Identification of DNA amplification fingerprinting (DAF) markers close to the symbiosis-ineffective sym31 mutation of pea (Pisum sativum L.)

 We demonstrate efficient genome mapping through a combination of bulked segregant analysis (BSA) with DNA amplification fingerprinting (DAF). Two sets of 64 octamer DAF primers, along with two PCR programs of low- and high-annealing temperatures (30°C and 55°C, respectively), appeared to be enough to locate molecular markers within 2–5 cM of a gene of interest. This approach allowed the rapid identification of four BSA markers linked to the pea (Pisum sativum L.) Sym31 gene, which is responsible for bacteroid and symbiosome differentiation. Three of these markers are shown to be tightly linked to the sym31 mutation. Two markers flanking the Sym31 gene, A21-310 and B1-277, cover a 4–5 cM interval of pea linkage group 3. Both markers were converted to sequence-characterized amplified regions (SCARs). The flanking markers may be potential tools for marker-assisted selection or for positional cloning of the Sym31 gene. Received: 2 July 1998 / Accepted: 8 October 1998     

Molecular markers residing close to the Rhg4 locus conferring resistance to soybean cyst nematode race 3 on linkage group A of soybean

 The restriction fragment length polymorphism (RFLP) clone pBLT65 is a 450-nt soybean cDNA encoding a portion of the bifunctional enzyme aspartokinase-homoserine dehydrogenase (AK-HSDH). pBLT65 maps within 3.5 cM of the i locus, conferring a pigmented seed coat, on linkage group A; hence, it is closely linked to the Rhg ₄ locus conferring resistance to race 3 of the soybean cyst nematode. From this useful RFLP we developed a PCR reaction yielding polymorphic bands for use in marker-assisted breeding programs to select progeny containing the Rhg ₄ allele. The polymorphic bands were sequenced to determine the cause of the polymorphisms. Using primers 548 and 563, PCR amplification of DNA from the soybean cultivar Peking (Rhg ₄ ) yielded three DNA fragments, 1a (1160 bp), 1b (1146 bp) and 3 (996 bp). Amplification of DNA from the cultivar Kent (rhg ₄) yielded DNA fragments 2 (1020 bp), 3 (996 bp) and 4 (960 bp). Fragments 1a, 1b, 2 and 4 were also polymorphic between the soybean lines PI 290136 and BARC-2(Rj ₄ ). A segregating population of 80 F₂ and F₃ plants derived from the cross PI 290136×BARC-2 (Rj ₄ ) was used to confirm the map position of the PCR polymorphisms near the i locus, and hence the Rhg ₄ locus on linkage group A. The nucleotide sequences of fragments 1b, 3 and 4 were determined. Large and small deletions in the intronic region were responsible for the size differences of the different fragments, whereas the exon was well conserved. Received: 8 January 1998 / Accepted: 15 July 1998     

Linkage relationships among molecular markers and storage root traits of carrot (Daucus carota L. ssp. sativus)

 A 109-point linkage map consisting of three phenotypic loci (P ₁, Y ₂, and Rs), six restriction fragment length polymorphisms (RFLPs), two random amplified polymorphic DNAs (RAPDs), 96 amplified fragment length polymorphisms (AFLPs), and two selective amplification of microsatellite polymorphic loci (SAMPL) was constructed for carrot (Daucus carota L. ssp. sativus; 2n=2x=18). The incidence of polymorphism was 36% for RFLP probes, 20% for RAPD primers, and 42% for AFLP primers. The overall incidence of disturbed segregation was 18%. Linkage relationships at a LOD score of 4.0 and θ=0.25 indicated 11 linkage groups. The total map length was 534.4 cM and the map was clearly unsaturated with markers spaced at 4.9 cM. AFLP P6B15 was 1.7 cM from P ₁, AFLP P1B34 was 2.2 cM from Y ₂, and AFLP P3B30XA was 8.1 cM from Rs. Received: 2 September 1998 / Accepted: 28 November 1998     

A Genetic linkage map of Pinyon pine (Pinus edulis) based on amplified fragment length polymorphisms

 Amplified fragment length polymorphisms (AFLP) were used to rapidly generate a dense linkage map for pinyon pine (Pinus edulis). The map population consisted of 40 megagametophytes derived from one tree at Sunset Crater, Arizona. A total of 78 primer combinations, each with three to five selective nucleotides, amplified 542 polymorphic markers. Of these, 33 markers showed significant deviation from the expected Mendelian genotypic segregation ratio of 1 : 1, and 164 showed complete linkage with another marker. This resulted in 338 unique markers mapping to 25 linkage groups, each of which ranged from 2 to 22 markers, averaging 80 centiMorgans (cM) in size and covering 2,012 cM (2,200 cM with the inclusion of 25 cM for each of 7 unlinked markers). Pairwise linkage values gave a genome size estimate of 2,390 cM, suggesting comprehensive coverage of the genome. A search for subsets of primer combinations giving the best map coverage found 10 primer combinations which together marked 72% of the linkage map to within 10 cM; an additional 10 primer combinations increased this percentage to 85%. Our map represents an initial step towards the identification of quantitative trait loci associated with pest resistance and water stress in pinyons and will further allow us to examine introgression rates between P. edulis and P. californiarum. Received: 14 October 1997 / Accepted: 29 April 1998     

FISH to meiotic pachytene chromosomes of tomato locates the root-knot nematode resistance gene Mi-1 and the acid phosphatase gene Aps-1 near the junction of euchromatin and pericentromeric heterochromatin of chromosome arms 6S and 6L, respectively

 The root-knot nematode resistance gene Mi-1 in tomato has long been thought to be located in the pericentromeric heterochromatin region of the long arm of chromosome 6 because of its very tight genetic linkage (approx. 1 cM) to the markers Aps-1 (Acid phosphatase 1) and yv (yellow virescent). Using Mi-BAC clones and an Aps-1 YAC clone in fluorescence in situ hybridisation (FISH) to pachytene chromosomes we now provide direct physical evidence showing that Mi-1 is located at the border of the euchromatin and heterochromatin regions in the short arm (6S) and Aps-1 in the pericentromeric heterochromatin of the long arm (6L) close to the euchromatin. Taking into account both the estimated DNA content of hetero- and euchromatin regions and the compactness of the tomato chromosomes at pachytene (2 Mb/μm), our data suggest that Mi-1 and Aps-1 are at least 40 Mb apart, a base pair-to-centiMorgan relationship that is more than 50-fold higher than the average value of 750 kb/cM of the tomato genome. An integrated cytogenetic-molecular map of chromosome 6 is presented that provides a framework for physical mapping. Received: 24 July 1998 / Accepted: 14 August 1998     

Two-trait selection response with marker-based assortative mating

 Marker-based assortative mating (MAM) – the mating of individuals that have similar genotypes at random marker loci – can increase selection response for a single trait by 3–8% over random mating (RM). Genetic gain is usually desired for multiple traits rather than for a single trait. My objectives in this study were to (1) compare MAM, phenotypic assortative mating (PAM), and RM of selected individuals for improving two traits and (2) determine when MAM will be most useful for improving two traits. I simulated 20 generations of selecting 32 out of 200 individuals in an F₂ population. The individuals were selected based on an index (SI) of two traits and were intermated by MAM, PAM, or RM. I studied eight genetic models that differed in three contrasts: (1) weight, number of quantitative trait loci (QTL), and heritability (h ²) for each trait; (2) linkage of QTL for each trait; and (3) trait means of the inbred parents of the F₂. For SI and the two component traits, MAM increased short-term selection response by 5–8% in six out of the eight genetic models. The MAM procedure was least effective in two genetic models, wherein the QTL for one trait were unlinked to the QTL for the other trait and the parents of the F₂ had divergent means for each trait. The loss of QTL heterozygosity was much greater with MAM than with PAM or RM. Consequently, the advantage of MAM over RM dissipated after 5–7 generations. Differences were small between selection responses with PAM and RM. The MAM procedure can enhance short-term selection response for two traits when selection is not stringent, h ² is low, and the means of the parents of the F₂ are equal for each trait. Received: 10 June 1998 / Accepted: 5 August 1998     

Chemical and genetic characterization of calli derived from somatic hybridization between tansy (Tanacetum vulgare L.) and pyrethrum (Tanacetum cinerariifolium (Trevir.) Schultz-Bip.)

 Pyrethrum (Tanacetum cinerariifolium (Trevir.) Schultz-Bip.) produces environmentally benign pesticides, the pyrethrins, and tansy (Tanacetum vulgare L.) lower terpenes of variable biological effectiveness. As an approach to improve the oil content and composition of tansy for enhanced biological activity, a somatic hybridization technique between tansy and pyrethrum was established. About 1×10⁶ of leaf-mesophyll protoplasts of both species were mixed and fused with a solution containing 15% polyethylene glycol. Light-green and yellowish calli developed from the fusion experiments. The fusion-derived calli grew vigorously on MS medium supplemented with 6.4 mg l^-1 of BAP, 0.8 mg l^-1 of NAA, and 30–40 g l^-1 of glucose. Nuclear DNA content, RAPD patterns, and volatile compounds were analyzed to determine the hybridity of the calli. The nuclear DNA content of the tansy and pyrethrum genotypes, and the protoplast-derived calli of tansy were 6.41, 7.39, 13.84, and 8.11 pg, respectively. The nuclear DNA content of individual calli derived from the protoplast fusion between tansy +tansy ranged from 8.84 (F43A) to 19.59 pg (F43C) while those of the tansy+pyrethrum fusions were 10.66 (F46A) and 31.87 pg (F46B). Using four 10-mer primers a total of 56 RAPD-PCR fragments were produced. The distance matrices of fragments were calculated by average linkage cluster analysis. Two visually separated clusters were observed. One cluster consisted of the two tansy genotypes and the fusion-derived callus F43A; the other consisted of pyrethrum and fusion-derived calli F46B and F46C. Volatile compounds, such as decadienal, artedouglasia oxide, heptadecane, syringaldehyde and coniferyl alcohol, analyzed by gas chromatography mass spectrometry, were found only in the protoplast fusion-derived calli F43A and F46B. Several less volatile compounds were also detected only in fusion calli. Hexadecanoic and linoleic acids were common to fusion-derived calli and tansy, and one unknown compound to fusion-derived calli and pyrethrum. Pyrethrins I and II were detected from pyrethrum, but not from the fusion-derived calli. The additive nuclear DNA content of protoplast fusion-derived calli and the results of the RAPDs suggest that interspecific fusions had occurred. The small number of volatile compounds detected from both the fusion calli and from the donor species indicates that the unorganized callus tissue is unable to produce tissue-specific volatile compounds. Received: 4 August 1998 / Accepted: 30 September 1998     

Genetic variation and differentiation in Nordic populations of Elymus alaskanus (Scrib. ex Merr.) Löve (Poaceae)

 To gain information on the extent and nature of genetic variation in Elymus alaskanus, levels and distribution of genetic variation were assessed within and among 13 populations originating from Iceland, Norway, Sweden and Russia using allozymes. The results showed that four (30.7%) of the 13 loci were polymorphic within the species, while the mean percentage of polymorphic loci within the populations was 1.9%. The mean number of alleles per locus for the species was 1.8 and 1.02 across the populations. Genetic diversity at the species level was low (H _es=0.135), and mean population diversity was notably lower (H _ep=0.005). A high degree of genetic differentiation was observed among populations. The salient points emerging from this study are: (1) statistically significant differences were found in allele frequencies among populations for every polymorphic locus (P<0.001), (2) the high mean coefficient of gene differentiation (G _ST) showed that 95% of the total allozyme variation was attributable to differences among populations, and (3) relatively high genetic distances between the populations were obtained (mean D=0.16). The Norwegian populations had the highest genetic diversity as compared with the other populations. Geographical comparisons revealed three different groups of populations clearly differentiated, i.e. Scandinavia (Norway and Sweden), Iceland and Russia. Cluster and principal coordinates analyses revealed the same genetic patterns of relationships among populations. Generally, this study indicates that E. alaskanus contains low allozymic variation in its populations. The implications of these results for the conservation of the species are discussed. Received: 23 October 1998 / Accepted: 19 December 1998     

Sex-specific population structure,natural selection,and linkage disequilibrium in a wild bird population as revealed by genome-wide microsatellite analyses

Background  

Sexual dimorphism in ecologically important traits is widespread, yet the differences in the genomic architecture between the two sexes are largely unexplored. We employed a genome-wide multilocus approach to examine the sexual differences in population subdivision, natural selection and linkage disequilibrium (LD) in a wild Siberian jay (Perisoreus infaustus) population, using genotypes at a total of 107 autosomal and Z-chromosomal microsatellites.     

Allele-specific hybridization markers for soybean

 Soybean (Glycine max) is one of the world’s most important crop plants due to extensive genetic improvements using traditional breeding approaches. Recently, marker-assisted selection has enhanced the ability of traditional breeding programs to improve soybeans. Most methods of assessing molecular markers involve electrophoretic techniques that constrain the ability to perform high-throughput analyses on breeding populations and germplasm. In order to develop a high-capacity system, we have developed allele-specific hybridization (ASH) markers for soybean. As one example, restriction fragment length polymorphism (RFLP) locus A519-1 (linkage group B) was converted into an ASH marker by (1) sequencing the pA519 cloned insert, (2) designing locus-specific PCR amplification primers, (3) comparative sequencing of A519-1 amplicons from important soybean ancestors, and (4) designing allele-specific oligonucleotide probes around single nucleotide polymorphisms (SNPs) among soybean genotypes. Two SNPs were identified within approximately 400 bp of the sequence. Allele-specific probes generated a 100-fold greater signal to target amplicons than to targets that differed by only a single nucleotide. The A519-1 ASH marker is shown to cosegregate with the A519-1 RFLP locus. In order to determine ASH usefulness, we genotyped 570 soybean lines from the Pioneer Hi-Bred soybean improvement using both A519-1 SNPs. Combined haplotype diversity (D) was 0.43 in this adapted germplasm set. These results demonstrate that ASH markers can allow for high-throughput screening of germplasm and breeding populations, greatly enhancing breeders’ capabilities to do marker-assisted selection. Received: 10 August 1998 / Accepted: 17 September 1998     

Cloning, quantification and characterization of a minisatellite DNA sequence from common bean Phaseolus vulgaris L.

 We describe the cloning and the characterization of a 130-bp DNA fragment, called OPG9-130, amplified from bean (Phaseolus vulgaris L.) genomic DNA. This fragment corresponds to a minisatellite DNA sequence containing seven repeats of 15 bp which differ slightly from each other in their sequence. Southern analysis showed that the core sequence of 15 bp is repeated in clusters dispersed throughout the genome. The use of this fragment as a probe allowed us to identify common bean lines by their DNA fingerprints. We suggest that OPG9-130 will be useful for line identification as well as for the analysis of genetic relatedness between bean species and lines. Received: 14 February 1998 / Accepted: 10 February 1998     

A composite map of expressed sequences and phenotypic traits of the sunflower (Helianthus annuus L.) genome

 A map of the sunflower genome, based on expressed sequences and consisting of 273 loci, was constructed. The map incorporates data from seven F₂ populations, for a total of 1115 individuals. Two hundred and fourty five loci corresponding to 170 anonymous cDNA markers and four loci for morphological markers were mapped. We also mapped 18 loci corresponding to previously described genes or to sequences obtained through homology cloning. The unit maps vary from 774 cM to 1060 cM, with an average value of 14 major linkage groups. The integrated map is arranged in 17 major linkage groups including 238 loci, plus four small segments with 2–5 marker loci; and covers 1573 cM with an overall average marker interval of 7 cM. Thirty five percent of the markers were dominant in nature and 30% showed inter-linkage group duplication without any indication of homoeologous linkage groups. Evidence is provided for the independence of two distinct fertility restoration genes, for the presence of two loosely linked branching loci, and for marker tightly linked to the Rf1 restoration locus. This map provides an efficient tool in breeding applications such as disease-resistance mapping, QTL analyses and marker-assisted selection. Received: 27 August 1998 / Accepted: 28 December 1998     

Genetic mapping in pea. 2. Identification of RAPD and SCAR markers linked to genes affecting plant architecture

 Random amplified polymorphic DNA (RAPD) markers linked to two morphological markers ( fa and det), three ramosus genes (rms2, rms3 and rms4) and two genes conferring flowering response to photoperiod in pea (sn, dne) were selected by bulk segregant analysis on F₂ populations. Two RAPD fragments were cloned and sequenced to generate the two SCAR markers V20 and S2 which are linked to rms3 and dne, respectively. All these genes, except rms2, were previously located on the pea classical linkage map. Rms2 mapped to linkage group IB which contains the afila gene. Precise genetic maps of the regions containing the genes were obtained and compared to the RAPD map generated from the recombinant inbred-lines population of the cross Térèse×K586. This cross was chosen because several mutants were obtained from cultivars Térèse and Torsdag (K586 was derived from Torsdag). This collection of isogenic lines was used for the construction of F₂ mapping populations in which polymorphic RAPD markers were already known and mapped. Moreover, the well-known problem in pea of variability in the linkage associations between crosses was avoided. This work contributes to the precise integration between the classical map and the molecular maps existing in pea. Received: 13 March 1998 / Accepted: 29 April 1998     

Allozymes and nDNA markers show different levels of population differentiation in the mussel Mytilus?edulis on British coasts

In order to test the hypothesis that natural selection can be operating on allozymes, six nuclear DNA markers were used to study population structure in six Mytilus edulis (Linnaeus, 1758) populations around the British Isles and the results compared with previous allozyme data from the same (two sites) or very close localities (the other four sites). Allozymes and nDNA markers show significant differentiation between populations at the P < 0.01 level, with the exception of the nDNA marker PLIIa. The results also indicate significant differences (modified version of the Lewontin–Krakauer test) between the two sets of markers which are about one order of magnitude higher for nDNA than for the allozyme loci. The bootstrap analysis was carried out to correct for the possibility that this difference is a sample size artefact. These results are consistent with the operation of balancing selection on allozymes or on loci in linkage disequilibrium with them. Handling editor: C. Sturmbauer     

Construction of an RFLP linkage map for cultivated sunflower

 An RFLP linkage map was constructed for cultivated sunflower Helianthus annuus L., based on 271 loci detected by 232 cDNA probes. Ninety-three F₂ plants of a cross between inbred lines RHA 271 and HA 234 were used as the mapping population. These genetic markers plus a fertility restoration gene, Rf ₁, defined 20 linkage groups, covering 1164 cM of the sunflower genome. Of the 71 loci 202 had codominant genotypic segregation, with the rest showing dominant segregation. Thirty-two of the 232 probes gave multiple locus segregation. There were 39 clusters of tightly linked markers with 0 cM distance among loci. This map has an average marker-to-marker distance of 4.6 cM, with 11 markerless regions exceeding 20 cM. Received: 17 June 1997 / Accepted: 19 June 1997     

Convergence of multilocus systems under weak epistasis or weak selection

 The convergence of multilocus systems under viability selection with constant fitnesses is investigated. Generations are discrete and nonoverlapping; the monoecious population mates at random. The number of multiallelic loci, the linkage map, dominance, and epistasis are arbitrary. It is proved that if epistasis or selection is sufficiently weak (and satisfies a certain nondegeneracy assumption whose genericity we establish), then there is always convergence to some equilibrium point. In particular, cycling cannot occur. The behavior of the mean fitness and some other aspects of the dynamics are also analyzed. Received: 15 November 1997 / Revised version: 25 May 1998     

The importance of slope aspect and stand age on the photosynthetic carbon fixation capacity of forest: a case study with black locust (<Emphasis Type="Italic">Robinia pseudoacacia</Emphasis>) plantations on the Loess Plateau

The black locust (Robinia pseudoacacia L.) is an important tree species not only for the vegetation rehabilitation but also for the photosynthetic carbon dynamics on the Loess Plateau. Slope aspect and stand age play important roles in the photosynthesis of the black locusts. To investigate the photosynthetic carbon fixation capacity (PCFC) of the juvenile and mature black locusts located on the sunny and shady slopes, we have analyzed the capacity and daily dynamics of photosynthesis of the whole canopy of juvenile (6-year-old) and mature (18-year-old) black locusts located on the sunny (southeast facing) and shady (northwest facing) slopes. Mature plantations on the sunny slopes have lower average daily E, VPD, CE, A _n, LAI and PCFC than those on the shady slopes. Juvenile plantations have higher average daily g _s, E, C _i/C _a, CE, A _n and PCFC compared to the mature plantations. It is concluded that the lower average daily A _n and PCFC of the mature black locust plantations on the sunny slopes may be due to variations in the microclimatic conditions between sunny and shady slope aspects. The higher average daily A _n and PCFC of the juvenile black locust plantations are likely associated with stand age-related differences in tree sizes.     

Identification of molecular markers associated with leptine production in a population of Solanum chacoense Bitter

  Solanum chacoense Bitter, a wild relative of the cultivated potato, produces several glycoalkaloids, including solanine, chaconine, and the leptines. The foliar-specific leptine glycoalkaloids are believed to confer resistance to the Colorado Potato Beetle (CPB). Using two bulked DNA samples composed of high- and low-percent leptine individuals from a segregating F₁ population of S. chacoense, we have identified two molecular markers that are closely linked to high percent solanine+chaconine and, conversely, to nil/low percent leptine. One of these, a 1,500-bp RAPD product (UBC370-1500), had a recombination value of 3% in the F₁ progeny, indicating tight linkage. UBC370-1500 mapped to the end of the short arm of potato chromosome 1, in the region of a previously mapped major QTL for solanidine, from a S. tuberosum (solanidine)×S. berthaultii (solasodine) cross. Taken together, these results suggest that either (1) a major locus determining solanidine accumulation in Solanum spp. is on chromosome 1 in the region defined by the RFLP markers TG24, CT197, and CT233, or (2) this region of chromosome 1 may harbor two or more important genes which determine accumulation of steroidal aglycones. These findings are important for the genetics of leptine (as well as other glycoalkaloid) accumulation and for the development of CPB-resistant potato varieties. Received: 5 March 1998 / Accepted: 28 July 1998     

Genetic analysis of inbreeding depression in plus tree 850.55 of Pinus radiata D. Don. I. Genetic map with distorted markers

 A genetic map of Pinus radiata plus tree 850.55 was constructed using megagametophytes of S₁ seeds. The map contained 19 linkage groups, with 168 RAPD and four microsatellite markers. The total map length was 1116.7 cM (Kosambi’s function) and was estimated to cover 56% of the genome. Of the 172 markers, 59 (34%) were distorted from the expected 1 : 1 ratio in megagametophytes (P<0.05). We show that if the distortion is caused by a single viability gene or by sampling error, the estimate of recombination frequency in megagametophytes of selfed seeds would not be affected. Received: 20 April 1998 / Accepted: 13 July 1998     

Transient dynamics in multilocus invasions by transgenic organisms

 With recent advances in molecular genetics, it is likely that releases of genetically modified organisms will be used for a variety of purposes. In many cases, such systems would utilize organisms that have been modified on multiple genetic loci. Predicting the effect of such releases will require an understanding of the transient dynamics in the system. However, theoretical understanding of transient dynamics in multilocus systems is limited, particularly for early generations when gametic disequilibrium is still high. I derive approximate expressions for marginal allele frequency and marginal two-locus disequilibrium that are applicable in this initial period, assuming infinite population size, two alleles per locus, and weak viability selection. I then apply these results to exploring the effect of parameters on the frequency of the resident gamete type in a release of organisms carrying an autocidal allele on multiple loci. This leads to simple approximate expressions for the optimal number of loci carrying the autocidal allele (as a function of release size and the degree of natural selection against the alleles) and the size of release needed to overcome a given level of selection against the released alleles. Received: 5 March 2001 / Revised version: 10 July 2002 / Published online: 18 December 2002 Current address: Department of Statistics, 204 Statistics Building, University of Georgia, Athens, GA 30602-1952, USA. e-mail: pdschlie@stat.uga.edu Key words or phrases: Multilocus – Selection – Transgenic – Pest control – Genetic control Acknowledgements. I thank Steve Ellner for insight and guidance throughout this project. This research was partially funded by a fellowship from the College of Agricultural and Life Sciences Foundation of North Carolina State University.