California Avocado
Society 1989 Yearbook 73:59-61
Molecular Genetics of Avocado
Michael
T. Clegg and Joel W. Davis
Department of Botany and Plant Sciences,
Genetic markers provide an important tool to address
a wide variety of practical problems in avocado improvement. Until recently, isozymes have provided the only source of genetic markers
in avocado. It is now possible to develop an almost unlimited number of genetic
markers using molecular techniques, and these can be used to address a number
of significant problems.
Some of the problems which we are currently working
on include: (1) A study of the genetic relationships between the major
varieties of avocado. The origins and genetic relationships among the major
botanical varieties are poorly understood and genetic markers are a major aid
in unraveling these relationships. (2) The specific parentage of important
cultivars is obscure in a number of cases, and detailed genetic markers will
allow us to identify pollen parents through the use of DNA fingerprints. (3) A
genetic map of the avocado genome can be constructed using molecular markers
and, once such a map is constructed, important practical breeding problems can
be addressed. Almost any character which has a strong genetic basis, ranging
from fruit quality, flowering times, root-rot resistance, salt tolerance,
drought tolerance, etc., could be analyzed and transferred if sufficient linked
markers were available.
In the last several years, the methods of molecular
genetics have provided genetic markers for mapping plant genomes. Molecular
methods have significant advantages over traditional approaches such as the use
of isozymes. Isozymes
provide a limited number of markers (typically a maximum of 20 to 30
polymorphic gene loci), whereas an unlimited supply of restriction fragment
length polymorphisms (RFLP's) can routinely be
obtained from most plant species. Second, isozyme
techniques have limited genetic resolution. Frequently, these methods are not
informative at or above the interspecific level. In
contrast, molecular markers can be selected that resolve genetic relationships
at virtually any taxonomic level, ranging from individuals within a family
through to subgeneric or higher level comparisons.
Finally, the use of molecular markers provides a foundation for further
biotechnological work on avocado improvement.
Project Objective
The primary objective of this project is to develop a
large number of molecular markers to address a number of practical problems in
avocado improvement.
Practical applications of this project include the
use of DNA markers to (1) identify the genetic relationships among the major
varieties of avocado; (2) to provide a precise genetic identification of
various avocado cultivars within varieties; (3) to identify the parentage of specific
cultivars; and (4) to begin a program to use RFLP's
to map the avocado genome.
Our current and preliminary work firmly establishes
the feasibility of molecular methods as applied to avocado genetics.
Progress Report
We have used cloned DNA probes for a cellulase gene, for the ribosomal RNA genes, and for
chloroplast genes to study the genetic relationships among avocado varieties.
Our previous work, which is now in press (Furnier et
al, Journal of Heredity, 1990), suggests that Persea
DNA has been prepared from 46 cultivars representing
all three major varieties of avocado. Twenty of these samples have previously
been screened using the cellulase probe. We found
that some, but not all, cultivars within varieties were identifiable based on
RFLP patterns. We are currently using anonymous single-copy clones (described
below) for further evaluation of cultivars.
A large number of DNA clones are required to provide
genetic markers for parental identification and for genome mapping. To begin
work towards these two objectives, we have cloned random avocado DNA fragments
into a plasmid vector and are characterizing these for their usefulness as
genetic markers. To date, 15 clones have been characterized. Eleven of these
clones represent single-copy sequences that are polymorphic when tested against
cultivars representing three major avocado varieties and are thus good RFLP
markers. Three of the remaining probes are multiple-copy sequences that appear
to be highly polymorphic between cultivars within varieties. These "hypervariable" probes may be especially useful in
cultivar identification. We are now characterizing 17 more clones and expect to
increase the number of single-copy probes to 20-25. In addition, there are
approximately 50 more clones yet to be screened and characterized.
Fig. 1. Relationships
among Persea.
We have now begun using our probes to screen a panel
of cultivars representing several commercial and experimental
selections in order to address certain questions of parentage and cultivar
identification which might aid the breeding program and the industry. DNA has
also been prepared from approximately 85 Hass seedlings that can be regarded as
F2 selfed progeny with respect to loci heterozygous
in the parent. This material is now being used to test for genetic linkage
among single copy probes.
1989-90 Objectives and Expected Accomplishments
Our objectives during the current project year are;
(1) To identify and characterize a large number of
genomic clones from avocado. Ultimately, we hope to characterize more than 100
clones in order to establish a large number of RFLP markers. The genetic characterization
requires that we hybridize each cloned fragment to a panel of avocado material
in order to identify so-called "single copy" genes and to further
identify those genes which are polymorphic; (2) We will also search among our
cloned fragments for potential hypervariable
sequences which will prove especially useful in determining genetic
fingerprints of cultivars; (3) We plan to continue making DNA preparations from
avocado cultivars and related Persea species;
and (4) we will continue to screen panels of various cultivars to address
certain problems in avocado breeding, as well as our F2 progeny to be used in
constructing a genomic map of the avocado.
The data obtained in our work will be analyzed to (1)
establish the genetic basis of each molecular clone (e.g., single copy,
multiple copy); (2) search for linkage relationships
among RFLP's based upon the analysis of segregating
F2 progenies; and (3) establish unique genetic identifications of all avocado
cultivars.
In future years we intend to assemble a detailed RFLP
map of the avocado genome where individual RFLP's
will mark segments of all 12 avocado chromosomes. This technology has proved
very powerful in tomato, maize, soybean, and other crop breeding efforts, and
it should prove even more powerful when applied to the genetic characterization
of long-lived crops such as avocado.