The genome of the non-cultured,bacterial-like organism associated with citrus greening disease contains thenusG-rplKAJL-rpoBC gene cluster and the gene for a bacteriophage type DNA polymerase

We have recently cloned three DNA fragments (In-2.6, In-1.0, and In-0.6) of the noncultured, bacterial-like organism (BLO) associated with citrus greening disease. Nucleotide sequence determination has shown that fragment In-2.6 is part of therplKAJL-rpoBC gene cluster, a well-known operon in eubacteria. The DNA fragment upstream of and partially overlapping with In-2.6 could be isolated and was shown to be thenusG gene. InEscherichia coli, nusG is also immediately upstream ofrplKAJL-rpoBC. Fragment In-1.0 carries the gene for a bacteriophage type DNA polymerase. Fragment In-0.6 could not be identified.When In-2.6 was used, at high stringency, as a probe to detect greening BLO strains in infected plants, hybridization was obtained with all Asian strains tested, but not with the African strain examined. At lower stringencies, In-2.6 was able to detect also the African strain. The implications of these reults in the taxonomical position of the greening BLO are discussed.     

Podospora anserina mutant defective in protoperithecium formation, ascospore germination, and cell regeneration.

A mutant (modx) was selected on the basis of the suppression of self-lysis due to a recessive mutation (modB). modx, a dominant mutation, reduced hyphal branching from nonapical cells, abolished protoperithecium formation, and induced the death of stationary cells only when these were isolated to obtain further development. Mutant ascospores, formed in the fruiting bodies which occasionally occur under specific conditions (32 degrees C on starved medium), showed a delay in the germination process (up to 3 months instead of about 5 h for wild-type ascospores) when submitted to incubation under standard conditions (26 degrees C on germination medium) and failed to germinate at 18 degrees C. Revertants from modx strains, selected on the basis of the suppression of the nonrenewal of growth from stationary cells, were wild type for all the other three defects. Indirect arguments suggested that the modx mutant strain might be defective in the control of a specific class of stable messenger ribonucleic acids which would be essential for the physiology of ascospores and stationary cells.     

Phylogenetic relationships of three porcine mycoplasmas, Mycoplasma hyopneumoniae, Mycoplasma flocculare, and Mycoplasma hyorhinis, and complete 16S rRNA sequence of M. flocculare.

The nucleotide sequence of the 16S rRNA gene of Mycoplasma flocculare was determined and was compared with the sequence of a related porcine mycoplasma, Mycoplasma hyopneumoniae. While the overall level of DNA-DNA homology was approximately 11%, sequence alignment of the two 16S rRNA genes yielded a homology value of more than 95%, emphasizing the highly conserved nature of the 16S rRNA gene. Multiple sequence alignments with other mollicutes indicated that M. flocculare, M. hyopneumoniae, and Mycoplasma hyorhinis form a subcluster within the fermentans phylogroup, and this subcluster is distinct from the Mycoplasma pneumoniae phylogroup. Thus, the three mycoplasmas isolated from porcine respiratory systems exhibit phylogenetic similarities.     

Characterization of a molecularly cloned retroviral sequence associated with Fv-4 resistance.

The murine leukemia virus (MuLV) sequence associated with the resistance allele of the Fv-4 gene (Fv-4r) was molecularly cloned from genomic DNA of uninfected mice carrying this allele. The 5.2-kilobase cloned EcoRI DNA fragment (pFv4) was shown by nucleotide sequencing to contain 3.4 kilobases of a colinear MuLV-related proviral sequence which began in the C-terminal end of the pol region and extended through the env region and the 3' long terminal repeat. Cellular sequences flanked the 3' as well as the 5' ends of the truncated MuLV sequence. Alignment of the N-terminal half of the pFv4 env sequence with ecotropic, mink cell focus-forming, and xenotropic MuLV env sequences established the relatedness of pFv4 and ecotropic MuLV env sequences. A subcloned 700-base pair segment (pFv4env) from the 5' env region of pFv4 was used as an Fv-4-specific probe; it hybridized specifically to the Fv-4r-associated proviral sequence but not to endogenous ecotropic MuLV proviral DNA under high stringency. All Fv-4-resistant mice contained the same retroviral segment associated with the same flanking cellular DNA. Expression of Fv-4r-specific mRNA was demonstrated in the spleens of Fv-4r mice but not Fv-4s mice, supporting the previously proposed resistance model based on interference.     

Inheritance and molecular variations of PCR-SSCP fragments in pedunculate oak (Quercus robur L.)

Single-strand conformaiton polymorphism (SSCP) profiles of six PCR-amplified fragments (250–800 bp) were analyzed in three full-sib families of pedunculate oak (Quercus robur L.) and their parents. Among the six fragments, four were polymorphic and one exhibited complex patterns that were not changed by varying the SSCP conditions. The number of bands for the analyzed fragments varied between two and four among individuals regardless of fragment size. As shown by segregation data, the variation in the number of bands between trees could only be attributed to the allelic composition (homozygotes vs heterozygotes): a genotype that exhibited two bands was presumptively homozygous, wheras a genotype exhibiting three or four bands was heterozygous. Mendelian proportions were observed in all crosses for each polymorphic fragment. In one cross, we could clearly identify a null allele due to a possible mutation at a primer site. Single-base mutations and short insertion-deletions were shown to be the molecular causes of the SSCP polymorphism observed between different alleles. The use of SSCP as a technique to identify co-dominant markers of PCR fragments (up to 800 bp) is recommended for gene diversity studies or for gene mapping.     

Characterization of mixed disomic and polysomic inheritance in the octoploid strawberry (<Emphasis Type="Italic">Fragaria</Emphasis> × <Emphasis Type="Italic">ananassa</Emphasis>) using AFLP mapping

A two-way pseudo-testcross strategy, combined with Single Dose Restriction Fragment (SDRF) marker analysis, was used for genetic mapping in the octoploid cultivated strawberry Fragaria x ananassa (2n = 8 x = 56). Based on a 113 full-sib progeny from a cross between the variety Capitola and the clone CF1116, we generated two parental maps using Amplified Fragment Length Polymorphism (AFLP) markers. Ninety two percent of the markers (727 out of 789) showed ratios corresponding to simplex markers (the majority being SDRF markers), and 8% (62 out of 789) fitted a multiplex ratio. Linkage maps were first established using SDRF markers in coupling phase. The female map comprised 235 markers distributed among 43 co-segregation groups, giving a map size of 1,604 cM. On the male map, 280 markers were assigned to 43 co-segregation groups, yielding a map size of 1,496 cM. Once the co-segregation groups were established, their association was tested using repulsion-phase markers. In total, taking into account associations representing the same linkage groups, 30 linkage groups were detected on the female side and 28 on the male side. On the female map, 68.3% of the pairwise marker linkages were in coupling versus 31.7% in repulsion phase, and the corresponding figures on the male map were 72.2% and 27.8%, respectively. In addition, both groups linked only in the coupling phase and groups linked in the repulsion phase were characterized. The observations suggest that the meiotic behavior of the F. x ananassa genome is neither fully disomic nor fully polysomic, but rather mixed. The genome may not be as completely diploidized as previously assumed.     

Location of independent root-knot nematode resistance genes in plum and peach

Prunus species express different ranges and levels of resistance to the root-knot nematodes (RKN) Meloidogyne spp. In Myrobalan plum (Prunus cerasifera), the dominant Ma gene confers a high-level and wide-spectrum resistance to the predominant RKN, Meloidogyne arenaria, Meloidogyne incognita, Meloidogyne javanica and the isolate Meloidogyne sp. Florida which overcomes the resistance of the Amygdalus sources. In Japanese plum (Prunus salicina), a similar wide-spectrum dominant resistance gene, termed R _jap , has been hypothesized from an intraspecific segregating cross. In peach, two crosses segregating for resistance to both M. incognita and M. arenaria were used to identify single genes that each control both RKN species in the Shalil (R _Mia557 ) and Nemared (R _MiaNem ) sources. Localisation of these genes was made possible using the RFLP and SSR- saturated reference Prunus map T×E, combined with a BSA approach applied to some of the genes. The Ma1 allele carried by the Myrobalan plum accession P.2175 was localised on the linkage group 7 at an approximate distance of 2 cM from the SSR marker pchgms6. In the Japanese plum accession J.222, the gene R _jap was mapped at the same position in co-segregation with the SSR markers pchgms6 and CPPCT022. The peach genes R _Mia557 and R _MiaNem , carried by two a priori unrelated resistance sources, were co-localized in a subtelomeric position on linkage group 2. This location was different from the more centromeric position previously proposed by Lu et al. (1999) for the resistance gene Mij to M. incognita and M. javanica in Nemared, near the SSR pchgms1 and the STS EAA/MCAT10. By contrast, R _Mia557 and R _MiaNem were flanked by STS markers obtained by Yamamoto and Hayashi (2002) for the resistance gene Mia to M. incognita in the Japanese peach source Juseitou. Concordant results for the three independent sources, Shalil, Nemared and Juseitou, suggest that these peach RKN sources share at least one major gene resistance to M. incognita located in this subtelomeric position. We showed that plum and peach genes are independent and, thus, can be pyramided into interspecific hybrid rootstocks based on the plum and peach species.Communicated by H.C. Becker     

Genetic relationships between diploid and allotetraploid cherry species (Prunus avium, Prunus x gondouinii and Prunus cerasus)

Prunus avium L. (diploid, AA, 2n=2x=16), Prunus cerasus L. (allotetraploid, AAFF, 2n=4x=32) species, and their hybrid Prunus x gondouinii Rehd., constitute the most widely cultivated cherry tree species. P. cerasus is supposed to be an hybrid species produced by the union of unreduced P. avium gametes and normal P. fruticosa gametes. A continuum of morphological traits between these three species makes their assignation difficult. The aim of this paper is to study the genetic relationships between tetraploid and diploid cherry species. In all, 114 genotypes belonging to these species were analyzed using 75 AFLP markers. The coordinates of these genotypes on the first axis of a correspondence analysis allowed us to clearly distinguish each species, to identify misclassifications and to assign unknown genotypes to one species. We showed that there are specific alleles in P. cerasus, which are not present in the A genome of P. avium and which probably come from the F genome of P. cerasus. The frequencies of each marker in the A and the F genomes were estimated in order to identify A and F specific markers. We discuss the utility of these specific markers for finding the origin of the A and F genomes in the allopolyploid species.     

Development of a second-generation genetic linkage map for peach [Prunus persica (L.) Batsch] and characterization of morphological traits affecting flower and fruit

An improved genetic linkage map was constructed from a peach Ferjalou Jalousia® × Fantasia (J×F) F₂ population. Ferjalou Jalousia® is a flat low-acidity clingstone peach, and Fantasia is a round, normally acidic freestone peach. This population is segregated for six Mendelian characters: pollen sterility, peach or nectarine fruit, flat or round fruit, clingstone, or freestone fruit. It also segregates for the D major gene controlling the fruit’s low acidity. A new character is reported here for the first time that segregates as a Mendelian character: trees bearing aborting fruits. These trees have flowers, but fruits start to fall 2 months after blooming. This recessive character has been named Af. We demonstrate that it is linked to the flat shape of the fruit. The previous map obtained from this cross was constructed using 63 individuals, whereas the present map was constructed using 207 individuals. Moreover, 82 simple-sequence repeat (SSR) markers, including 10 expressed sequence tag-SSRs, and 43 amplified fragment length polymorphism (AFLP) markers were added. Molecular markers linked to the six Mendelian characters were identified, and one of them has already been used for marker-assisted selection. This map will be used for detection of quantitative trait loci controlling organoleptic and nutritional fruit quality in peach.