Implications for resource management as well as for breeding and clonal selection strategies are considered.     

A SEX DETERMINING MECHANISM IN THE CLOSTERIUM EHRENBERGII (CHLOROPHYTA) SPECIES COMPLEX1

Homozygous mt^?/mt^? diploid clones of the Closterium ehrenbergii Menegh. ex Ralfs species complex were obtained by hypertonic treatment from minus vegetative cells, and mating type segregation ratios in the F₁ progeny of “triploid” zygospores between wild type mt⁺ haploid and mt^?/mt^? homozygous diploui were analyzed. The ratio of plus to minus individuals was 1:4.8, and the ratio of the pairs of opposite mating types to those of minus mating type was 1:2.1. The results clearly show that mt^? is dominant to mt⁺ and that the mating type inheritance in these zygospores follows the triploid-like pattern. The validity of our assumption that the two mating types are determined by one genetic factor (mt^? allele dominant) was confirmed in B₁ progeny analyses as well. The results suggest that this sex determining mechanism is working effectively in the C. ehrenbergii species complex, in which several biological species have evolved through polyploidization.     

Development of a ploidy series from a single interspecific Trifolium repens L.Ā . nigrescens Viv. F1 hybrid

 The objective of the current research was to generate a ploidy series of backcross progenies from a single triploid (2n=3x=24) Trifolium repens×T. nigrescens F₁ hybrid (3x H-6909-5). The 3x H-6909-5 plant was highly sterile and produced no seeds from approximately 3000 reciprocal backcrosses to both parental species. Chromosome doubling by an in vitro colchicine method resulted in a marked increase in fertility. Pollen stainability was increased from 9.9% in 3x H-6909-5 to an average of 89.2% (range 87.7–90.9%) in the three chromosome-doubled 6x H-6909-5 plants. Subsequent backcrosses of 6x H-6909-5 and interbreeding of backcross derivatives resulted in an array of fertile hybrids at 4x, 5x and 7x levels and some aneuploids. The occurrence of 7x BC₁F₁ progeny from the T. repens×6x H-6909-5 (4x×6x) cross is the first unequivocal evidence of functional female 2n gametes in white clover. Meiotic pairing in F₁ and BC₁F₁ progeny indicated the presence of allosyndetic pairing, suggesting that genetic exchange between the two species is possible. Received: 17 October 1996 / Accepted: 8 November 1996     

Doubled haploids of novel trigenomic Brassica derived from various interspecific crosses

To develop doubled haploid (DH) mapping populations of hexaploid Brassica, 10 F₁ hybrids derived from crosses between allohexaploid Brassica parents were evaluated in this study. The allohexaploid Brassica parents were selfed progenies of unique interspecific crosses between Brassica rapa (genome AA) × B. carinata (BBCC), B. nigra (BB) × B. napus (AACC), and a complex cross between B. juncea (AABB), B. napus and B. carinata, with relatively stable chromosome number (2n = 54). Hexaploid status and chromosome behavior during meiosis I in four promising F₁ hybrids were assessed using microscopy and flow cytometry, and progeny were obtained following microspore culture. Hybrids H11-2 and H16-1 demonstrated higher amenability for embryo generation, plantlet regeneration, and frequency of production of DH microspore-derived progeny of hexaploid DNA content (6x) compared to hybrids H08-1 and H24-1. A total of 370 6x DH progeny were selected out of 693 plantlets from H11-2, 241/436 from H16-1, 23/54 from H08-1, and 21/56 from H24-1. DH progenies of hybrids H11-2 and H16-1 were then designated as promising mapping populations of a new hexaploid Brassica species.     

Inheritance of Cry1Ac resistance and associated biological traits in the cotton bollworm, Helicoverpa armigera (Lepidoptera: Noctuidae)

The analysis of reciprocal genetic crosses between resistant Helicoverpa armigera strain (BH-R) (227.9-fold) with susceptible Vadodara (VA-S) strain showed dominance (h) of 0.65-0.89 and degree of dominance (D) of 0.299-0.782 suggesting Cry1Ac resistance as a semi-dominant trait. The D and h values of F₁ hybrids of female resistant parent were higher than female susceptible parent, showing maternally enhanced dominance of Cry1Ac resistance. The progeny of F₂ crosses, backcrosses of F₁ hybrid with resistant BH-R parent did not differ significantly in respect of mortality response with resistant parent except for backcross with female BH-R and male of F₁ (BH-R × VA-S) cross, suggesting dominant inheritance of Cry1Ac resistance. Evaluation of some biological attributes showed that larval and pupal periods of progenies of reciprocal F₁ crosses, backcrosses and F₂ crosses were either at par with resistant parent or lower than susceptible parent on treated diet (0.01 μg/g). The susceptible strain performed better in terms of pupation and adult formation than the resistant strain on untreated diet. In many backcrosses and F₂ crosses, Cry1Ac resistance favored emergence of more females than males on untreated diet. The normal larval period and the body weight (normal larval growth) were the dominant traits associated with susceptible strain as contrast to longer larval period and the lower body weight (slow growth) associated with resistance trait. Further, inheritance of larval period in F₂ and backcross progeny suggested existence of a major resistant gene or a set of tightly linked loci associated with Cry1Ac sensitivity.     

Potential gene flow of two herbicide-tolerant transgenes from oilseed rape to wild B. juncea var. gracilis

Four successive reciprocal backcrosses between F₁ (obtained from wild Brassica juncea as maternal plants and transgenic glyphosate- or glufosinate-tolerant oilseed rape, B. napus, as paternal plants) or subsequent herbicide-tolerant backcross progenies and wild B. juncea were achieved by hand pollination to assess potential transgene flow. The third and forth reciprocal backcrosses produced a number of seeds per silique similar to that of self-pollinated wild B. juncea, except in plants with glufosinate-tolerant backcross progeny used as maternal plants and wild B. juncea as paternal plants, which produced fewer seeds per silique than did self-pollinated wild B. juncea. Germination percentages of reciprocal backcross progenies were high and equivalent to those of wild B. juncea. The herbicide-tolerant first reciprocal backcross progenies produced fewer siliques per plant than did wild B. juncea, but the herbicide-tolerant second or third reciprocal backcross progenies did not differ from the wild B. juncea in siliques per plant. The herbicide-tolerant second and third reciprocal backcross progenies produced an amount of seeds per silique similar to that of wild B. juncea except for with the glufosinate-tolerant first and second backcross progeny used as maternal plants and wild B. juncea as paternal plants. In the presence of herbicide selection pressure, inheritance of the glyphosate-tolerant transgene was stable across the second and third backcross generation, whereas the glufosinate-tolerant transgene was maintained, despite a lack of stabilized introgression. The occurrence of fertile, transgenic weed-like plants after only three crosses (F₁, first backcross, second backcross) suggests a potential rapid spread of transgenes from oilseed rape into its wild relative wild B. juncea. Transgene flow from glyphosate-tolerant oilseed rape might be easier than that from glufosinate-tolerant oilseed rape to wild B. juncea. The original insertion site of the transgene could affect introgression.     

Cytoplasmic effects on activity of individual anionic isoenzymes of peroxidase in crosses between two genotypes of flax (Linum usitatissimum L.)

Four anionic peroxidase isoenzymes in main stem tissues of flax (Linum usitatissimum L.) were separated electrophoretically on acrylamide gels and their individual activities measured spectrophotometrically in the gels. Activities were expressed in terms of areas of optical density peaks corresponding to the locations of the isoenzymes. Four isoenzymes were assayed for activity in this way in two inbred genotypes and their F₁, F₂, and first backcross progeny. Crosses were made in all combinations between the two parental genotypes and their reciprocal F₁ hybrids to produce 16 progenies of the generations above. Isoenzyme separations and assays for activity were carried out on individual plants of each of the 16 progenies. A model estimating cytoplasmic effects across segregating generations as differences between all progeny of the one compared to the other F₁ reciprocal hybrid, either in a male or a female direction, was fitted to the activity data by weighted least-squares procedures. Cytoplasmic effects transmitted through male gametes from the F₁ reciprocals were demonstrable for three of the four anionic peroxidase isoenzymes.Supported in part by the National Research Council of Canada.     

A newly identified chemotactic sexual pheromone from Closterium ehrenbergii

 In sexual reproduction of Closterium ehrenbergii, pairing with the sexual partner cells is the first process observed. A cell migration-inducing activity, specific for mating-type plus (mt⁺; NIES-228) cells, was detected in the culture medium of mating-type minus (mt^–; NIES-229) cells. Light was necessary for production of the active substance by mt^– cells and for migration of mt⁺ cells. The active substance was heat-labile and had an apparent molecular mass of 20 kDa, as determined by gel filtration. A protein of 20 kDa was detected in the active fraction of gel filtration after sodium dodecyl sulfate polyacrylamide gel electrophoresis. Based on these results, it is proposed that a chemotactic sexual pheromone involved in the formation of sexual pairs of cells is secreted by mt^– cells of C. ehrenbergii and is proteinaceous, like other sexual pheromones secreted by Closterium species. Received: 31 July 1997 / Revision accepted: 25 November 1997     

Genetic Control of Resistance to the Sterol 14α-Demethylase Inhibitor Fungicide Prochloraz in the Cereal Eyespot Pathogen Tapesia yallundae

Sexual crosses were used to determine the genetic basis of resistance to the sterol 14 α-demethylase inhibitor fungicide prochloraz in the cereal eyespot pathogen Tapesia yallundae. Three different crosses between sensitive parental strains (22-432 and 22-433 [the concentration required to inhibit growth by 50% {IG₅₀} for each was ≤0.03 mg/liter]) and field isolates from France and New Zealand with differing levels of resistance (PR11 [IG₅₀ = 0.5 mg/liter], PR1 [IG₅₀ = 1.0 mg/liter], and 11-3-18 [IG₅₀ = 2.4 mg/liter]) yielded progeny showing a bimodal distribution, with an even number of sensitive and resistant progeny. This indicated the segregation of a single major gene for resistance in each cross, which was confirmed by the use of backcrosses, crosses between F₁ progeny, and control crosses between sensitive parents. However, there was also evidence of additional quantitative genetic components responsible for the increased IG₅₀s of the more resistant isolates. A further cross was made between isolate PR11 and an F₁ progeny arising from isolate 11-3-18, and this also yielded progeny which were entirely prochloraz resistant. This suggested that resistance genes were allelic in these two isolates, with resistance conferred by a gene at the same locus (or closely linked loci), despite the fact that the isolates (PR11 and 11-3-18) originated from different continents.     

Inheritance of chromosome length polymorphisms inCoprinus cinereus

The sexually compatible strains ofCoprinus cinereus 5302 and Dd 13 revealed chromosome length polymorphisms in their electrophoretic karyotypes. The dikaryon derived from two monokaryons contained a mixture of the two electrophoretic patterns. F₁ progenies were isolated by crossingC. cinereus 5302 and Dd 13 strains and it showed unique karyotypes. Chromosome length polymorphisms of both parental strains were inherited at random in the F₁ progenies. As a result, several novel electrophoretic karyotypes which had not been observed in either parental strains were found in the F₁ progeny. The rDNA probe hybridized with one chromosome in both parental strains, with two chromosomes in the hybridization pattern of both parental strains in the dikaryon, and with one to two chromosomes in the F₁ progenies. The relation between mating type and hybridization pattern has thus not been made clear in the case of F₁ progeny.     

Genetics of self incompatibility in diploid Ageratum houstonianum mill

Summary Diallels and backcrosses among self-incompatible (SI) clones and progeny of Ageratum houstonianum Mill. could be organized into intra-incompatible classes. Four of 5 progenies segregated in expected ratios of S genotypes. Ageratum expressed a one-locus incompatibility system of the sporophytic type with a linear dominance series of multiple alleles and complete allelic dominance in both pollen and stigma. In the second part of the study, a high percentage of self-seed set was observed during the first flowering of a progeny from a pseudo-self compatible (PSC) seed source. Six progenies were derived from the PSC seed source. Five of the 6 segregated PSC SI plants, 4 of which fit a 3 1 ratio of PSC SI plants. All plants of the sixth progeny were SI. Two F₁ progenies with the same PSC pollen parent produced significantly different segregations of PSC SI plants. It appeared that PSC acted as a major gene when the most recessive S allele was also present, but PSC was not expressed when the most dominant S allele was present. Clones propagated from PSC plants were SI and cross incompatible with a related S-allele tester. Thus, PSC was transient in that it was apparent in seed-propagated plants but not in plants clonally propagated from the PSC individuals.Scientific Journal Series Paper Number 12,299 of the Minnesota Agricultural Experiment Station     

Isogenic Strains of PHYCOMYCES BLAKESLEEANUS Suitable for Genetic Analysis

The progeny of crosses between wild-type strains of Phycomyces usually do not exhibit all of the expected genotypes from meiosis. By backcrossing, we have isolated a new (+) mating-type strain, A56, which is nearly isogenic with the (-) wild-type NRRL1555 commonly used in Phycomyces research. Tetrad analysis of the backcrosses shows that meiosis becomes more regular as the parental (+) and (-) strains become more isogenic. In our two-factor crosses with unlinked markers, the regularity of meiosis is measured as the percent of reciprocal ditypes plus tetratypes in the progeny. We have shown that this percentage increases from about 15% for crosses between nonisogenic parents to 90% in the eighth backcross. The results indicate that routine, reliable recombination analyses are possible in P. blakesleeanus.     

马铃薯杂种F1无性株系的ISSR鉴定

为给马铃薯新品种选育提供可靠材料,采用ISSR分子标记对2个马铃薯杂交组合‘J07-4’ב陇薯6号’和‘J07-6’ב陇薯6号’杂交种F₁无性繁殖株系的真实性进行了鉴定。结果从152个ISSR引物中筛选出适于‘J07-4’ב陇薯6号’杂种F₁ 5个无性株系鉴定的3个ISSR引物AF18550、AW75511和AW20617及‘J07 6’ב陇薯6号’杂种F₁ 7个无性株系鉴定的2个ISSR引物AW20607和AF18549;以子代中含有父本特征带为主要依据,鉴定出杂种F₁共12个无性繁殖株系均为真实的杂交种,并建立了杂种F₁不同无性株系间的ISSR指纹图。表明ISSR分子标记技术用于马铃薯杂种F₁无性株系鉴定是可行的。     

Biochemical genetics of hybrid sunfish: Differential survival of heterozygotes

Allelic segregation in reciprocal backcrosses involving the largemouth bass (Micropterus salmoides) and the F₁ hybrid (largemouth bass × smallmouth bass, M. dolomieui) was investigated to determine the extent of euheterosis and luxuriance. The frequencies of allelic isozymes encoded in the lactate dehydrogenase E, malate dehydrogenase B, and isocitrate dehydrogenase loci were determined for reciprocal backcross progeny subjected to different selection pressures. The progeny of the backcross (male F₁ × female largemouth bass) underwent a rapid loss of heterozygous individuals in a natural pond environment. When the offspring of this same mating were placed in artificial pools, where cannibalism is the main source of mortality, heterozygosity was advantageous. There was a marked correlation of increased heterozygosity at these enzyme loci with an increased growth rate. None of the above responses to selection was observed when the F₁ hybrid served as the maternal parent in the reciprocal backcross. A maternal factor in the egg cytoplasm may influence the expression of heterosis.     

Inheritance of bluesac disease for hatchery charr of the genus Salvelinus

Synopsis Bluesac disease is one of the major causes of post-hatching embryo mortality in salmonids. The incidence of bluesac is strongly inherited in splake (Salvelinus fontinalis × S. namaycush hybrid) under hatchery conditions. Lake charr, F₂ splake, F₆ splake, and lake charr backcrosses of F₆ splake had incidences of 7%, 23%, 5% and 40% respectively. For the backcrosses, which are of practical interest because they are used for experimental plantings in the Great Lakes and northern Ontario inland lakes, the variation among families in the incidence of bluesac is primarily determined by the splake parent. Hence the incidence of bluesac disease in the progeny of splake and lake chair is considered to be a character of the splake parent. The heritability of this character in the splake is about 0.74. The level of bluesac in the backcrosses can, therefore, be significantly reduced in one generation by selecting as breeders those splake that produce low levels of bluesac in test backcrosses to lake charr.     

Natural populations of the Closterium ehrenbergii (Desmidiales, Chlorophyta) species complex in Nepal

Several natural populations of the Closterium ehrenbergii Meneghini ex Ralfs species complex were collected in Nepal, in October–December 1982. Water temperature and pH were also recorded. Clonal isolates from these populations were identified to one of four mating groups (H, I, J and M) by test crossing with standard mating-type strains of known mating groups. Groups H and M have smooth walled zygospores, while Groups I and J have scrobiculated zygospore walls. Several undetermined isolates were found in some population samples. In contrast to the previously reported population samples from Nepal, especially from dried soil samples, some of these populations appeared to be rather heavily loaded with mutations that are deleterious to the sexual cycle (i.e. sexual compatibility, zygospore formation and germination). By genetic analysis, a zygote maturation-defective mutation (zym) was detected. One reason for such a heavy genetic load was suggested to be that most population samples had been maintained exclusively by asexual reproduction for a long period in large lakes and nearby ponds, or left-over vegetative populations in paddy fields after other members entered into dormancy through sexual reproduction. The significance of studying such mutations at sexual gene loci is discussed in the light of speciation problems in microalgae.     

Some genetic aspects of the resistance of Spodoptera frugiperda to a nuclear polyhedrosis virus

A probit analysis of the dosage-response of two isolates of Spodoptera frugiperda to a single nuclear polyhedrosis virus revealed a greater than 5-fold difference in the LD₅₀. The response of F₁ progeny of reciprocal crosses between the two parental isolates demonstrated that resistance to the virus is not sex-linked. The LD₅₀ values of F₁ and F₂ backcrosses suggest that the major variation in resistance is due to a single gene or genes that lack dominance.     

Protein and RNA Synthesizing Activities during Sexual Process of Heterothallic Closterium

Protein and RNA synthesizing activities increased markedly during the mating process and decreased during the maturation stage of zygotes in heterothallic strains of Closterium peracerosum-strigosum-littoale, KAS-4-29 (mating-type minus) and KAS -4-30 (mating-type plus) and a homothallic Closterium acerosum. Different proteins were synthesized at the different stages of the mating process, suggesting that a sequential expression and repression of mating genes occur for the mating-specific protein synthesis during the sexual reproduction.