Fingerprinting and Markers for Floral Crop Improvement

James W. MoyerDept. of Plant Pathology

North Carolina State University, Raleigh, NC 27695

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

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

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

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

Crop Values

Crop 2000 2001Potted poinsettias 246,263 256,211Geranium 207,928 202,728Chrysanthemums 205,504 197,080Impatiens 163,713 163,111Petunia 128,663 137,101Pansy/viola 106,343 126,731Orchids 99,158 108,397Lily 97,089 101,179Begonias 96,787 100,583Roses 83,164 94,071

Top ten:

New Guinea Impatiens was 11th at 75,219,000Carnation was 27th at 6,430,000

Value x $1000

Other high priority crops:

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

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

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

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

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)

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

SSR Strategies

• Database mining

• Library enrichment

• Library screening– Hybridization– PCR

• High throughput sequencing

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

SSR Results: Strategy 13 repeats 4 repeats 6 repeats 7 repeats 8 repeats

22 repeats

Total

Di-nucleotide 184 14 1 1 200

Tri-nucleotide 27 4 1 1 33

• 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

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

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 repeats 3 repeats 4 repeats 6 repeats Total

Di-nucleotide

TNTC 122 6 128

Tri-nucleotide

330 35 3 368

4-nucleotide

103 4 2 109

5-nucleotide

20 20

6-nucleotide

10 10

7-nucleotide

1 1

Retrotransposons

Identified in:– Poinsettia ( 11 cultivars)

– Chrysanthemum (1 cultivar)

– African violet ( 2 cultivars)

– Petunia (1 cultivar)

LTR Gag LTRPR INT RT RNASE H

Pol

5’ 3’300 bp

LTR Gag LTRPR INT RT RNASE H

Pol

5’ 3’300 bp300 bp

Typical Retrotransposon:

300 bp

300 bp

Reverse Transcriptase Gene:

Retrotransposon Application

Inverse PCR

Sequence

Primer Design

PCR Amplification

Genetic Markers

Clone PCR product

Inverse PCR

Sequence

Primer Design

PCR Amplification

Genetic Markers

Clone PCR product

Retrotransposon Analysis

Eckespoint Pink PeppermintSonora Jingle Bells

Freedom MarbleWinter RoseFreedom Jingle BellsEckespoint Jingle Bells

PetuniaPink African VioletPeterstar Jingle BellsPetuniaPurple African VioletCoral Davis Mum

Coral Davis MumCoral Davis MumCoral Davis MumPetuniaPink African VioletPurple African Violet

Eckespoint Jingle BellsEckespoint Pink PeppermintPeterstar Jingle BellsSonora Jingle BellsFreedom MarbleWinter Rose

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