Rhonda J. Honeycutt

Parentage determination in maize hybrids using the arbitrarily primed polymerase chain reaction (AP-PCR)

SummaryUsing a novel procedure based on the polymerase chain reaction, we have developed a rapid, efficient, and economical method for identifying plant genotypes. The arbitrarily primed polymerase chain reaction (AP-PCR) generates reproducible fingerprints from any organism, without the need for DNA sequence information. These fingerprints include DNA fragment polymorphisms that can be (1) used for varietal identification and parentage determination, (2) followed in segregating populations produced by crosses, (3) used as markers for the construction of genetic maps, and (4) used to generate dendograms of phylogenetic relationships, especially at the intraspecific level. AP-PCR requires only minute quantities of DNA (10–25 ng per reaction) and therefore can be used in situations in which DNA is limiting. We demonstrate the use of AP-PCR to identify inbred parents of hybrid maize plants in double-blind experiments.

Saccharum spontaneum L. ‘SES 208’ genetic linkage map combining RFLP- and PCR-based markers

A 527 marker linkage map ofSaccharum spontaneum L. ‘SES 208’ (2n = 64) was established by analyzing 208 single-dose (SD) arbitrarily primed PCR polymorphisms, 234 SD RFLPs, 41 double-dose (DD) and one triple-dose (TD) polymorphisms. A map hypothesis constructed using these markers (minimum LOD = 4.00,θ = 0.25 M) had 64 linkage groups with 13 SD, nine DD, and one TD markers unlinked. Eight chromosome homology groups were identified by using DD fragments as well as SD RFLPs that identified more than one linkage group. Linkages in repulsion phase were absent from the map, as found in two previous genetic studies of this species. Together, these data demonstrate that SES 208 displayed polysomic segregation, a genetic behavior typical of autopolyploid species. As with previous studies, it was concluded that SES 208 behaved like an auto-octoploid, which was also in agreement with the number of homology groups observed. Aχ2 was used to test whether the 527 markers were randomly distributed throughout the genome: both arbitrarily primed PCR markers and RFLPs had a distribution that was statistically indistinguishable from random. The integrated arbitrarily primed PCR-RFLP map had a predicted genomic coverage of 93% (considering only 442 SD polymorphisms) and an average interval between markers of 6 cM. SD markers were used to estimate the genome size of SES 208 at ca. 33 00 cM.

High output genetic mapping of polyploids using PCR-generated markers

SummaryThe polymerase chain reaction (PCR) with arbitrarily selected primers has been established as an efficient method to generate fingerprints that are useful in genetic mapping and genomic fingerprinting. To further increase the productivity of mapping and fingerprinting efforts, we have altered existing protocols to include the use of the Stoffel fragment, which is derived from genetically engineered Taq polymerase. We also optimized the thermal profile of the reaction to increase the number of useful primers. In mapping of the genome of Saccharum spontaneum ‘SES 208’, a polyploid wild relative of sugarcane, these modifications allowed for an increase of 30% in the number of loci screened per primer, and an 80% increase in the number of polymorphisms per primer. Furthermore, the enzyme cost per reaction was decreased approximately 1.6-fold. Finally, there was an increase from about 70% to about 97% in the number of primers that were useful (i.e., gave a reproducible fingerprint) using our protocol. We have placed some of these markers into linkage groups.

Chromosome assortment in Saccharum.

Recent work has revealed random chromosome pairing and assortment in Saccharum spontaneum L., the most widely distributed, and morphologically and cytologically variable of the species of Saccharum. This conclusion was based on the analysis of a segregating population from across between S. spontaneum ‘SES 208’ and a spontaneously-doubled haploid of itself, derived from anther culture. To determine whether polysomic inheritance is common in Saccharum and whether it is observed in a typical biparental cross, we studied chromosome pairing and assortment in 44 progeny of a cross between euploid, meiotically regular, 2n=80 forms of Saccharum officinarum ‘LA Purple’ and Saccharum robustum ‘ Mol 5829’. Papuan 2n=80 forms of S. robustum have been suggested as the immediate progenitor species for cultivated sugarcane (S. officinarum). A total of 738 loci in LA Purple and 720 loci in Mol 5829 were amplified and typed in the progeny by arbitrarily primed PCR using 45 primers. Fifty and 33 single-dose polymorphisms were identified in the S. officinarum and S. robustum genomes, respectively (χ2 at 98%). Linkage analysis of single-dose polymorphisms in both genomes revealed linkages in repulsion and coupling phases. In the S. officinarum genome, a map hypothesis gave 7 linkage groups with 17 linked and 33 unlinked markers. Four of 13 pairwise linkages were in repulsion phase and 9 were in coupling phase. In the S. robustum genome, a map hypothesis gave 5 linkage groups, defined by 12 markers, with 21 markers unlinked, and 2 of 9 pairwise linkages were in repulsion phase. Therefore, complete polysomic inheritance was not observed in either species, suggesting that chromosomal behavior is different from that observed by linkage analysis of over 500 markers in the S. spontaneum map. Implications of this finding for evolution and breeding are discussed.

Genetics, Plants, and the Polymerase Chain Reaction

The polymerase chain reaction (PCR) has given plant geneticists, ecologists, evolutionary, and population biologists a powerful new tool for studying their favorite organisms. In this chapter, we will use specific PCR to mean a standard, two-primer amplification that has as a target a specific genomic region, or gene, and therefore requires specific primers to be designed based on knowledge of DNA sequence. We differentiate this from PCR that uses primers of arbitrary sequence to specifically amplify a set of arbitrary loci in any genome, without the requirement for prior sequence knowledge. This is usually referred to as arbitrarily primed PCR or random amplified polymorphic DNA (RAPD) markers; herein, we will use the term arbitrarily primed PCR.

Analysis of rice (Oryza sativa L.) genome using pulsed-field gel electrophoresis and rare-cutting restriction endonucleases.

TheOryza sativa (rice) genome is small (600 to 900 megabase pairs) when compared to that of other monocotyledonous plants. Rice was the first of the major cereals to be successfully transformed and regenerated. An RFLP map with approximately 300 markers is readily available, and the DNA content per map unit is only two to three times that ofArabidopsis thaliana. Rice is also the main staple food for the majority of peoples in the world. We developed techniques for the preparation of intact genomic DNA from Indica and Japonica subspecies of rice, used statistical methods to determine which restriction endonucleases are rare-cutting, and used pulsed-field gel electrophoresis (PFE) to separate large fragments of rice DNA. Southern hybridization to blotted rice PFE gels was used to show that the digests were complete. The long-term goal of our work is to generate an integrated genetic/physical map for the rice genome, as well as helping to establish rice as a model for map-based gene cloning and genome analysis.

Applications of DNA and RNA Fingerprinting by the Arbitrarily Primed Polymerase Chain Reaction

The related methods Arbitrarily Primed Polymerase Chain Reaction (AP-PCR) and Random Amplified Polymorphic DNA (RAPD) were developed independently by our group (Welsh and McClelland, 1990) and by Williams et al. (1990). These methods were based on the observation that PCR performed at relatively low stringency (in the case of AP-PCR) or with low selectivity primers at high stringency (in the case of RAPD) yield a reproducible collection of products that depend on the template and primer sequences. Arrayed on an agarose or acry1-amide gel, this collection of products can be viewed as a “fingerprint” or “bar code” for the DNA template. Because of their dependence on template sequence, AP-PCR and RAPD can be used as methods for sampling in sequence space; diverse applications can be imagined, many of which have been demonstrated in the literature.

Application of the Polymerase Chain Reaction to the Detection of Plant Pathogens

This chapter will focus on polymerase chain reaction (PCR)-based techniques applied to plant pathogens. Readers are also referred to the chapter by Kate J. Wilson on the subject of molecular approaches to the field ecology of microorganisms.

Screening differentially displayed PCR products by single-strand conformation polymorphism gels

Publisher Summary Differential display and ribonucleic acid (RNA) arbitrarily primed PCR have been extensively used to investigate differential gene expression and coordinate regulation in a wide variety of situations, including cell responses to different treatments and growth conditions and comparisons of different developmental stages. The RNA fingerprinting approach has also found many applications in cancer research. Several modifications to the original protocols have been reported, and the development of new technologies, such as automated sequencing or the use of fluorescent-tagged primers, is facilitating the use of this approach RNA fingerprinting has three steps: (i) cDNA synthesis, (ii) isolation and characterization of differentially amplified transcripts, and (iii) confirmation of differential expression. The step of isolation and characterization of the PCR fragments representing differentially amplified products is often a problem. This is so because its laborious or gives products that are not differentially expressed because they are not the ones being targeted. These problems are associated with the comigration of other PCR products in the fingerprint gel. This chapter focuses on the use of single-strand polymorphism gels to facilitate the isolation and purification of differentially amplified cDNAs from differential display and RAP-PCR fingerprinting experiments. The two protocols that are currently in use are also provided.