Variation in vigour and in RFLP-estimated heterozygosity by selfing tetraploid alfalfa: new perspectives for the use of selfing in alfalfa breeding

The effect of selfing on vigour and heterozygosity was analysed in six independent families obtained by recurrent cycles of self-fertilization and selection until the S₃ generation. The heterozygosity level, estimated by means of 11 homologous probes, decreased from the S₀ to S₃ generations and was partially restored in S₂×S₂ polycrosses. The decreasing trend was influenced by the inbreeding level of the chosen mother plant in each self generation to advance in subsequent selfing. Plant vigour estimated by Dry Matter Yield (DMY) decreased during inbreeding, and phenotypic selection of S₂ individuals seemed to prove effective as the differences in DMY between vigorous and weak plants were maintained in S₂×S₂ crosses. No correlation between plant vigour and heterozygosity was found between subgroups of vigorous and weak plants selected within the same S₂ family. Results are discussed with a view to selecting the best performing and least heterozygous plants during inbreeding to isolate useful genes and linkats in superior partially inbred parental lines. Received: 4 August 1999 / Accepted: 6 December 1999     

Trait mean depression for second-generation inbred strawberry populations with and without parent selection

 Strawberry genotypes selected for superior fruit yield or chosen at random from first-generation self, full-sib, and half-sib populations were crossed to provide second-generation inbred progenies and composite cross-fertilized control populations. Mean yields for inbred offspring from crosses among selected parents exceeded those from the offspring of unselected parents by 87%, 23%, and 37% for self, full-sib, and half-sib populations, respectively; yields for offspring from unrelated crosses among selected parents were 54% larger than those for crosses among unselected parents. Selection for yield also resulted in significant correlated response for fruit number and plant diameter. Mean yields for second-generation half-sib and full-sib offspring from selected parents were greater than those for offspring from the unselected but non-inbred control population. This suggests that selection can be a powerful force in counteracting most of the inbreeding depression expected in cross-fertilized strawberry breeding programs. Selection treatment× inbreeding rate interactions were non-significant for all traits; thus, selection among partially inbred offspring did not have a large effect on the rate of genetic progress. Differential realized selection intensity among individuals with differing levels of homozygosity accumulated due to inbreeding is suggested as the most likely explanation for the absence of association between pedigree inbreeding coefficients and cross performance detected previously in strawberry. Received: 21 July 1996 / Accepted: 7 March 1997     

On estimating the linkage of marker genes to viability genes controlling inbreeding depression

Statistical properties and extensions of Hedrick and Muona's method for mapping viability alleles causing inbreeding depression are discussed in this paper. Their method uses the segregation ratios among selfed progeny of a marker-locus heterozygote to estimate the viability reduction, s, of an allele and its recombination fraction, c, with the marker. Explicit estimators are derived for c and s, including expressions for their variances. The degree of estimation bias is examined for cases when (1) the viability allele is partially recessive and (2) the marker locus is linked to two viability loci. If linkage or viability reduction is moderate, very large sample sizes are required to obtain reliable estimates of c and s, in part because these estimates show a statistical correlation close to unity. Power is further reduced because alleles causing viability reduction often occur at low frequency at specific loci in a population. To increase power, we present a statistical model for the joint analysis of several selfed progeny arrays selected at random from a population. Assuming a fixed total number of progeny, we determine the optimal number of progeny arrays versus number of progeny per array under this model. We also examine the increase of information provided by a second, flanking marker. Two flanking markers provide vastly superior estimation properties, reducing sample sizes by approximately 95% from those required by a single marker.     

The performance of microgametophytes is affected by inbreeding depression and hybrid vigor in the sporophytic generation

Inbreeding reduces the level of heterozygosity, thereby exposing deleterious recessives to selection and simultaneously reducing the number of loci expressing heterosis (overdominance). In contrast, hybridization increases the level of heterozygosity, thereby masking deleterious recessives and simultaneously increasing the number of loci expressing heterosis. Most studies of inbreeding depression/hybrid vigor have focused on sporophytic performance such as survivorship, vegetative growth rates, fruit and seed production and (rarely) pollen production. Because the genetic mechanisms that underlie inbreeding depression/hybrid vigor are relevant only to the diploid stage of the life cycle, most studies have tacitly assumed that they have no effects on pollen performance (pollen germination, pollen tube growth rate, ability to achieve fertilization under conditions of pollen competition). However, we reasoned that because pollen is dependent upon the sporophyte for the resources necessary to develop, germinate and initiate tube growth, the level of heterozygosity (vigor) in the pollen-producing parent can affect pollen performance by affecting the ability of the sporophyte to provision its pollen. In a series of studies conducted under field conditions over 7 years, we experimentally varied the level of heterozygosity in wild gourd (Cucurbita pepo) plants (four levels of inbreeding, f = 0.75, 0.50, 0.25, 0 and a zucchini × wild gourd F1). We found that sporophytic vigor (e.g., flower and fruit production) increased with the level of heterozygosity and that the level of heterozygosity of the sporophyte affects the in vitro and in vivo performance of the microgametophytes it produces. These findings are analogous to the ”maternal environmental effects” frequently observed in seeds. Received: 29 October 2000 / Accepted: 3 May 2001     

Genetic analysis of inbreeding depression in plus tree 850.55 of Pinus radiata D. Don. II. Genetics of viability genes

 Most coniferous species exhibit severe inbreeding depression. Selfed individuals usually have decreased viability, reduced vigour and morphological defects. The number of filled seeds after selfing Pinus radiata plus tree 850.55 was 48% that of the outcrossing, and 26.1% of the selfed seedlings died at an early stage. The segregation of 172 markers (covering 56% of the genome) in selfed progenies of radiata pine plus tree 850.55 was studied. Based on the segregation ratio of the markers, genes associated with inbreeding depression on viability were identified (P<0.05). Using the Expectation/Conditional Maximization (ECM) algorithm, we estimated the location, degree of dominance and selection coefficient of viability genes. Nine viability genes were discovered. Seven of them appeared to be dominant and one partially dominant (degree of dominance=0.4). The other gene was overdominant or pseudo-overdominant, with selection coefficients for the two homozygotes of 0.4 and 0.42, respectively. Of the genes showing dominance or partial dominance, seven were sub-lethal with selection coefficients ranging from 0.55 to 0.79; one gene (SDPr), which was responsible for seedling death within the first month following germination, was lethal. Received: 13 December 1998 / Accepted: 28 December 1998     

Levels of genetic diversity at different stages of the domestication cycle of interior spruce in British Columbia

Concerns over the reductionist nature of the domestication of forest-tree species focus on the possibility of potential genetic erosion during this process. To address these concerns, genetic diversity assessments in a breeding zone the Province of British Columbia “interior” spruce (Picea glauca×engelmanni) program was conducted using allozyme markers. Genetic-variation comparisons were made between natural and production (seed orchard) populations as well as seed and seedling crops produced from the same breeding zone’s seed orchard. The natural population sample consisted of a total of 360 trees representing three stands within each of three watersheds present in the Shuswap-Adams low-elevation zone of interior British Columbia. Small amounts of genetic differentiation were observed among the nine natural populations (4%) and this was attributable to extensive gene flow Consequently, the sum of these nine populations was considered as a baseline for the genetic variation present in the breeding zone. The comparisons between the seed orchard and the breeding zone produced a similar percentage of polymorphic loci while the expected hetrozygosity (0.207 vs 0.210) and the average number of alleles per locus (2.7 vs 2.4) were slightly lower in the seed orchard. A total of seven natural populations’ rare alleles were not present in the orchard population, while one allele was unique to the orchard. The %P increased to 70.6% in the seedlot, but dropped to the natural populations level (64.7%) in the plantation. The observed increase in %P was a result of pollen contamination in the orchard. It is suspected that the reduction in the plantation was caused by an unintentional selection in the nursery. Simulated roguing in the orchard did not drastically reduce even if up to 50% of the orchard’s clones were rogued. However, roguing was associated with a reduction in the average number of alleles per locus (i.e., sampling effect). Received: 2 January 1996 / Accepted: 24 May 1996     

Specifying the introgressed regions from H. argophyllus in cultivated sunflower (Helianthus annuus L.) to mark Phomopsis resistance genes

A method based upon targetting of intro-gressed markers in a Phomopsis-resistant line (R) of cultivated sunflower, issuing from a H. argophyllus cross was used to mark the Phomopsis resistance regions. Our study was based upon 203 families derived from a cross between an inbred line susceptible to Phomopsis (S1) and the introgressed resistant line (R). Families were checked for Phomopsis resistance level in a design with replicated plots and natural infection was re-inforced by pieces of contaminated stems. Thirty four primers were employed for RAPD analysis. Out of 102 polymorphic fragments between (S1) and H. argophyllus, seven were still present in (R) suggesting that they marked introgressions of H. argophyllus into (R). The plants were scored for the presence or absence of 19 fragments obtained from five primers, and the relationships between the presence/absence of fragments in plants and Phomopsis resistance/susceptiblity in the progenies was determined by using an analysis of variance. We found that at least two introgressed regions, as well as favourable factors from sunflower, contributed to the level of Phomopsis resistance in cultivated sunflower. Received: 28 June 1996 / Accepted: 5 July 1996     

Evaluation of experimental designs and spatial analyses in wheat breeding trials

Thirty-three wheat breeding trials were conducted from 1994 to 1996 in the Northern Grains Region (QLD and Northern NSW) of Australia to evaluate the influence of experimental designs and spatial analyses on the estimation of genotype effects for yield and their impact on selection decisions. The relative efficiency of the alternative designs and analyses was best measured by the average standard error of difference between line means. Both more effective designs and spatial analyses significantly improved the efficiency relative to the randomised complete block model, with the preferred model (which combined the design information and spatial trends) giving an average relative efficiency of 138% over all 33 trials. When the Czekanowski similarity coefficient was used, none of the studied models were in full agreement with the randomised complete block model in the selection of the top lines. The agreement was influenced by selection proportions. Hence, the use of these methodologies can impact on the selection decisions in plant breeding. Received: 17 December 1998 / Accepted: 29 July 1999     

Highly polymorphic microsatellites of rice consist of AT repeats, and a classification of closely related cultivars with these microsatellite loci

Microsatellites consisting of AT repeats are highly polymorphic in rice genomes and can be used to distinguish between even closely related japonica cultivars in Japan. Polymorphisms of 20 microsatellite loci were determined using 59 japonica cultivars, including both domestic and modern Japanese cultivars. Although the polymorphisms of these 20 microsatellite loci indicated that the Japanese cultivars were genetically quite similar, microsatellites consisting of AT repeats showed high gene diversity even among such closely related cultivars. Combinations of these hypervariable microsatellites can be employed to classify individual cultivars, since the microsatellites were stable within each cultivar. An identification system based on these highly polymorphic microsatellites could be used to maintain the purity of rice seeds by eliminating contamination. A parentage diagnosis using 17 polymorphic microsatellite loci clearly demonstrated that plants which carried desired chromosome regions had been selected in breeding programs. Thus, these hypervariable microsatellites consisting of AT repeats should promote the selection of plants which carry desired chromosomes from genetically similar parents. Backcrossing could also help to eliminate unnecessary chromosome regions with microsatellite polymorphisms at an early stage in breeding programs. Received: 8 July 1996 / Accepted: 12 July 1996     

Inbreeding in Pinus radiata. I. The effect of inbreeding on growth, survival and variance

 The effects of inbreeding on growth, survival and variance in a 12-year-old radiata pine trial were studied in five populations each inbred to one of five different levels: outcross (F=0), half-sib (F=0.125), full-sib (F=0.25), selfing (S₁, F=0.5), and two-generations of selfing (S₂, F=0.75). These five populations were derived from a founder population of eight clones. Inbreeding reduced diameter, growth, and survival but increased the variance for diameter. Inbreeding depression at F=0.125, 0.25, 0.5, and 0.75 was 5%, 6%, 15%, and 19% respectively for DBH; −3%, 1%, 7%, and 11% respectively, for survival. The standard deviation for diameter increased by 10%, 10%, 30%, and 25% respectively for F=0.125, 0.25, 0.5, and 0.75 and, similarly, the coefficient of variation increased by 17%, 16%, 53%, and 55% respectively. There were significant differences among the eight founder clones in their response to inbreeding. The best clone in the trial showed no inbreeding depression. Overall, inbreeding depression was found to be linearly related to the inbreeding coefficient F with no significant quadratic effects for any trait at any population level. However, two individual clones had a quadratic relationship with F for DBH and one clone had a similar relationship for survival. A significant correlation (r=0.96) between S₂ and the breeding values of founder clones was observed while the correlation (r=0.58) between S₁ and breeding values was insignificant. The low inbreeding depression in radiata pine relative to other conifers may indicate that historical purging of detrimental alleles through small geographic populations, a higher degree of population subdivision, and the relative high fecundity of inbred progenies has rendered radiata pine an ideal species to use inbreeding as a breeding tool. Received: 10 March 1998 / Accepted: 19 May 1998     

Positive assortative mating with selection restrictions on group coancestry enhances gain while conserving genetic diversity in long-term forest tree breeding

Selection and mating principles in a closed breeding population (BP) were studied by computer simulation. The BP was advanced, either by random assortment of mates (RAM), or by positive assortative mating (PAM). Selection was done with high precision using clonal testing. Selection considered both genetic gain and gene diversity by "group-merit selection", i.e. selection for breeding value weighted by group coancestry of the selected individuals. A range of weights on group coancestry was applied during selection to vary parent contributions and thereby adjust the balance between gain and diversity. This resulted in a series of scenarios with low to high effective population sizes measured by status effective number. Production populations (PP) were selected only for gain, as a subset of the BP. PAM improved gain in the PP substantially, by increasing the additive variance (i.e. the gain potential) of the BP. This effect was more pronounced under restricted selection when parent contributions to the next generation were more balanced with within-family selection as the extreme, i.e. when a higher status effective number was maintained in the BP. In that case, the additional gain over the BP mean for the clone PP and seed PPs was 32 and 84% higher, respectively, for PAM than for RAM in generation 5. PAM did not reduce gene diversity of the BP but increased inbreeding, and in that way caused a departure from Hardy-Weinberg equilibrium. The effect of inbreeding was eliminated by recombination during the production of seed orchard progeny. Also, for a given level of inbreeding in the seed orchard progeny or in a mixture of genotypes selected for clonal deployment, gain was higher for PAM than for RAM. After including inbreeding depression in the simulation, inbreeding was counteracted by selection, and the enhancement of PAM on production population gain was slightly reduced. In the presence of inbreeding depression the greatest PP gain was achieved at still higher levels of status effective number, i.e. when more gene diversity was conserved in the BP. Thus, the combination of precise selection and PAM resulted in close to maximal short-term PP gain, while conserving maximal gene diversity in the BP.Communicated by O. Savolainen     

Conservation and divergence in the control of yolk protein genes in dipteran insects

 We have investigated the conservation of regulatory elements for sex- and tissue-specific gene expression in three dipteran species, Drosophila melanogaster, Musca domestica and Calliphora erythrocephala, using the yolk protein (yp) genes. Yolk proteins of the fruitfly, medfly, housefly and blowfly are very well conserved both in their sequence and their expression in ovarian follicle cells and in fat bodies of adult females. Furthermore, yp regulation by both hormonal and nutritional factors shows similar features in all four species. To study conservation of yp regulation in dipteran insects, we tested 5′ flanking regions from one Musca yp gene and one Calliphora yp gene for enhancer functions in D. melanogaster. Two fragments of 823 and 1046 bp isolated from Musca and Calliphora yp genes, respectively, are able to direct correct expression of a reporter gene in the ovarian follicle cells of transformed Drosophila at specific stages during oogenesis. Surprisingly, these enhancers do not confer sex-specific reporter gene expression in the fat body, as expression was found in both sexes of the transformed flies. None-the-less by in vitro DNA/protein interaction assays, a 284-bp DNA region from the Musca yp enhancer was able to bind the Drosophila DOUBLESEX (DSX) protein, which in D.melanogaster confers sex-specific expression of yp. We speculate that the sex-determining pathway is not directly involved in yp regulation in Musca or Calliphora adult females, but depends instead on hormonal controls to achieve sex-specific expression of yp genes in the adult. Received: 17 April 1997 / Accepted: 12 July 1997     

An allele responsible for seedling death in Pinus radiata D. Don

 When inbred, most outcrossing species show high mortality, manifested at several life stages. The occurrence of homozygotes for deleterious or lethal alleles is believed to be responsible. Here, we report the identification of an allele responsible for the death of selfed Pinus radiata D. Don seedlings in their first month after germination. Among 291 S₁ seedlings of plus-tree 850.55, 76 died within 1 month of emergence. Their death appears to be caused by a single recessive lethal allele, SDPr (seedling death in Pinus radiata). SDPr is located in a linkage group with 28 RAPD markers, the closest of which is ai05800a. Of the 76 seedlings that died, megagametophytes of 73 could be genotyped. Of these, 71 had the null (no band) allele of ai05800a; only two had the band allele. Of the 190 surviving S₁ diploids that were genotyped, only two individuals were homozygous for the null allele of ai05800a. By two different methods, the map distance between SDPr and ai05800a was estimated to be between 1.0 and 2.7 cM respectively. The frequency of band and null alleles in the combined population of dead and surviving seedlings and in un-sown seeds shows no evidence of selection at this locus prior to germination. Received: 30 September 1997 / Accepted: 29 October 1997     

The evolution of paired appendages in vertebrates: T-box genes in the zebrafish

The presence of two sets of paired appendages is one of the defining features of jawed vertebrates. We are interested in identifying genetic systems that could have been responsible for the origin of the first set of such appendages, for their subsequent duplication at a different axial level, and/or for the generation of their distinct identities. It has been hypothesized that four genes of the T-box gene family (Tbx2–Tbx5) played important roles in the course of vertebrate limb evolution. To test this idea, we characterized the orthologs of tetrapod limb-expressed T-box genes from a teleost, Danio rerio. Here we report isolation of three of these genes, tbx2, tbx4, and tbx5. We found that their expression patterns are remarkably similar to those of their tetrapod counterparts. In particular, expression of tbx5 and tbx4 is restricted to pectoral and pelvic fin buds, respectively, while tbx2 can be detected at the anterior and posterior margins of the outgrowing fin buds. This, in combination with conserved expression patterns in other tissues, suggests that the last common ancestor of teleosts and tetrapods possessed all four of these limb-expressed T-box genes (Tbx2–Tbx5), and that these genes had already acquired, and have subsequently maintained, their gene-specific functions. Furthermore, this evidence provides molecular support for the notion that teleost pectoral and pelvic fins and tetrapod fore- and hindlimbs, respectively, are homologous structures, as suggested by comparative morphological analyses. Received: 14 July 1999 / Accepted: 4 September 1999     

Erythropoiesis and unexpected expression pattern of globin genes in the salamander Hynobius retardatus

Transition of hemoglobin (Hb) from larval to adult types during the metamorphosis in a salamander Hynobius retardatus has been reported to occur almost independently of thyroid activity, in contrast to the case with many amphibians. In order to obtain further information on the mechanism of the transition in H. retardatus, larval and adult globin cDNAs were cloned, and the globin gene expression was analyzed in normally developing and metamorphosis-arrested animals. Northern hybridization and RT-PCR revealed that larval globin genes were initially expressed 5 days before hatching, and unexpectedly remained expressed even in juveniles 2 years old. The adult globin gene was expressed 19 days after hatching, much earlier than the initiation of morphological metamorphosis. Furthermore, the pattern of globin gene expression in metamorphosis-arrested larvae was almost identical to that in normal controls, suggesting that the transition occurs independently of thyroid hormones. In larvae recovering from anemia, precocious Hb transition, which occurs in Xenopus laevis and Rana catesbeiana, did not occur in H. retardatus. In situ hybridization convincingly demonstrated that the erythropoietic sites are the ventral blood island and the dorsolateral plate at the prehatching stage. During the ontogeny they changed to the liver, kidney, and spleen and were finally restricted to the spleen. Single erythroid cells expressed concurrently larval and adult globin genes, as demonstrated by double in situ hybridization. Thus the transition occurred within a single erythroid cell population, a unique characteristic of H. retardatus. Received: 5 August 1999 / Accepted: 14 October 1999     

Variation in the number of expressed MHC genes in different cattle class I haplotypes

 Analysis of cattle major histocompatibility complex (MHC) (BoLA) class I gene expression using serological and biochemical methods has demonstrated a high level of polymorphism. However, analysis of class I cDNA sequences has failed to produce conclusive evidence concerning the number and nature of expressed genes. Such information is essential for detailed studies of cattle immune responses, and to increase our understanding of the mechanisms of MHC evolution. In this study a selective breeding programme has been used to generate a number of MHC homozygous cattle expressing common serologically defined class I specificities. Detailed analysis of five class I haplotypes was carried out, with transcribed class I genes identified and characterized by cDNA cloning, sequence analysis, and transfection/expression studies. Surface expression of the gene products (on lymphocytes) was confirmed using monoclonal antibodies of defined BoLA specificity. Phylogenetic analysis of available transcribed cattle MHC class I sequences revealed complex evolutionary relationships including possible evidence for recombination. The study of individual haplotypes suggests that certain groupings of related sequences may correlate with loci, but overall it was not possible to define the origin of individual alleles using this approach. The most striking finding of this study is that none of the cattle class I genes is consistently expressed, and that in contrast to human, haplotypes differ from one another in both the number and composition of expressed classical class I genes. Received: 15 February 1999 / Revised: 23 June 1999     

Growth, metastasis, and invasiveness of Drosophila tumors caused by mutations in specific tumor suppressor genes

 More than 50 genes have been identified in Drosophila by loss-of-function mutations that lead to overgrowth of specific tissues. Loss-of-function mutations in the lethal giant larvae, discs large, or brain tumor genes cause neoplastic overgrowth of larval brains and imaginal discs. In the present study, the growth and metastatic potential of tumors resulting from mutations in these genes were quantified. Overgrown brains and imaginal discs were transplanted into adults and β-galactosidase accumulation was used as a marker to identify donor cells. Mutations in these three genes generated tumors with similar metastatic patterns. For brain tumors, the metastatic index (a measure we defined as the fraction of hosts that acquired secondary tumors normalized for the amount of primary tumor growth) of each of the three mutants was similar. Analysis of cell proliferation in mutant brains suggests that the tumors arise from a population of several hundred cells which represent only 1–2% of the cells in third instar larval brains. For imaginal disc tumors from lethal giant larvae and brain tumor mutants, it is shown for the first time that they can be metastatic and invasive. Primary imaginal disc tumors from lethal giant larvae and brain tumor mutants formed secondary tumors in 43 and 53% of the hosts, respectively, although the secondary tumors were, in general, smaller than the secondary tumors derived from primary brain tumors. Received: 18 August 1997 / Accepted: 16 October 1997     

Reversed sexual size dimorphism in raptors: evaluation of the hypotheses in kestrels breeding in a temporally changing environment

Reversed sexual size dimorphism (RSD, females larger than males) is commonly found in birds of prey. We used kestrels (Falco tinnunculus), breeding in western Finland in a temporally varying environment of 3-year vole cycles, to assess current hypotheses for the evolution and maintenance of RSD. Our 12-year data showed only weak correlations between parental size and breeding parameters (laying date, clutch size and the number of fledglings produced). The degree of RSD per se was unrelated to breeding success, contrary to the prediction of the female dominance hypothesis. Females with small males produced larger clutches in low-vole years, independently of laying date, which supports the small male (or its equivalent inter-sexual selection) hypothesis. Small females tended to have more fledglings, particularly in low-vole years, which is inconsistent with the hypotheses for an advantage of large female size (the starvation, intra-sexual selection, reproductive effort, and supplementary feeding hypotheses). As for males, smaller females may be more efficient hunters, the importance of which should be most pronounced under harsh breeding conditions. Our results suggest that the directional selection on a particular size in kestrels may be under contrasting selection pressures by the environment, and, at least in breeding females, the advantages of large size can actually be counterbalanced during harsh environmental conditions. Received: 7 May 1999 / Accepted: 20 January 2000     

Identifying different types of de-differentiated microspores from indica-japonica F1 hybrids with subspecies-differentiating RFLP probes in rice

The indica, japonica and intermediary types of de-differentiated microspores from indica-japonica hybrids were identified with 11 subspecies-differentiating RELP probes in rice (Oryza sativa L.). The results showed that the distribution of indica, japonica and intermediary types of de-differentiated microspores could be easily detected in a simple and quick way using the RFLP method. Moreover, the microspores from the same hybrid but inoculated onto different media, or microspores from different hybrids when inoculated onto the same medium, often displayed distinctive distribution curves of de-differentiated microspores types, indicating that the media employed in this experiment had high selectivity for the de-differentiation of certain types of microspores. The application of the RELP method to de-differentiated microspore identification is of great theoretical and practical significance in rice doubled-haploid breeding. Received: 27 February 1996 / Accepted: 14 June 1996