Purity control of seedlots or seedling batches of hybrid larch
(Larix decidua x L.kaempferi)
Prepared by Luc E.Pâques1 (INRA-Orléans)
June 8, 2009
Genetic control of seed quality has for larch a special interest for interspecific hybrids seed
lots. Indeed due to non- or only partial-overlapping of flowering phenology between pure
parental species (namely European (EL) and Japanese larches (JL)), seedlots – mostly
collected from open-pollinated mother trees in seed orchards- comprise a more or less
important proportion of hybrids seeds mixed up with either EL or JL or both according to
orchard designs, ways of seed collection, etc. Therefore foresters who are planting hybrid
larch seedlings have no guarantee on the hybrid purity and as a result, plantation growth and
yield may be disappointing with regards to the expected and promoted hybrid vigour, creating
thereby a loss of income. This raises the question of how suited if the certification of forest
reproductive material, in particular when hybrids are concerned (Myking and Skroppa, 2007).
To answer to this question, isozyms markers were first developed by Bergmann and Ruetz
(1987). Based on a couple of isozyms (SKDH and NDH) specific to the parental species
clones of one orchard in Germany, Bergmann and Ruetz (1987) and Häcker and Bergmann
(1991) could determine the hybrid proportion of seedlots from different crops of that orchard.
On one side, they showed that the hybrid purity was sometimes far from 100%; on the other
side, they showed that it was also variable from year to year (69-85%) and according to the
species collected (19-35%), reflecting the quality of the overlapping of flowering between
species.
In order to further improve the quality of the diagnostic and to make it more reliable and
‘universal’ whatever the orchards (various origins, various numbers of clones) - INRA
developed new molecular markers based on cytoplasmic DNA (Achère, 1999; Achère et al.
2004) (LL and F-13). The interest of these markers in conifers is due to the way they are
inherited: the mitochondrial DNA being of mother origin, while the chloroplastic DNA is of
male origin. Therefore the combination of these markers allows a clear distinction of the
European larch (EL), the Japanese larch (JL) and their hybrid (HL), and thereby a precise
determination of hybrid purity in a given lot. In addition, it allows a precise identification of
the female and male parents, permitting for example to distinct ELxJL hybrids from JLxEL
hybrids. Advantageously, these markers can be used on any type of tissues (needle, seed, bud,
embryogenic mass, etc).
Since their development in 1999, these diagnostic markers have been routinely used in France
firstly for expertise of seedlots purity and seedlings purity, but also for control of pollution in
pure species seed stands or seed orchards. They are also used to detect errors in established
seed orchards due to mislabelling of clones collected, graft reject, clone selection errors, etc,
but also to assist in establishment of new hybrid seed orchards (control of clone taxa). They
also allow detecting errors in seed collection process (when seed should be collected on one
given species and not on the two) and in seed/plant handling (mixtures). They can be used at a
1
paques@orléans.inra.fr
very early developmental stage of embryos in cones and in any case prior to any commercial
seed collection as an early diagnostic test: according to hybrid purity results, seed orchard
managers can decide to proceed or not to the cones harvesting: some years, it may avoid
costly collection of seedlots with a too low hybridity.
These markers have also been useful
- to compare seed orchard designs or to monitor ways of flowering management (such
as flower induction, supplemental pollination, etc) enhancing interspecies
hybridisation,
- to develop morphological markers to sort out seedlings in nursery batches (Pâques,
2002; Pâques et al. 2006),
- to approach heterosis studies by a cheap way (Pâques, 2009),
- to validate growth models or any kind of studies relying on so-called hybrid stands,
- etc.
So far, their validity is confirmed only for hybrids between EL and JL and therefore should be
used carefully if any other species is susceptible to be present, and only on first-generation
hybrids: here again caution should be taken if hybrids are already –usually by mistakepresent in the orchard as parent.
Since January 1, 2003, the Council Directive 1999/105/EC demands that the percentage of
hybrids in the reproductive material from a hybrid seed orchard must be determined by a
verification test. The diagnostic markers developed for EL and JL larches and their hybrids
allow now this verification routinely.
Literature
Achère V. Recherche de marqueurs moléculaires pour l’identification des mélèzes hybrides
(Larix x eurolepis). DEA Biologie forestière. INRA-Univ. Nancy I. 20pp + annexes.
Acheré V., Faivre-Rampant P., Pâques L.E., Prat D. 2004. Chloroplast and mitochondrial
molecular tests identify European x Japanese larch hybrids. Theor.Appl.Genet. 108: 16431649.
Bergmann F., Ruetz W. 1987. Identifizierung von Hybridlärchensaatgut aus Samenplantagen
mit Hilfe eines Isoenzym-Markers. Silvae Genet. 36: 102-105.
Häcker M., Bergmann F. 1991. The proportion of hybrids in a seed orchard composed of two
larch species (L.europaea and L.leptolepis). Ann.Sci.For. 48: 631-640.
Myking T., Skroppa T. 2007. Certification of forest reproductive material – is present practice
sufficient? Issue n° 33- IPGRI Newsletter for Europe.
Pâques L.E. 2002. Détermination de la pureté spécifique dans les descendances de mélèze
hybride issues de vergers à graines d’hybridation : Evaluation de l’efficacité du tri en
pépinière selon des critères morphologiques. DERF-INRA 01-40-12/99 final report,
28pp.
Pâques L.E. 2009. Growth rhythm parameters as components of hybrid vigour in young
seedlings of hybrid larch (Larix decidua x L.kaempferi), Silvae Genet. 58 (1-2): 42 :53.
Pâques L.E., Philippe G., Prat D. 2006. Identification of European and Japanese larch and
their interspecific hybrid with morphological markers: application to young seedlings.
Silvae Genet. 55 (3): 123-134.