K. P. Pauls

Rapid estimation of genetic relatedness among heterogeneous populations of alfalfa by random amplification of bulked genomic DNA samples

A procedure which involves the use of RAPD markers, obtained from bulked genomic DNA samples, to estimate genetic relatedness among heterogeneous populations is demonstrated in this study. Bulked samples of genomic DNA from several alfalfa plants per population were used as templates in polymerase chain reactions with different random primers to produce RAPD patterns. The results show that the RAPD patterns can be used to determine genetic distances among heterogeneous populations and cultivars which correspond to their known relatedness. The results also indicate that, by using ten primers with bulked DNA samples from ten individuals, 18–72 populations or cultivars can be distinguished from each other on the basis of at least one unique RAPD marker. We anticipate that DNA bulking and methods for comparing RAPD patterns will be very useful for identifying cultivars, for studying phylogenetic relationships among heterogeneous populations and for selecting parents to maximize heterosis in crosses.

A comparison of anther and microspore culture as a breeding tool in Brassica napus

SummaryA direct comparison of microspore culture and anther culture was made in Brassica napus using F1 crosses of Regent (canola) by Golden (rapeseed), and their reciprocals, as well as a hybrid between Reston and a highly embryogenic, canola-quality breeding line (G231) as donor plants. The study confirmed that microspore culture can be ten times more efficient than anther culture for embryo production. Embryo yields from cultures initiated from the Reston x G231 were four-fold greater than those initiated from the Regent x Golden crosses, and significant differences were also detected among cultures initiated from the different Regent x Golden crosses. These results illustrate the influence that donor plant genotype has on embryo production. However, superior embryogenic potential among donor material was not always coincident with superior plant production. The average haploid-todiploid ratio in microspore-derived regenerates was 2∶1 for the population obtained from the Regent x Golden crosses but 1∶1 for the Reston x G231 cross. For both types of material, the frequency of diploids increased upon repeated cycles of explanting. A field study showed that there were no differences between the populations of anther-derived and microspore-derived spontaneous diploid and doubled haploid lines, with respect to the days required for them to flower or to mature. The information is valuable for canola breeding programs considering the use of haploidy.

The combination of polima cms and cytoplasmic triazine resistance in Brassica napus.

SummaryProtoplast fusion was used to combine cytoplasmic triazine resistance (ctr) and Polima type cytoplasmic male sterility (cms) in Brassica napus. The cybrids produced constitute the major biological input required for the production of commercial single-cross hybrid rapeseed bearing cytoplasmic triazine resistance. The results also indicate that Polima cms is associated with the mitochondrial genome.

Microsatellite and RAPD polymorphisms in Ontario corn hybrids are related to the commercial sources and maturity ratings

Random-amplified polymorphic DNA (RAPD) and microsatellite markers were used to estimate the genetic relationships among 37 Ontario corn hybrids. Almost all (95%) of the 160 RAPD fragments and all of the 79 microsatellite alleles were polymorphic across the 37 hybrids. Similarity values among the hybrids ranged from 31% to 86% when based on the RAPD data. The similarities based on microsatellite markers ranged from 12% to 77%. The genetic diversity revealed by microsatellite marker analysis was higher than that obtained from RAPD analysis. The similarity matrices for the microsatellite data and the RAPD data were moderately correlated (0.43). Cluster analyses based on either type of marker showed that most of the hybrids from the same company were closely related to each other. Both dendrograms clustered similar pairs or groups of hybrids. A principal component analysis, based on the combined RAPD and microsatellite data, yielded a good separation of the hybrids with Ontario Corn Heat Unit (OCHU) values <2800 from those with OCHU values >2800. Seventeen RAPD markers and 5 microsatellite markers were significantly associated with the OCHU ratings of the hybrids.

The use of haploidy to develop plants that express several recessive traits using light-seeded canola (Brassica napus) as an example.

SummaryThe use of haploidy to introgress recessive traits into Brassica napus canola is illustrated by describing the properties of doubled haploids obtained by microspore culture from crosses between a yellow-seeded rapeseed line (low erucic acid, high glucosinolate) and black-seeded canola. Of the 99 doubled haploid lines that were produced, 3 were yellow-seeded canola lines. This result was not significantly different than the predicted frequency of 1 in 64 for the homozygous recessive phenotype in a doubled haploid population segregating for six recessive genes. Thus, the study supports previous models of inheritance determined for yellow seededness and glucosinolate content in Brassica napus. Also, since the chances of obtaining a plant with the same characteristics in a F2 population are 1 in 4,096, the underscore results the advantages of using haploidy to introgress recessive traits into Brassica napus canola.

Inheritance patterns of erucic acid content in populations of Brassica napus microspore-derived spontaneous diploids

SummaryThe levels of erucic acid and other fatty acids in seeds of microspore-derived spontaneous diploid plants from crosses between low and high erucic acid parents were examined. The analysis confirmed that erucic acid is simply inherited and is determined by two genes that act in an additive manner. The effects of the genes for erucic acid on the levels of the other fatty acids was also determined and many significant correlations were found. In particular, erucic acid levels were negatively correlated with oleic acid and linoleic acid levels. The study also illustrates several advantages of using haploidy to analyze the inheritance of agronomically important traits. In particular, the number of phenotypic classes is smaller in androgenic populations and differences between classes are greater than in an F2 population.

Inheritance of plant regeneration from maize (Zea mays L.) shoot meristem cultures derived from germinated seeds and the identification of associated RAPD and SSR markers

The inheritance of shoot regeneration through shoot-tip meristem culture derived from maize seedling was evaluated, and the markers (RAPD and SSR) associated with this regeneration character were identified both in a group of North American maize inbreds and a crossing population. A discrete distribution of percent regeneration and no. of shoots per explant was observed in the inbred group and the F2 population. The results suggested that this regenerable trait was controlled by several major genes. Five RAPD markers were identified to be relevant to percent regeneration in maize shoot-tip culture system. One RAPD marker and three SSR markers were associated with no. of shoot per explant and its relevant traits. Of them marker BC603-1600 explained 18% of the variation for no. of shoot per explant and 16% of the variation for callus size. The BC603-1600 was sequenced and assigned in linkage group 7 based on a NCBI blast search. The information provided here should benefit to determine the genetic mechanisms involved in the maize regeneration response related to shoot meristem culture pathway and benefit to select high regenerable germplasm by using marker assisted selection.

THE USE OF HAPLOID PROTOPLAST FUSION TO COMBINE CYTOPLASMIC ATRAZINE RESISTANCE AND CYTOPLASMIC MALE STERILITY IN BRASSICA NAPUS.

This report describes the use of haploid protoplast fusion to create cytoplasmic atrazine resistant (CATR) and cytoplasmic male sterile (CMS) B. napus plants. The study was carried out in parallel with the diploid/diploid protoplast fusion work described by Barsby et al. (1987). Protoplasts from haploids were used because the fusion process reestablishes the diploid condition and precludes the need for nuclear inactivation or cytoplast production in one of the parental protoplast populations. The haploid protoplasts were also well suited for use with a flow cytometer/cell sorter to isolate heterokaryons from the fusion mixtures. The success of this approach was dependent on the development of procedures to isolate haploid lines from CMS and CATR B. napus (Chuong et al., 1987a) and the development of plant regeneration protocols from haploid B. napus protoplasts (Chuong et al., 1987b,c).

Effects of tissue culture on a highly repetitive DNA sequence (E180 satellite) in Medicago sativa

Genomic DNA extracted from leaves of sixteen alfalfa genotypes, six-week-old callus cultures derived from them and from a suspension culture was used in Southern blots and slot blots probed with alfalfa E180 satellite DNA. The restriction patterns revealed by the E180 probe did not differ among alfalfa genotypes and no differences between the leaf and callus restriction patterns were seen. However, the number of copies of the E180 satellite was lower in the callus samples than in the corresponding leaf samples and significant differences in copy number among callus samples were detected. Genomic stress induced by tissue culture may have caused the reduction in copy number. This phenomenon may be important in the generation of somaclonal variation in alfalfa.