Location,expression and orientation of the putative chlororespiratory enzymes,Ndh and IMMUTANS,in higher-plant plastids

The chloroplasts of many plants contain not only the photosynthetic electron transport chain, but also two enzymes, Ndh and IMMUTANS, which might participate in a chloroplast respiratory chain. IMMUTANS encodes a protein with strong similarities to the mitochondrial alternative oxidase and hence is likely to be a plastoquinol oxidase. The Ndh complex is a homologue of complex I of mitochondria and eubacteria and is considered to be a plastoquinone reductase. As yet these components have not been purified to homogeneity and their expression and orientation within the thylakoid remain ill-defined. Here we show that the IMMUTANS protein, like the Ndh complex, is a minor component of the thylakoid membrane and is localised to the stromal lamellae. Protease digestion of intact and broken thylakoids indicates that both Ndh and IMMUTANS are orientated towards the stromal phase of the membrane in Spinacia oleracea L. Such an orientation is consistent with a role for the Ndh complex in the energisation of the plastid membrane. In expression studies we show that IMMUTANS and the Ndh complex are present throughout the development of both Pisum sativum L. cv Progress No. 9 and Arabidopsis thaliana (L.) Heynh. leaves, from early expansion to early senescence. Interestingly, both the Ndh complex and the IMMUTANS protein accumulate within etiolated leaf tissue, lacking the photosystem II complex, consistent with roles outside photosynthetic electron transport.Abbreviations PQ plastoquinone - PSI, PSII photosystem I, II     

The development and evaluation of consensus chloroplast primer pairs that possess highly variable sequence regions in a diverse array of plant taxa

Although universal or consensus chloroplast primers are available, they are limited by their number and genomic distribution. Therefore, a set of consensus chloroplast primer pairs for simple sequence repeats (ccSSRs) analysis was constructed from tobacco (Nicotiana tabacum L.) chloroplast sequences. These were then tested for their general utility in the genetic analysis of a diverse array of plant taxa. In order to increase the number of ccSSRs beyond that previously reported, the target sequences for SSR motifs was set at A or T (n 7) mononucleotide repeats. Each SSR sequence motif, along with ±200-bp flanking sequences from the first of each mononucleotide base repeat, was screened for homologies with chloroplast DNA sequences of other plant species in GenBank databases using BLAST search procedures. Twenty three putative marker loci that possessed conserved flanking sequence spans were selected for consensus primer pair construction using commercially available computer algorithms. All primer pairs produced amplicons after PCR employing genomic DNA from members of the Cucurbitaceae (six species) and Solanaceae (four species). Sixteen, 22 and 19 of the initial 23 primer pairs were successively amplified by PCR using template DNA from species of the Apiaceae (two species), Brassicaceae (one species) and Fabaceae (two species), respectively. Twenty of 23 primer pairs were also functional in three monocot species of the Liliaceae [onion (Allium cepa L.) and garlic (Allium sativum L.)], and the Poaceae [oat (Avena sativa L.)]. Sequence analysis of selected ccSSR fragments suggests that ccSSR length and sequence variation could be useful as a tool for investigating the genetic relationships within a genus or closely related taxa (i.e., tribal level). In order to provide for a marker system having significant coverage of the cucumber chloroplast genome, ccSSR primers were strategically "recombined" and named recombined consensus chloroplast primers (RCCP) for PCR analysis. Successful amplification after extended-length PCR of 16 RCCP primer pairs from cucumber (Cucumis sativus L.) DNA suggested that the amplicons detected are representative of the cucumber chloroplast genome. These RCCP pairs, therefore, could be useful as an initial molecular tool for investigation of traits related to a chloroplast gene(s) in cucumber, and other closely related species.Communicated by C. Möllers     

Mapping QTLs influencing rice floral morphology using recombinant inbred lines derived from a cross between <Emphasis Type="Italic">Oryza sativa</Emphasis> L. and <Emphasis Type="Italic">Oryza rufipogon</Emphasis> Griff

To understand the genetic basis of floral traits associated with the mating system in rice, we analyzed pistil, stamen and glume traits using a recombinant inbred line population, derived from a cross between an Asian cultivated rice ( Oryza sativa L.), Pei-kuh, and a wild rice ( Oryza rufipogon Griff.), W1944. Quantitative trait loci (QTLs) affecting floral morphology were detected by composite interval mapping using a linkage map constructed using 147 markers, mostly RFLPs. A total of 7, 4, 14 and 6 QTLs were detected for traits related to pistil, stamen, and size and shape of the glume, respectively. Comparison of 31 QTLs affecting these organs revealed ten QTLs affecting the different organs in four adjacent regions on chromosomes 2, 4, 5 and 10, but most QTLs (68%) were located separately on the whole chromosomes. Although four QTLs for stigma breadth, anther length and thickness of lemma and palea explained more than 25% of the total phenotypic variance, most QTLs (87%) had smaller effects. These results suggest that quantitative variation observed for pistil, stamen and glume traits is controlled by several distinct genes with small effects.     

Comparative genetic linkage maps of <Emphasis Type="Italic">Eucalyptus grandis</Emphasis>, <Emphasis Type="Italic">Eucalyptus globulus</Emphasis> and their F<Subscript>1</Subscript> hybrid based on a double pseudo-backcross mapping approach

Comparative genetic mapping in interspecific pedigrees presents a powerful approach to study genetic differentiation, genome evolution and reproductive isolation in diverging species. We used this approach for genetic analysis of an F₁ hybrid of two Eucalyptus tree species, Eucalyptus grandis (W. Hill ex Maiden.) and Eucalyptus globulus (Labill.). This wide interspecific cross is characterized by hybrid inviability and hybrid abnormality. Approximately 20% of loci in the genome of the F₁ hybrid are expected to be hemizygous due to a difference in genome size between E. grandis (640 Mbp) and E. globulus (530 Mbp). We investigated the extent of colinearity between the two genomes and the distribution of hemizygous loci in the F₁ hybrid using high-throughput, semi-automated AFLP marker analysis. Two pseudo-backcross families (backcrosses of an F₁ individual to non-parental individuals of the parental species) were each genotyped with more than 800 AFLP markers. This allowed construction of de novo comparative genetic linkage maps of the F₁ hybrid and the two backcross parents. All shared AFLP marker loci in the three single-tree parental maps were found to be colinear and little evidence was found for gross chromosomal rearrangements. Our results suggest that hemizygous AFLP loci are dispersed throughout the E. grandis chromosomes of the F₁ hybrid.Communicated by O. Savolainen     

Sequence variation and genomic organization of fatty acid desaturase-2 (fad2) and fatty acid desaturase-6 (fad6) cDNAs in maize

Increased levels of oleic acid may enhance the nutritional and functional value of corn. Corn oil is primarily composed of palmitic, stearic, oleic, linoleic and linolenic fatty acids. Delta-12 desaturase in plants converts oleic acid (18:1) to linoleic acid (18:2) by inserting a double bond at the delta-12 position. Fatty acid desaturase-2 (fad2) encodes delta-12 desaturase that functions in the endoplasmic reticulum while fatty acid desaturase-6 (fad6) encodes delta-12 desaturase that functions in plastids. Complementary DNA (cDNA) clones from putative maize homologs for fad2 and fad6 were identified and the entire clones DNA sequenced. The maize fad2 cDNAs showed an amino-acid identity of 67-77% to fad2 of Glycine, Arabidopsis and Brassica species. Our cDNA sequence comparisons suggested that more than one fad2 gene is transcribed in maize embryos. Two different fad2 cDNAs from an embryo cDNA library map to separate chromosomal positions, providing evidence consistent with two different isoforms of fad2 expressed in the embryo. The fad2 cDNAs from multiple tissue sources clustered into three groups on a phenogram, and map to different positions on chromosomes 4, 5 and 10, which suggests at least three different isoforms of fad2 may be expressed in the maize plant. The two maize fad6 cDNAs share 81% amino-acid identity with the Arabidopsis fad6 and map to chromosome 1. Northern analysis revealed that fad2 is transcribed in embryos at 14, 21, 28 and 35 days after pollination, with the highest level observed at day 14. None of the fad2 or fad6 clones mapped to maize chromosome bins associated with QTLs for the ratio of oleic/linoleic acid, notably bin 6.04 which contains the linoleic1 locus and the largest reported QTL for the oleic/linoleic ratio. This suggests, but does not prove, that some of the QTLs for the oleic/linoleic acid ratio do not involve allelic variants of fad2 or fad6 but rather involve other genes that may influence flux through the enzymes encoded by fad2 or fad6.     

Synthesis of intergeneric hybrids and establishment of genomic affinity between <Emphasis Type="Italic">Diplotaxis catholica</Emphasis> and crop <Emphasis Type="Italic">Brassica</Emphasis> species

Intergeneric hybrids of the wild crucifer Diplotaxis catholica (2n = 18, D(C)D(C)) as female with two crop Brassica species, namely Brassica rapa (2n = 20; AA) and Brassica juncea (2n = 36; AABB) as male, were developed, using ovary and sequential culture. Reciprocal crosses were not successful, suggesting unilateral cross incompatibility. Morphologically, the hybrid plants resembled the crop brassica parents, but were nearly male- as well as female-sterile. Induction of amphiploidy helped to improve pollen fertility for the D. catholica x B. rapa cross (73%), but less so for the D. catholica x B. juncea cross (35-40%). Female fertility was also higher in both the amphiploids. Cytological analysis of the F(1) hybrids revealed aberrant meiosis with predominant occurrence of the univalents. Partial genomic homoeology between the A genome of B. rapa and the D(C) genome of D. catholica was indicated by the presence of up to five bivalents in 14.7% of the PMCs in the D. catholica x B. rapa hybrid, and 1-2 trivalents or a quadrivalent in nearly 44% of the PMCs in the derived amphiploid. In the second cross, D. catholica x B. juncea, up to six bivalents and one trivalent were observed indicating homoeology between the A/B genomes of B. juncea and the D(C) genome of D. catholica. The possibility of introgression of desirable genes from D. catholica into crop Brassica species exists in view of significant affinity between the D(C) and A/B genomes.     

Gene flow estimation with microsatellites in a Malagasy seed orchard of Eucalyptus grandis

Eucalyptus grandis has a mixed-mating reproductive system. Malagasy Eucalyptus seed orchards were established 15 years ago with two aims both based on panmixia: open-pollinated seed production and genetic improvement. The panmixia hypothesis has never been confirmed in the seed orchard. From a seedling seed-orchard stand comprising 349 trees and using data obtained with six selected microsatellite markers, paternity analysis was performed for 724 offspring collected on 30 adult trees. Paternity assignment, based on exclusion procedures and likelihood-ratio method, was achieved with high accuracy; the exclusion probability value was 0.997. The outcrossing rate was very high (96.7%). More than 50% of potential male trees (199 out of 349) in the seed orchard contributed to pollination for 440 offspring of 30 progenies (8.6% of the basic population). The pollination rate from outside the seed orchard was high (39.2%), but might be due to the small size of this seed orchard. This study showed that "panmixia-like pollination" can be assumed.     

Genetic mapping of QTLs affecting productivity and plant architecture in a full-sib cross from non-inbred parents in Cassava (<Emphasis Type="Italic">Manihot esculenta</Emphasis> Crantz)

An attempt was made to identify quantitative trait loci (QTLs) for several productivity and plant architecture traits in a full-sib progeny of 144 individuals from two non-inbred parents in cassava. A molecular linkage map of this cross constructed previously with over 250 markers was the source of molecular markers. The progeny were grown under field conditions at two locations (Palmira and Quilichao) in Colombia and evaluated in 2 years (1998 and 1999) for architecture and productivity traits. Architecture traits evaluated were plant height (PH), branching height (BH), branching levels (BL), branching index (BI), stem portion with leaves (SPL) and leaf area index (LAI). Productivity traits were those related to total dry matter production and distribution, namely fresh root yield (FRY), fresh shoot yield (FSY), harvest index (HI) and the number of storage roots (NR). Phenotypic evaluation of the traits in this population revealed continuous variation for all traits. Broad-sense heritability estimates, ranged from 36% (for NR) to 94% (for BH). Several significant phenotypic correlations were observed between architecture and productivity traits. Primary QTLs, using the single-QTL model, and secondary QTLs, by a primary QTL interaction model, were detected by interval mapping. A total of 30 primary QTLs and 84 secondary QTLs were detected. We identified 35% of detected QTLs in two or more trials, the other QTLs were environment-specific. These results underscore the significant genotype × environment interactions found for most of the traits. Several genomic segments affecting multiple traits were identified and were in agreement with correlation among traits. All QTLs identified for FRY were found associated with either component traits of productivity or architecture traits. This study suggests that QTLs for plant architecture can be used to improve productivity. However an exhaustive search and analysis of QTLs controlling architecture is required before marker-assisted selection (MAS) for increasing productivity can be initiated.Communicated by H. C. Becker