NUCIS number 10. December 2001. 48 pages (full ... - IAMZ - ciheam

RNase polyacrylamide gel
Agarose PCR gel
leading to year to year fluctuations. Nevertheless,
field-testing is required to verify
the best crossing combinations to
achieve self-compatible progenies or to
identify production differences among related
S-genotypes. To improve the efficiency
of the field methods, autogamy levels
for the same cultivars should be recorded
over several years. Similarly, pollen-growth
studies in the laboratory suffer
from moisture stress in flowers removed
from the tree, and consequently growth
rates may be adversely affected. Pollen
tube growth in laboratory should be tested
at least for two years to be confident
on results. Only by field assays it is impossible
to predict the best crosses to
achieve 100% self-compatible progenies
or to identify genetic differences between
related individuals.
Table 1 summarises different aspects of
the biological and molecular approaches
for the assessment of self-incompatibility
in almond. Applicability for routine work,
usefulness, preciseness, rapidity and cost
of the different methods has been rated.
RNases analysis provides important advantages:
• It tests the enzyme directly associated
with self-incompatibility.
• Since all self-incompatible alleles are visible
after gel staining, it allows differentiation
between self-compatible and selfincompatible
cultivars in segregation progenies
as well as the detection of new S-
alleles.
• It can provide S-genotype information to
Table 1. Ratings of biological and molecular approaches for the assessment
of self-incompatibility in almond.
Aspects rated 1 Biological methods Molecular methods
Field crosses Microscopy S-RNases PCR
Availability of facilities ++++ +++ +++ ++
Repetitiveness ++ ++ +++ ++++
Interpretation of results +++ ++ +++ ++++
Ease of routine application ++ ++ ++++ ++++
Speed in obtaining results + ++ ++++ ++++
Initial investments +++ +++ ++ +
Cost of development ++++ +++ ++ +
Cost per sample ++++ ++++ ++ +
1
Rates given as a scale between ++++ (good) and + (bad).
assist early field selection of parents to
obtain 100% self-compatible offspring
(Batlle et al., 1997; López et al., 2001;
Mnejja et al., 2002).
• Testing of only 5-10 pistils is required
(Batlle et al., 1999).
• It allows modification of electrophoresis
conditions in order to improve discrimination
between alleles.
• It presents a high reproducibility.
• It involves moderate costs for equipment
and consumables and so less initial
investment than PCR and other molecular
techniques.
Limitations of the RNases approach include:
• Pistils are required during the blooming
period, thus flowering trees of at least 2-3
years old are needed.
• Protocol development is arduous and
specific only for RNases assessment.
• Many factors influence protein migration
during electrophoresis.
• The electrophoresis process is tedious
and time-consuming, particularly for vertical
polyacrilamide gels, which are very
thin (0.75-1 mm), and posterior staining.
• Difficult zymograms interpretation.
The main advantages provided by PCR
approaches include:
• It allows the use of vegetative tissues,
which are available since first sprout, therefore
it is not necessary to wait for the
blooming period. Seedlings can be grown
easily in greenhouses to provide leaves,
selecting only the self-compatible ones to
plant in the field.
• Only 0.1g of leaf tissue is necessary.
• Easy preparation and management of
horizontal agarose gels, and good applicability
for routine work.
10 FAO-CIHEAM - Nucis-Newsletter, Number 10 December 2001