D. Prat

Application of AFLP, RAPD and ISSR markers to genetic mapping of European and Japanese larch.

Abstract Genetic linkage maps have been increasingly developed for a wide variety of plants, using segregating populations such as F2s or backcrosses between inbred lines. These pedigrees are rarely available in outbred species like forest trees which have long generation times. Thus genetic mapping studies have to use peculiar pedigrees and markers in appropriate configurations. We constructed single-tree genetic linkage maps of European larch (Larix decidua Mill.) and Japanese larch [Larix kaempferi (Lamb.) Carr.] using segregation data from 112 progeny individuals of an hybrid family. A total of 266 markers (114 AFLP, 149 RAPD and 3 ISSR loci) showing a testcross configuration, i.e.heterozygous in one parent and null in the other parent, were grouped at LOD 4.0, θ=0.3. The maternal parent map (L. decidua)consisted of 117 markers partitioned within 17 linkage groups (1152 cM) and the paternal parent map (L. kaempferi) had 125 markers assembled into 21 linkage groups (1206 cM). The map distance covered by markers was determined by adding a 34.7-cM independence distance at the end of each group and unlinked marker. It reached 2537 cM and 2997 cM respectively for European larch and Japanese larch, and represented respectively a 79.6% and 80.8% coverage of the overall genome. A few 3:1 segregating markers were used to identify homologous linkage groups between the European larch and the Japanese larch genetic maps. The PCR-based molecular markers allowed the construction of genetic maps, thus ensuring a good coverage of the larch genome for further QTL detection and mapping studies.

Heritable Nuclear DNA Changes in Doubled Haploid Plants Obtained by Pollen Culture of Nicotiana Sylvestris

Summary Heritable quantitative and qualitative changes can be observed in the nuclear DNA of doubled-haploid (D.H.) plants obtained by pollen culture of Nicotiana sylvestris . D.H. plants resulting from consecutive androgenic cycles contain on the average increasing amounts of total DNA and increasing proportions of highly repeated sequences. All the results suggest that both A-T rich and G-C rich regions are amplified in the D.H. genome. Some hypotheses on the origin of the changes are discussed.

Genetic variability induced in Nicotiana sylvestris by protoplast culture

SummaryPlants were regenerated from axenic plantlets by mesophyll protoplast culture, without mutagenic treatment. Two different lines of Nicotiana sylvestris were used: an original line, and a diploid androgenetic line derived from it. The regenerated plants were either diploid and phenotypically similar to their respective protoplast source line, or tetraploid. Genetic studies carried out on several diploid regenerated plants revealed genetic variability. Eight of 13 selfed progenies of plants regenerated from the original line, and 1 of 8 selfed progenies of plants regenerated from the androgenetic line, produced new mutant phenotypes never observed in the protoplast source lines. Two plants regenerated from the same protoplast-derived callus produced different mutations. Selfed progenies without a recognizable mutant phenotype were also different from their respective protoplast source line for quantitative characters; protoplast culture induced a depressive effect on the size of plants derived from protoplasts at younger and older stages of development. The origin of this depression and of the mutations is discussed.

Several nuclear genes control both male sterility and mitochondrial protein synthesis in Nicotiana sylvestris protoclones.

SummaryMale sterile plants appeared in the progeny of three fertile plants obtained after one cycle of protoplast culture from a fertile botanical line and two androgenetic lines ofNicotiana sylvestris. These plants showed the same foliar and floral abnormalities as the cytoplasmic male sterile (cms) mitochondrial variants obtained after two cycles of culture. We show that male sterility in these plants is controlled by three independent nuclear genes,ms1, ms2 andms3, while no changes can be seen in the mitochondrial genome. However, differences were found between thein organello mitochondrial protein synthesis patterns of male sterile and parent plants. Two reproducible changes were observed: the presence of a new 20 kDa polypeptide and the absence of a 40 kDa one. Such variations were described previously in mitochondrial protein synthesis patterns of the cms lines. Fertile hybrids of male sterile plants showed normal synthesis patterns. The male sterile plants are thus mutated in nuclear genes involved in changes observed in mitochondrial protein synthesis patterns.

Heterozygosity and hybrid performance in larch.

Random Amplified Polymorphic DNAs (RAPD) were used for estimating genetic distances between 12 European larches (Larix decidua) and 12 Japanese larches (L. kaempferi) that were the parents in a factorial mating design. One hundred and eleven fragments were used for establishing genetic distances based on Jaccards coefficient between parents. Thirteen fragments differentiated the larch species. The genetic distance between individuals of the same species (DJ=0.39 in the Japanese larch and 0.45 in the European larch) was lower than the genetic distance between species (DJ=0.72). A UPGMA dendrogram based on genetic distances clearly clustered each larch species, confirming the speciation at a molecular level. Correlations between genetic distances of the parents and performances of the hybrid families were established for various quantitative traits. Significant values were found for growth characters and branch insertion angle, which suggested an effect of general heterozygosity level on hybrid traits. These correlations also evolved with tree age: the maximal correlation was noticed on 6-year-old trees for height. The lack of correlation between parental genetic distances and hybrid performances for the other quantitative traits suggested that these characters were controlled by fewer genes. The results of this study show that crosses between genetically distant parents produce hybrids with excellent growth performances; this represents a potential selection criterion of the genitors.

Differences in the physiological responses of two clones of Eucalyptus microtheca selected for their salt tolerance

Abstract Two Eucalyptus microtheca clones (clones 42 and 43) suspected to be salt tolerant were rooted and grown in a greenhouse. Five-month old cuttings were watered for 2 months with and without 200 mM NaCl. During the salt stress period, mineral (Na, Ca, K and Cl) and organic (soluble amino acids and proline) compounds were determined in roots, stems and leaves. Changes in protein profiles induced by salt stress were investigated after 2 weeks of salt treatment. Clone 42 demonstrated a delayed growth during salt stress while clone 43 showed a complete inhibition of shoot length. Salinity had a significant effect on mineral compounds: whatever the duration of the salt treatment, the uptake of sodium in roots was 2.5 times higher in the more tolerant clone 42 than in clone 43. Regarding potassium and calcium contents in roots, clone 42 demonstrated an increase in its amount whereas clone 43 showed a decrease. The increase in soluble amino acids induced by the stress in the different organs of the two clones was not significantly different whereas a higher content of proline was determined in clone 42 relative to clone 43. Salinity had significant effects on the content of one predominat polypeptide with an apparent molecular weight of 18 kDa which was specifically induced under salt stress in roots of clone 43. In clone 42, this polypeptide was present in low amounts in control conditions and salt treatment increased its synthesis.

Identification of European and Japanese Larch and Their Interspecific Hybrid with Morphological Markers: Application to Young Seedlings

Abstract Open-pollinated hybridisation seed orchards of European and Japanese larches produce mixed progenies combining a highly variable proportion of hybrids along with pure parental species. For several reasons, it is desirable to identify and to sort out hybrids from pure species at the seedling stage. Taxa identification of 1-2 yr-old seedlings was attempted using non-destructive assessment of several traits, including morphology, phenology, growth and architecture parameters. Two sets of progenies originating from 10 open-pollinated hybridisation seed orchards were used, relying in a first step on taxa identification of individual seedlings with diagnostic molecular markers. Based on 21 traits assessed, some clear trends in pure species and hybrid features were apparent but due to the large and overlapping ranges of taxa characteristics, no single parameter allowed unambiguous identification of taxa. Combination of traits through linear discriminant analysis made possible correct classification of 90.2% to 98.6% of individuals depending on the orchard although there were a few problematic orchards. Two traits appeared particularly pertinent for discriminating young plants taxa, namely 1st-yr leaf retention (marcescence) and the bark colour of 2nd-year shoot increments. Results were corroborated using progenies from several orchards and over two experimental periods.

Alterations of leaf characteristics and peroxidase activity in diploid androgenetic lines of Nicotiana sylvestris spegaz. et comes

Abstract Two series of diploid androgenetic lines of Nicotiana sylvestris and their original line were investigated for leaf characteristics and peroxidase activity. The first series included two androgenetic lines (‘flat’ and ‘crumpled’ phenotypes named after the leaf aspect) derived from the same doubled haploid regenerated plant. Compared to the original line, the leaves were less developed, although cell size and peroxidase activity was higher. The second series comprised androgenetic lines obtained by consecutive cycles of androgenesis. These plants had smaller leaves, larger cells, and increasing peroxidase activity through consecutive cycles of androgenesis. The ‘crumpled’ phenotype was also more and more marked with each cycle. Results are discussed in relation to other analyses performed on these plants (inheritance, DNA analysis, DNA content).

Conservation recommendations from a large survey of French orchids

The French Atlas of Orchids published in autumn 2010 is a large-scale and intensive survey of orchid populations throughout the France territory. The project was directed by the French Orchid Society and involved 3000 specialists in orchids. Overall, more than 110 000 stations were referenced and 160 taxa were reported with their range on 154 maps. This project is original by synthesizing, at large scale, much accurate naturalistic information from local surveys, thanks to a participative approach and on the basis of scientific objectives. Some orchid populations have been recorded for more than 20 years, revealing demographic trends at the scale of the country. Guidelines for application IUCN Red List criteria at regional level have been applied in order to establish The French Red List of orchid species. This represents one of the few examples of IUICN criteria being applied at a regional scale for a plant family. Based on the IUCN categorization about 1 in 5 species is threatened, one as Regionally Extinct (RE), 4 as Endangered (EN) and 23 as Vulnerable (VU). The main extinction risk consists of population size decrease, mostly due to habitat change. Further investigations are needed for the 33 taxa classified as Data Deficient. Most threatened species benefit already from protection regulations. Some recommendations will be drawn in order to conserve orchid species, paying attention to patrimonial and threatened species.

Quantitative Traits and Genetic Markers: Analysis of a Factorial Mating Design in Larch

A factorial mating design involving European and Japanese larches was carried out in order to produce interspecific hybrids. Hybrid families have been measured for 11 years for quantitative traits including growth characters, morphological characters and wood quality. Parent trees were genetically assessed using RAPD markers. Ninety-eight markers showing intraspecific polymorphism in one species at least were used in this study. Firstly, family frequencies for a marker were deduced from parental frequencies for this marker, and statistical relationship between marker frequency and performance of the family was investigated. Significant relationships were determined for 19 markers and quantitative characters including height, branch angle, stem straightness and wood specific gravity. This suggests that these markers are linked to QTL involved in these traits. Secondly, correlations between hybrid family performances and genetic distance of respective parents were investigated. A positive correlation between genetic distance and growth characters (height, volume, B.H. girth) and a negative correlation between genetic distance and branch angle were observed, and demonstrated the effect of heterozygosity level on these characters. Thus, genetic distance between parents is a first selection criteria if hybrid growth is a requested characteristic. More precisions (complete parent genotype, genetic map, QTLs distribution) are required for a better understanding of QTLs effects and expression and a possible application in marker-assisted selection.