Maine Potato Board Project Report 2004

Development of genetic fingerprinting strategy for the Maine potato breeding program

Benildo G. de los Reyes, Principal Investigator

Dept. of Biological Sciences, University of Maine-Orono

Zenaida Ganga, Co-Investigator

Aroostook Research Farm, Presque Isle

Executive Summary

Efficient means to assess genetic diversity and identify genotypes is essential for continued success of the potato breeding and production programs in Maine. In order to address these needs, we adopted the previously published methodologies for generating Simple Sequence Repeat (SSR) and Inter-Simple Sequence Repeat (ISSR) genetic markers in potato and tested their relative efficiency in distinguishing genotypes within a relatively narrow range of genetic diversity. Both the SSR and ISSR profiles were able to distinguish a newly released Russet-type cultivar from Aroostook Farm (AF1753-16) from one of its Russet-type parents (CF7608-19) and from the commercial Russet Burbank. The DNA-fingerprint will be used as a ‘molecular ID’ for this cultivar and will be included in the variety release report to be published in the American Potato Journal. The simple DNA-fingerprinting techniques developed from this project were also used to confirm the genetic purity of selected donor genotypes that are currently being used in potato breeding program in Maine. The DNA marker capability established by this project will serve as a platform for the integration of conventional and molecular marker-based potato breeding strategies in Aroostook Research Farm and for genetic research at the University of Maine-Orono.

Project Accomplishments

The potato breeding program in Maine has been producing new promising lines with potential as breeding donors and for commercial release. A routine method for identifying these genotypes is an important aspect of efficient handling and exchange among scientists and for subsequent varietal control, protection and registration. A current major limitation of the program is the lack of access to a robust and unbiased means of cataloging these lines and method to differentiate them with traditional and other improved varieties. Therefore, a good pipeline for the efficient maintenance, use and distribution of these lines must integrate DNA-based fingerprinting strategy with current genotyping procedures based on morphological features. As an initial effort to address this limitation, we conducted a small study with the goal of:

(a) evaluating the applicability of two types of DNA markers (Simple Sequence Repeats and Inter-Simple Sequence Repeats) for genetic identification/fingerprinting of selected lines from the Maine potato breeding nursery; and (b) assessing the genetic homogeneity of selected commercial potato cultivars from independent seed sources.

1) Experimental Strategy. Total genomic DNA was isolated from pulverized periderm and cortex tissues of mature potato tubers with 2% CTAB pH. 8 and 0.1% (w/v) PVP-400, according to the procedure described by Wulff et al. (2002). The SSR and ISSR primers that have been previously reported to be polymorphic in Solanum tuberosum were used to generate the DNA fingerprints (Bornet et al., 2002; Provan et al., 1996). The SSR and ISSR fingerprints were generated as previously described. Briefly, 20 ng of genomic DNA was used as template for 38-cycle amplification of the SSR region at an annealing temperature equivalent to the Tm of the primers (e.g., SSR-LERBCO was amplified at an annealing temperature of 48^oC). The fragments were labeled during amplification by incorporation of ³³P-dATP. Amplification products were fractionated in standard denaturing polyacrylamide sequencing gels with 8M urea and autoradiographed for 2-3 days at -80^oC. The ISSR fingerprints were generated by amplification of the target regions from 10 ng of genomic DNA at primer-specific annealing temperatures (e.g., ISSR-CCA= 62^oC, ISSR-GCA= 64^oC, ISSR-CAC= 60^oC; Bornet et al., 2002). The amplification product was fractionated in 1.5% TBE-agarose gel and visualized by ethidium bromide staining.

Figure 1. Comparison of ISSR profiles of AF1753-16 (1), CF7608-19 (2), Russet Burbank (3) and S. commersonii (4). Band pairs that exhibit either intraspecific or interspecific polymorphism (presence or absence of band) are indicated with arrowheads.

2) Evaluation of DNA-marker systems. Simple Sequence Repeats (SSR) and Inter-Simple Sequence Repeats (ISSR) are two of the most commonly used DNA marker systems for various applications in potato breeding and genetic research. Using the previously published protocols and primer kits (Provan et al., 1996; Bornet et al., 2002), we compared the efficiency of the SSR and ISSR marker systems to distinguish different potato genotypes within a narrow range of genetic diversity. For this initial study we focused on generating DNA fingerprints that distinguish AF1753-16, a Russet-type cultivar released recently by the breeding program from its Russet-type parent (CF7608-19) and from the commercial Russet Burbank with completely different pedigree. In order to assess the extent of intraspecific allelic polymorphism for each marker, we also included in all analysis the diploid Solanum commersonii as an outlier.

The ISSR profiles generated by primers ISSR-CCA, ISSR-GCA, ISSR-CAC, and ISSR-GT on conventional agarose gels showed between two to six polymorphic loci (average of 15% of the total band pairs scored) among the tetraploid cultivated potato genotypes (AF1753-16, CF7608-19 and Russet Burbank; Example of polymorphic DNA profiles shown in Fig. 1). Of the five ISSR primers tested initially, only one (ISSR-ACA) was monomorphic among the tetraploid cultivated potatoes and also with respect to the diploid outlier S. commersonii. In all four polymorphic ISSRs, AF1753-16 (new release) can be distinguished from CF7608-19 and Russet Burbank by the presence or absence of at least one band. The overall similarity based on dominant ISSR markers was higher between AF1753-16 and CF7608-19 (>80% of bands are common) than between AF1753-16 and Russet Burbank (60-70% of bands are common). This result was expected considering that CF7608-19 is one of the parents of AF1753-16. ISSR markers also revealed high level of interspecific polymorphism based on the comparative analysis of the S. tuberosum (overall) and S. commersonii DNA fingerprints. These results indicate the potential of this simple system for the identification of species-specific DNA markers for future use in marker-assisted wide-introgression.

Figure 2. Comparison of SSR profiles of AF1753-16 (1), CF7608-19 (2), Russet Burbank (3) and S. commersonii (4). Examples of SSR fingerprints showing the occurrence of genotype-specific alleles (A) and hetorozygosity (B) are shown.

Both intraspecific and interspecific genetic polymorphism was revealed by the SSR profiles. Of the six primer pairs tested (Provan et al., 1996), five revealed polymorphic loci (STRBCS3, STCPKIN3, LERBCO, STPRINPSG) that distinguish a small subset of tetraploid cultivated potatoes from each other including AF1753-16, CF7608-19, and Russet Burbank. All of the loci revealed by these markers also distinguished the cultivated species from the diploid wild species S. commersonii. Only one of the six primer pairs (STGLGPB) tested so far was monomorphic within the cultivated potatoes but polymorphic between the cultivated species and the wild species S. commersonii. All of the five SSR markers that exhibit intraspecific polymorphism were able to distinguish AF1753-16 (new release) from its Russet-type parent CF7608-19 and common Russet Burbank (Examples of SSR fingerprint shown in Fig. 2). As a co-dominant marker, the SSR profiles were able to differentiate potato genotypes more precisely than ISSR markers because of its ability to reveal all the alternative alleles that define each genotype (Fig. 2A). For example, the profile generated by the STCPKIN3 primer pair showed only one allele in common between AF1753-16 and CF7608-19 and no allele in common between these two genotypes and Russet Burbank. Furthermore, AF1753-16 has two unique alleles while CF7608-19 has only one unique allele. Another aspect of the co-dominant nature of SSR marker is its ability to reveal heterozygosity at specific loci (Fig 2B). For example, the primer pair LERBCO showed that CF7608-19 is heterozygous with regard to the respective alleles from AF1753-16 and Russet Burbank. The fact that AF1753-16 (new release) is homozygous for a locus that is heterozygous in one of its parents (CF7608-19), and where Russet Burbank has a different allele is another unique feature of this new potato variety from Maine.

The limited survey of genetic polymorphism in commercial cultivars and wild species of potato presented here shows that both SSR (co-dominant) and ISSR (dominant) markers can be used to address the various issues and questions relevant to the activities and goals of the Maine potato breeding program in Presque Isle. Both marker types were able to distinguish the newly released Russet-type Maine potato (AF1753-16) from its Russet-type parent (CF7608-19) and common Russet Burbank. The DNA fingerprints generated so far provide a measure of its ‘distinctness’ with respect to a small set of Russet-type potato genotypes. This fingerprint can be used as a reference or standard for subsequent assessment of the ‘uniformity’ and ‘stability’ of this variety. The information from this study can also be used as a platform for more precise identification by comparison with a larger set of Russet-type potato genotypes.

3) Assessment of the genetic homogeneity of selected commercial cultivars. The second objective of this project was to assess the genetic purity of selected commercial cultivars that are currently being used as donors in the breeding program. The focus of this study was to compare different samples of the same cultivar from different sources. Samples of Red La Soda (Stock No. 2505, 2517, 2506), Russet Burbank (Stock No. 2507, 2518) and Russet Narkotah (Stock No. 2509, 2519) were compared using five ISSR primers (ISSR-CCA, ISSR-GCA, ISSR-CAC, ISSR-GT, ISSR-ACA). Our current results showed that all of the clones for each variety have identical ISSR profiles indicating high purity and efficient maintenance of these lines.

Conclusion

With the use of two types of DNA markers (SSR and ISSR), this study was able to differentiate newly released Maine potato cultivar (AF1753-16) from its Russet-type parent (CF7608-19) and commercial Russet Burbank. The DNA fingerprints generated will be used as a ‘molecular ID’ for this cultivar and will be included in the variety release report to be published in American Potato Journal.

The DNA fingerprinting strategy that we developed is suitable for genomic DNA samples from both leaf and tuber tissues. The use of tuber tissues as alternative to leaf tissues as source of DNA makes this technique handy for routine application since growing of plants is not required. Given the specific strengths and relative technical simplicity of the two marker strategies used in this survey, the choice of which method to adopt for the Maine potato breeding program should be based on a number of factors and should consider the type of application. ISSR is a ‘quick and dirty’ method with enough resolution to distinguish genotypes within a relatively narrow range of genetic diversity. It is cheap and simple (fingerprints can be generated with simple agarose gel electrophoresis) and therefore can be used for routine variety identification. On the other hand, SSR is more robust and exhibit higher resolution by virtue of its ability to reveal heterozygosity and genotype-specific or species-specific alleles. However, it requires the use of expensive and more technically demanding radioisotope or fluorescent detection methods. The Maine potato breeding program is heading towards the integration of conventional and marker-based breeding strategies, particularly for the interspecific introgression of novel genes of agronomic importance (e.g. resistance to diseases and insects and tolerance to abiotic stresses). The information generated by this project will serve as the basis for future use of SSR and ISSR marker systems for monitoring introgression and perhaps map-based cloning.

References

Bornet B, Goraguer F, Joly G, Branchard M (2002) Genetic diversity in European and Argentinian cultivated potatoes (Solanum tuberosum subsp. tuberosum) detected by inter-simple sequence repeats (ISSRs). Genome 45:481-484.

Provan J, Powell W, Waugh R (1996) Microsatellite analysis of relationships within cultivated potato (Solanum tuberosum). Theor Appl Genet 92:1078-1084.

Wulff EG, Torres S, Gonzalez-Vigil E (2002) Protocol for DNA extraction from potato tubers. Plant Mol Biol Rep 20:187a-187e.