1. Fingerprinting and Markers for Floral Crop Improvement James W. Moyer Dept. of Plant Pathology North Carolina State University, Raleigh, NC 27695

2. Introduction Floral industry has experienced significant growth –Industry production –Introduction of new products Cultivars of existing crops New species Rapid expansion brings new issues –Breeder’s rights –Grower confidence in cultivar identity –Improved plant quality Disease and insect resistance Heat and drought tolerance Longer shelf life

3. DNA Fingerprinting and Molecular Markers DNA fingerprinting is a useful tool in floral crop genetics –Cultivar identification –Maintenance of breeding lines –Protecting breeders’ rights Molecular markers can facilitate the identification and introgression of genes for cultivar improvement Methods for generating genetic markers include: –AFLP –SSR –Retrotransposons

4. Objectives Identify and prioritize commercially important crops –Survey the industry Develop core tools for priority crops –Research available technologies –Select a method and develop a strategy –Generate polymorphisms useful for fingerprinting or other marker assisted breeding applications Develop high-throughput technologies for efficient processing

5. Survey Prioritizing a list of crops according to: –Breeding effort: could support development and use of molecular tools –Competitiveness: would benefit from fingerprinting for patenting and monitoring of license agreements Responses: –Highest priority crops are chrysanthemum, petunia, geranium, carnation, and New Guinea Impatiens

6. Crop Values Crop20002001 Potted poinsettias246,263256,211 Geranium207,928202,728 Chrysanthemums205,504197,080 Impatiens163,713163,111 Petunia128,663137,101 Pansy/viola106,343126,731 Orchids99,158108,397 Lily97,089101,179 Begonias96,787100,583 Roses83,16494,071 Top ten: New Guinea Impatiens was 11 th at 75,219,000 Carnation was 27 th at 6,430,000 Value x $1000 Other high priority crops:

7. AFLP Fingerprinting Used to generate molecular markers for fingerprinting without prior knowledge of the genome Successful for poinsettia and NGI –Databases created for both crops Progressed from manual radioactive techniques (above) to semi-automated fluorescent techniques (below) F-AFLP utilized for genetic analysis in several plant species –Barley, wheat, and azalea

8. F-AFLP Fingerprinting Advantages of fluorescent-based methods over traditional AFLP: –Fluorescent label replaces the radioactive label, making this procedure safer and less expensive –Scoring is more accurate and more reproducible Better separation of fragments on the gel Internal size standard present in every lane –Fragment scoring is based on a numerical representation of the fragment intensity Compared to conventional 33P-labeled AFLP, this technique: –Increased the number of detectable fragments –Showed higher resolution of amplification products –Made scoring faster and more objective

9. Fingerprinting in Poinsettia Poinsettia database: –117 cultivars –41 AFLP fragments Successfully distinguishes most cultivars –Multiple plants from representative cultivars used for validation studies –Plants from the same breeding family cluster together –Color sports cluster together as the same cultivar

10. Fingerprinting in NGI NGI database: –168 cultivars –95 AFLP fragments Successfully distinguishes cultivars –Samples collected from multiple breeders –Duplicate samples used for validation studies Always cluster together –Larger number of fragments used in order to account for genetic variation

11. AFLP Fingerprinting Disadvantages: –Technology is patented –Many polymorphisms may be needed to distinguish closely related cultivars or cultivars with higher levels of genetic variation (40 – 80 fragments)

12. Microsatellites (SSRs) Genetic markers used for genotype identification and marker-assisted breeding in a wide range of crops including: –Non-floral crops Soybean, rice, apple, pine, mango, cotton –Floral crops Chrysanthemum, Dianthus Fewer high quality markers are needed to differentiate genotypes System is patented but licensable, and could be used on a larger scale than AFLP technology

13. SSR Strategies Database mining Library enrichment Library screening –Hybridization –PCR High throughput sequencing

14. Strategy 1: Library Screening and PCR Genomic DNA from poinsettia was partially digested with a restriction enzyme to generate ~1200bp fragments Fragments were ligated to a plasmid vector and transformed to make a library The library was screened by PCR using primers complementary to the repetitive sequence with vector primers PCR positive primers were sequenced and analyzed

15. SSR Results: Strategy 1 3 repeats4 repeats6 repeats7 repeats8 repeats 22 repeats Total Di- nucleotide 1841411200 Tri- nucleotide 2741133 Number of plates sequenced = 3 Number of repeats identified = 233 Number of polymorphic repeats = 1 Change strategies to cover more of the genome and identify more potential markers

16. Strategy 2: Library Sequencing Partially digest genomic DNA to generate 1200bp fragments Ligate fragments into a plasmid vector to create a library Use high-throughput methods to sequence the library, and therefore more of the poinsettia genome –Plate has 96 wells: 700bp per well = 67200bp per plate –Literature indicates that one SSR will be present every 6000bp –Could theoretically identify 11 SSRs per sequencing plate

17. SSR Results: Strategy 2 Number of repeats identified to date = 636 The larger repeat sequences will be analyzed for possible polymorphism Additional colonies will be sequenced to identify additional microsatellites 2 repeats3 repeats4 repeats6 repeatsTotal Di- nucleotide TNTC1226128 Tri- nucleotide 330353368 4- nucleotide 10342109 5- nucleotide 20 6- nucleotide 10 7- nucleotide 11

18. Retrotransposons Identified in: –Poinsettia ( 11 cultivars) –Chrysanthemum (1 cultivar) –African violet ( 2 cultivars) –Petunia (1 cultivar) LTRGagLTRPRINTRTRNASE H Pol 5’3’ 300 bp LTRGagLTRPRINTRTRNASE H Pol 5’3’ 300 bp Typical Retrotransposon: 300 bp Reverse Transcriptase Gene:

19. Retrotransposon Application

20. Retrotransposon Analysis Eckespoint Pink Peppermint Sonora Jingle Bells Freedom Marble Winter Rose Freedom Jingle Bells Eckespoint Jingle Bells Petunia Pink African Violet Peterstar Jingle Bells Petunia Purple African Violet Coral Davis Mum Petunia Pink African Violet Purple African Violet Eckespoint Jingle Bells Eckespoint Pink Peppermint Peterstar Jingle Bells Sonora Jingle Bells Freedom Marble Winter Rose

21. Summary Accomplishments –Fluorescent technologies were adapted for use with microsatellite markers –Input was collected from the industry and important crops were identified –Strategies for finding SSR markers were developed Methods currently being tested and refined on poinsettia Techniques will be applied to other floral crops –Designed strategies for locating retrotransposons Tested in several crops –Implemented high-throughput methods DNA extraction from cloning experiments Examine possibility of multiplexing fluorescent SSR primers in future experiments