Isolation and characterization of microsatellite markers for Carolina hemlock (Tsuga caroliniana).

We describe the isolation and characterization of 31 polymorphic di- and trinucleotide microsatellite marker loci for Carolina hemlock (Tsuga caroliniana Englem.). In addition, primer pairs for 16 loci amplified scoreable alleles in six other Tsuga species. In eastern North America, both Carolina hemlock and eastern hemlock (Tsuga canadensis [L.] Carr.) populations are declining due to infestation by hemlock woolly adelgid, Adelges tsugae. The markers described here should enhance population genetic studies of hemlocks, providing valuable information for conserving and restoring these important forest tree species.

A framework linkage map of perennial ryegrass based on SSR markers.

A moderate-density linkage map for Lolium perenne L. has been constructed based on 376 simple sequence repeat (SSR) markers. Approximately one third (124) of the SSR markers were developed from GeneThresher libraries that preferentially select genomic DNA clones from the gene-rich unmethylated portion of the genome. The remaining SSR marker loci were generated from either SSR-enriched genomic libraries (247) or ESTs (5). Forty-five percent of the GeneThresher SSRs were associated with an expressed gene. Unlike EST-derived SSR markers, GeneThresher SSRs were often associated with genes expressed at a low level, such as transcription factors. The map constructed here fulfills 2 definitions of a "framework map". Firstly, it is composed of codominant markers to ensure map transferability either within or among species. Secondly, it was constructed to achieve a level of statistical confidence in the support-for-order of marker loci. The map consists of 81 framework SSR markers spread over 7 linkage groups, the same as the haploid chromosome number. Most of the remaining 295 SSR markers have been placed into their most likely interval on the framework map. Nine RFLP markers and 1 SSR marker from another map constructed using the same pedigree were also incorporated to extend genome coverage at the terminal ends of 5 linkage groups. The final map provides a robust framework with which to conduct investigations into the genetic architecture of trait variation in this commercially important grass species.

Cross-species transferability and mapping of genomic and cDNA SSRs in pines.

Two unigene datasets of Pinus taeda and Pinus pinaster were screened to detect di-, tri- and tetranucleotide repeated motifs using the SSRIT script. A total of 419 simple sequence repeats (SSRs) were identified, from which only 12.8% overlapped between the two sets. The position of the SSRs within their coding sequences were predicted using FrameD. Trinucleotides appeared to be the most abundant repeated motif (63 and 51% in P. taeda and P. pinaster, respectively) and tended to be found within translated regions (76% in both species), whereas dinucleotide repeats were preferentially found within the 5'- and 3'-untranslated regions (75 and 65%, respectively). Fifty-three primer pairs amplifying a single PCR fragment in the source species (mainly P. taeda), were tested for amplification in six other pine species. The amplification rate with other pine species was high and corresponded with the phylogenetic distance between species, varying from 64.6% in P. canariensis to 94.2% in P. radiata. Genomic SSRs were found to be less transferable; 58 of the 107 primer pairs (i.e. 54%) derived from P. radiata amplified a single fragment in P. pinaster. Nine cDNA-SSRs were located to their chromosomes in two P. pinaster linkage maps. The level of polymorphism of these cDNA-SSRs was compared to that of previously and newly developed genomic-SSRs. Overall, genomic SSRs tend to perform better in terms of heterozygosity and number of alleles. This study suggests that useful SSR markers can be developed from pine ESTs.

Testing for linkage disequilibrium in the New Zealand radiata pine breeding population.

Linkage analysis is commonly used to find marker-trait associations within the full-sib families of forest tree and other species. Study of marker-trait associations at the population level is termed linkage-disequilibrium (LD) mapping. A female-tester design comprising 200 full-sib families generated by crossing 40 pollen parents with five female parents was used to assess the relationship between the marker-allele frequency classes obtained from parental genotypes at SSR marker loci and the full-sib family performance (average predicted breeding value of two parents) in radiata pine ( Pinus radiata D. Don). For alleles (at a marker locus) that showed significant association, the copy number of that allele in the parents was significantly correlated, either positively or negatively, with the full-sib family performance for various economic traits. Regression of parental breeding value on its genotype at marker loci revealed that most of the markers that showed significant association with full-sib family performance were not significantly associated with the parental breeding values. This suggests that over-representation of the female parents in our sample of 200 full-sib families could have biased the process of detecting marker-trait associations. The evidence for the existence of marker-trait LD in the population studied is rather weak and would require further testing. The exact test for genotypic disequilibrium between pairs of linked or unlinked marker loci revealed non-significant LD. Observed genotypic frequencies at several marker loci were significantly different from the expected Hardy-Weinberg equilibrium. The possibilities of utilising marker-trait associations for early selection, among-family selection and selecting parents for the next generation of breeding are also discussed.

Molecular cloning, structure and expression of an elicitor-inducible chitinase gene from pine trees.

We have cloned, sequenced, and examined the expression of genes from pine trees that appear to encode extracellular class II chitinase. Nucleotide sequence analysis indicates a coding sequence composed of three exons interrupted by two introns at locations identical to those found in other chitinase genes that possess introns. One of the genes, Pschi4, potentially encodes a protein that shares 62% amino acid sequence identity through the catalytic domain with class II chitinase from tobacco. In contrast, Pschi1 contains a stop codon in the first exon and may be a pseudogene. Pschi4 genes are conserved in several species of pine, and appear to comprise a small multigene family. Treatment of pine cell suspension cultures with the general elicitor chitosan induced Pschi4 expression. The regulatory sequences associated with the Pschi4 gene were sufficient to direct chitosan-inducible expression of Pschi4 in transgenic tobacco plants, which indicates that Pschi4 is an actively expressed member of the multigene family. The observation that the Pschi4 gene from pine (a gymnosperm) was appropriately regulated by chitosan in tobacco (an angiosperm) suggests that the signaling pathways that mediate chitosan-induced transcription are highly conserved in the plant kingdom.

Survey of microsatellite DNA in pine.

A large insert genomic library from eastern white pine (Pinus strobus) was probed for the microsatellite motifs (AC)n and (AG)n, all 10 trinucleotide motifs, and 22 of the 33 possible tetranucleotide motifs. For comparison with a species from a different subgenus, a loblolly pine (Pinus taeda) genomic library was also probed with the same set of di- and tri-nucleotide repeats and 11 of the tetranucleotide repeats. The four most abundant microsatellite motifs in both species were (AC)n, (AG)n, (AAT)n, and (ATC)n, which as a group accounted for over half the microsatellite sites investigated. The two dinucleotide repeats were the most abundant microsatellite motifs tested in both species, each at 2-4.5 sites/megabase pair (Mbp), but the two trinucleotide motifs were nearly as abundant and are considered good candidates for pine microsatellite marker development efforts. Eastern white pine had more than twice as many (AC)n as (AG)n loci, in contrast with loblolly pine and most other plant species in which (AG)n is more abundant. In both pine species the minimum estimated genome density for all microsatellites, excluding (AT)n repeats, was 16 sites/Mbp.

Characterization of microsatellite markers in eastern white pine.

An enrichment cloning method was evaluated for the isolation of microsatellite loci from eastern white pine and the resulting markers were examined for polymorphisms. A 200-fold enrichment was achieved for highly abundant (AC)n repeats, but for much less abundant (ACAG)n repeats an enrichment of only 20-fold was obtained. Using a single set of PCR conditions, 19 microsatellite loci were identified from 77 primer pairs evaluated. Genotyping of 16 (AC)n loci in 16 unrelated white pines from the north-central United States revealed an average of 5.4 alleles per locus and an average observed heterozygosity of 0.515. Five loci were scored among megagametophytes from a single pine to obtain a haploid genotype of the segregating female meiotic products. All loci segregated according to Mendelian expectations and linkage was established for two of the loci. It was concluded that (AC)n loci are highly variable in this species and that SSR (simple sequence repeat) markers can be efficiently developed for genome mapping and population genetics studies.

Linkage mapping in diploid alfalfa (Medicago sativa).

A genome map of cultivated alfalfa was constructed using segregating restriction fragment length polymorphisms (RFLPs) and random amplified polymorphic DNAs (RAPDs) in a diploid backcross population generated from noninbred parents. Among the 153 loci scored in 87 progeny, four segregation ratios were observed for codominant and dominant markers: 1:1, 1:2:1, 1:1:1:1, and 3:1. Deviations from expected Mendelian ratios (p < 0.05) were observed for 34% of the loci studied. A genome map was assembled from two separate linkage maps, each constructed from a subset of the segregation data. One linkage map was constructed from 46 RFLP and 40 RAPD markers segregating 1:1 from the F1 parent of the backcross and the other linkage map was constructed from 33 RFLP and 28 RAPD markers segregating 1:1 from the recurrent parent. Sixteen loci with alleles segregating 1:1 from both parents were used as locus bridges to align individual linkage groups between the two maps. The combined use of RFLPs and RAPDs was an effective method for developing an alfalfa genome map.

Introduction and expression of an insect proteinase inhibitor in alfalfa Medicago sativa L.

As one approach to alleviating the need for insecticide spraying, our objective is to express protein insecticides in transgenic alfalfa. To initiate these studies, a cDNA encoding the protease inhibitor (PI) anti-elastase from Manduca sexta was placed under the control of the CaMV 35S promoter, inserted into pAN 70, and transferred into leaf and petiole sections of alfalfa (Medicago sativa L.) using Agrobacterium tumefaciens mediated gene transfer. Transformation rates were 10% of all explants exposed to Agrobacterium. More than 1000 transgenic plants containing the PI have been recovered. Transgenic plants were initially identified when leaf explants from the regenerated plants formed callus in the presence of 50 µg/ml kanamycin, and subsequently the presence of the PI gene was confirmed by southern analysis. The 35S promoter-PI fusion produced up to 0.125% of total protein as PI protein in leaves, roots, and flowers. Progeny analysis demonstrated Mendelian segregation of the NPTII gene (observed as kanamycin resistance) and the PI (confirmed by southern analysis). Accumulation of the anti-elastase PI insecticide in transgenic alfalfa reduced the onset of thrip predation, suggesting that this methodology can establish insect resistance within this agronomically important legume.

Potential of trispecies bridge crosses and random amplified polymorphic DNA markers for introgression of Medicago daghestanica and M. pironae germplasm into alfalfa (M. sativa).

This report describes the production and cytology of the first interspecific hybrids between cultivated alfalfa (Medicago sativa L.) at the diploid level (2n = x = 16) and the diploid (2n = 2x = 16) perennial species M. daghestanica and M. pironae. An ovule-embryo culture technique was required to rescue hybrid embryos and all hybrids were diploid. Predominately bivalent chromosome pairing was observed at meiotic metaphase. All F1 hybrids were male and female sterile and no species backcross progeny could be produced. We discovered that trispecies hybrids could be efficiently recovered via crossing diploid F1 interspecific hybrids of M. sativa x M. rupestris with either M. daghestanica or M. pironae. Ovule-embryo culture was also required to recover these trispecies hybrids with recovery efficiency of trispecies hybrids about 10 times greater than for bispecies hybrids. Most chromosomes paired as bivalents in the trispecies hybrids. Importantly, progeny can be recovered from crossing the trispecies hybrids with M. sativa. Therefore, the M. sativa x M. rupestris hybrids provide a bridge cross to potential introgression of M. daghestanica or M. pironae germplasm. Analysis of randomly amplified polymorphic DNA (RAPD) markers in the trispecies hybrids indicates that RAPD markers offer considerable potential for assaying germplasm introgression following complex hybridization of the type reported here.

Genetic segregation of random amplified polymorphic DNA in diploid cultivated alfalfa.

Polymerase chain reaction was used, with single 10-mer primers of arbitrary sequence, to amplify random regions of genomic DNA from a diploid cultivated alfalfa backcross population. Segregation of the random amplified polymorphic DNA (RAPD) fragments was analysed to determine if RAPD markers are suitable for use as genetic markers. Of the 19 primers tested, 13 amplified a total of 37 polymorphic fragments, of which 28 (76%) segregated as dominant Mendelian traits. RAPD markers appear useful for the rapid development of genetic information in species like alfalfa where little information currently exists or is difficult to obtain.

Molecular analysis of the maize wx-B3 allele indicates that precise excision of the transposable Ac element is rare.

The somatic and germinal behavior of the maize wx-B3 mutation indicates that this Ac allele rarely reverts. Endosperms containing wx-B3 display tiny and infrequent Wx revertant sectors while no significant reversion is detected when wx-B3 pollen is stained with I/KI. Previous studies of other transposable element alleles that revert infrequently have implicated low levels of element excision. Unlike these other alleles, the wx-B3 Ac element is indistinguishable from fully active Ac elements with respect to its structure, and its ability to transpose from the Wx gene or to trans-activate a Ds element. Characterization of somatic and germinal excision events lead us to conclude that excision of the wx-B3 Ac element almost always produces null alleles. Furthermore, the excellent correlation between the position of the wx-B3 mutation on the physical and genetic maps indicates that the Ac insertion is the only lesion of wx-B3. As a result, precise excision of this Ac should restore Wx function. The fact that revertant sectors and pollen grains are rare indicates that precise excision of Ac is also rare. The finding that the wx-B3 reversion frequency is comparable whether wx-B3 is hemizygous or over a wx allele with a wild-type insertion site illustrates a fundamental difference between the excision mechanisms of Ac and Drosophila P elements.

A Comparison of Two Sucrose Synthetase Isozymes from Normal and shrunken-1 Maize.

The maize sucrose synthetase isozyme (SS2) present in sh1 endosperm, sh1 seedlings, and in suspension culture cells was purified to homogeneity from each of these tissues by sequential ammonium sulfate fractionation, diethylaminoethyl-cellulose chromatography, gel filtration chromatography, and affinity elution with UTP from a carboxymethyl-cellulose column. Cyanogen bromide digests were used to demonstrate that the SS2 isozymes in these different tissues are structurally identical and are therefore the product of the same gene. The sucrose synthetase produced by the Sh1 gene (SS1) was purified by modification of the SS2 procedure and was used in comparative analyses of the two isozymes. Ouchterlony assays demonstrated that SS1 and SS2 have partial antigenic identity. The two isozymes have similar enzyme kinetics in the sucrose cleavage reaction but differ in their relative activities with ADP and TDP. The amino acid compositions of SS1 and SS2 are similar, and proteolytic digests revealed that they share limited structural homologies.

Evidence for the Inclusion of Controlling Elements within the Structural Gene at the Waxy Locus in Maize.

Minimal limits for the structural gene at the waxy locus have been set by investigations of the protein product of the gene. An altered protein is produced by four of the waxy mutants including B3, a controlling-element mutation. All are similar to wild type in molecular weight as determined by electrophoresis in SDS acrylamide gels. At least three of the five wx controlling-element mutations studied have been shown to lie within the limits of the structural gene.

Evaluation of megestrol acetate and ethinyl estradiol as an oral contraceptive.

PIP: Evaluation of a new sequential oral contraceptive, 16 tablets of .1 mg ethinyl estradiol plus 5 tablets of .1 mg ethinyl estradiol and 5 mg compound with a structure similar to to progesterone. 74 patients dropped out, 18 of the 32 did not respond to personal communications; of the remaining 14 who discontinued the medication, 4 became pregnant but not from failure of the pill, 5 left because of nausea, and 5 left for personal reasons. There was a total of 515 completed cycles. Nausea occurred in 14.8% of the cycles with a marked decrease after the first cycle; breast soreness occurred in 5.8% of the cycles, intermenstrual bleeding and spotting in 6.4% of the cycles. The endometrium showed no adverse effects, and except for shortened bleeding times, no significant changes in laboratory values, including clotting mechanisms, were noted.^ieng