Identification and mapping of resistance gene analogs (RGAs) in Prunus: a resistance map for Prunus.

The genetically anchored physical map of peach is a valuable tool for identifying loci controlling economically important traits in Prunus. Breeding for disease resistance is a key component of most breeding programs. The identification of loci for pathogen resistance in peach provides information about resistance loci, the organization of resistance genes throughout the genome, and permits comparison of resistance regions among other genomes in the Rosaceae. This information will facilitate the breeding of resistant species of Prunus. A candidate gene approach was implemented for locating resistance loci in the genome of peach. Candidate genes representing NBS-LRR, kinase, transmembrane domain classes, as well as, pathogen response (PR) proteins and resistance-associated transcription factors were hybridized to a peach BAC library and mapped by using the peach physical map database and the Genome Database for Rosaceae (GDR). A resistance map for Prunus was generated and currently contains 42 map locations for putative resistance regions distributed among 7 of the 8 linkage groups.

Microsatellite and AFLP markers in the Prunus persica [L. (Batsch)]xP. ferganensis BC(1)linkage map: saturation and coverage improvement.

A set of 146 single sequence repeats (SSRs) and 14 amplified fragment length polymorphism (AFLP) primer combinations were used to enrich a previously developed linkage map obtained from a (Prunus persicaxP. ferganensis)xP. persica BC(1) progeny. Forty-one SSR primer pairs gave polymorphic patterns detecting 42 loci. The restriction/selective primer AFLP combinations produced a total of 79 segregating fragments. The resulting map is composed of 216 loci covering 665 cM with an average distance of 3.1 cM. Novel regions were covered by the newly mapped loci for a total of 159 cM. Eight linkage groups were assembled instead of the earlier 10 as two small groups (G1a and G8b), previously independent, were joined to their respective major groups (G1b and G8a). Several gaps were also reduced resulting in an improved saturation of the map. Twelve gaps >or=10 cm are still present. A comparative analysis against the Prunus reference map (71 anchor loci) pointed out an almost complete synteny and colinearity. Six loci were not syntenic and only two were not colinear. Genetic distances were significantly longer in our map than in the reference one.

A deletion affecting several gene candidates is present in the Evergrowing peach mutant.

Evergrowing (EVG) peach is one of only two described mutants affecting winter dormancy in woody perennial species. EVG peach does not set terminal buds, cease new leaf growth, nor enter into a dormant resting phase in response to winter conditions. The EVG mutation segregates in F2 progeny as a single recessive nuclear gene. A local molecular genetic linkage map around EVG was previously developed using amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) markers, and a bacterial artificial chromosome (BAC) contig that contains the EVG mutation was assembled. A MADS box coding open reading frame (ORF) was found in a BAC of this contig and used as a probe. The probe detected a polymorphism between the wild-type and mutant genomes, and the polymorphism is indicative of a deletion in EVG peach. The EVG gene region contained six potential MADS-box transcription factor sequences, and the deletion in EVG affected at least four of these. The deletion was bracketed using RFLP analysis, which showed that it is contained within a segment of the genome no greater than 180 kb.

The phylogenetic relationship of possible progenitors of the cultivated peanut.

The cultivated peanut (Arachis hypogaea L.) is an allotetraploid composed of A and B genomes. The phylogenetic relationship among the cultivated peanut, wild diploid, and tetraploid species in the section Arachis was studied based on sequence comparison of stearoyl-ACP desaturase and oleoyl-PC desaturase. The topology of the trees for both fatty acid desaturases displayed two clusters; one cluster with A genome diploid species and the other with B genome diploid species. The two homeologous genes obtained for each of the two fatty acid desaturases from the tetraploid species A. hypogaea and A. monticola were separated into the A and B genome clusters, respectively. The gene phylogenetic trees showed that A. hypogaea is more closely related to the diploid species A. duranensis and A. ipaensis than to the wild tetraploid species A. monticola, suggesting that A. monticola is not a progenitor of the cultivated peanut. In addition, for the stearoyl-ACP desaturase, the A. duranensis sequence was identical with one of the sequences of A. hypogaea and the A. ipaensis sequence was identical with the other. These results support the hypothesis that A. duranensis and A. ipaensis are the most likely diploid progenitors of the cultivated tetraploid A. hypogaea.

An apricot (Prunus armeniaca L.) F2 progeny linkage map based on SSR and AFLP markers, mapping plum pox virus resistance and self-incompatibility traits.

A genetic linkage map of apricot ( Prunus armeniaca L.) was constructed using AFLP and SSR markers. The map is based on an F(2) population (76 individuals) derived from self-pollination of an F(1) individual ('Lito') originated from a cross between 'Stark Early Orange' and 'Tyrinthos'. This family, designated as 'Lito' x 'Lito', segregated for two important agronomical traits: plum pox virus resistance (PPV) and self-incompatibility. A total of 211 markers (180 AFLPs, 29 SSRs and two agronomic traits) were assigned to 11 linkage groups covering 602 cM of the apricot genome. The average distance (cM/marker) between adjacent markers is 3.84 cM. The PPV resistance trait was mapped on linkage group G1 and the self-incompatibility trait was mapped on linkage group G6. Twenty two loci held in common with other Prunus maps allowed us to compare and establish homologies among the respective linkage groups.

Construction and application of a bacterial artificial chromosome (BAC) library of Prunus armeniaca L. for the identification of clones linked to the self-incompatibility locus.

To facilitate gene discovery in the Rosaceae, a bacterial artificial chromosome (BAC) library was constructed using high-molecular-weight (HMW) DNA from apricot leaves (Prunus armeniaca L.). The library contains 101,376 clones (264, 384-well plates) with an average insert size of 64 kb, equivalent to 22-fold genome coverage. In the first application of this library, high-density filters were screened for self-incompatibility genes using apricot DNA probes. Eight positive BAC clones were detected and fingerprinted to determine clone relationships and assemble contigs. These results demonstrate the suitability of this library for gene identification and physical mapping of the apricot genome.

Simple sequence repeat (SSR) analysis for assessment of genetic variability in apricot germplasm.

Thirty SSR primer combinations, developed from peach SSR-enriched genomic libraries and BAC libraries of peach [ Prunus persica (L.) Batsch.], were tested for cross amplification with 74 apricot ( Prunus armeniaca L.) germplasm accessions. Twelve primer pairs amplified 14 polymorphic SSR loci useful for discriminating most apricot cultivars, as well as for investigating patterns of variation in apricot germplasm. Levels of polymorphism were higher than the levels described using other codominant marker systems (i.e., isozymes, RFLP markers). Overall, 107 alleles were identified, and all but 11 accessions were unambiguously discriminated. Genetic differentiation of native germplasm into traditional ecogeographical groups was low, with a high level of genetic identity (> 0.75) between the groups. However, neighbor joining cluster analysis of marker distances between cultivars reflected the complex history of apricot domestication, producing groupings not evidently based on the geographical origin of the cultivars. Distant positioning of Chinese cultivars on UPGMA and neighbor joining dendrograms supports the authors' consideration of Chinese apricots as subspecies, Prunus armeniaca var. ansu Maxim., rather than a separate species.

Genetic mapping of the evergrowing gene in peach [Prunus persica (L.) Batsch].

In temperate locations, terminal apices on evergrowing (also called evergreen) peach trees keep growing in winter until killed by low temperatures, while the lateral buds go into dormancy. A recessive allele of a single gene (evergrowing or evg) controls this trait in peach. The amplified fragment length polymorphism (AFLP) technique and bulked segregant analysis were applied to construct a local genetic linkage map for the evg gene from the cross Empress op op dwarf x Evergrowing (P.I. 442380). This map, comprising nine AFLP markers and the evg locus, covers a total genetic distance of 79.3 cM. Four dominant AFLP markers (EAT/MCAC, ETT/MCCA2, EAT/MCTA, and ETT/MACC) were linked to the evg locus at distances of 1, 5.3, 6.7, and 11.7 cM, respectively. EAT/MCAC and EAT/MCTA were converted into polymorphic sequence-tagged sites. Microsatellite markers in the evg region were developed from peach bacterial artificial chromosome (BAC) clones that hybridized to the AFLP marker fragments. Using three microsatellite anchor markers (pchgms12, pchgms17, and pchgms19), the local genetic linkage map was integrated into one minor linkage group of a previously constructed peach rootstock genetic linkage map. Three AFLP markers from the rootstock genetic linkage map were found linked to the evg locus.

Endoplasmic oleoyl-PC desaturase references the second double bond.

The regiospecificity for the gene product of fad2,(1) the microsomal oleoyl-PC desaturase from higher plants, differs from some previous suggestions. Rather than only referencing the carboxyl group (a Delta(12) desaturase) or the methyl terminus (an omega-6 desaturase), this desaturase locates the second double bond in its substrates by first referencing the existing double bond. This specificity was demonstrated for the oleoyl-PC desaturase cDNA from the developing seeds of peanut (Arachis hypogaea L) expressed in yeast (Saccharomyces cerevisae). The expressed enzyme was capable of desaturating monounsaturated fatty acyl groups in membrane lipids. Endogenous palmitoleate was desaturated to cis, cis 9,12 hexadecadienoate (9(Z)12(Z)C16:2), endogenous oleate to linoleate (9(Z)12(Z) octadecadienoate), and cis 10-nonadecenoate (provided as a supplement in the growth medium) to 10(Z)13(Z)C19:2. The rule, Delta(x+3) where x=9 is the double bond location in the substrate, best describes the consistent placement of the second double bond in the above monounsaturated substrates for the oleoyl-PC desaturase of higher plants.

Molecular properties of the oleoyl-phosphatidylcholine desaturase from Arachis hypogaea L.

Deficiencies in the activity of the microsomal oleoyl phosphatidylcholine (oleoyl-PC) desaturase from peanuts are the basis of the high oleate oil. Mutation of aspartate-150 to asparagine and the attendant decrease in activity, together with the loss in expression of the higher activity transcript, was the molecular basis of the high oleate trait. The ability of oleoyl-PC desaturase to desaturate palmitoleate, oleate and 10(Z) nonadecenoate to methylene-interrupted products was not consistent with description of this activity as a Delta(12) or omega-6 desaturase. Electrospray MS was used to examine the intact phospholipid products of desaturation by the oleoyl-PC desaturase. PC and phosphatidylinositol containing unsaturated moieties could be desaturated. The enzyme can act on either sn-1 or sn-2 moieties. Phosphatidylethanolamine was a poor substrate.

Intra- and Interpopulation Genome Variation In Meloidogyne arenaria.

The genetic heterogeneity of two M. arenaria race 2 populations (designated Pelion and Govan) was examined using RFLP analysis of 12 clonal lines established from single egg masses (six distinct clonal lines from each population). These populations are essentially identical by traditional biochemical and race identification schemes; however, the Govan population is more aggressive than the Pelion population, producing larger galls and exhibiting greater reproductive capabilities on many soybean cultivars and experimental accessions. Variation at the genomic DNA level was examined using probes representative of expressed DNA sequences present in the eukaryotic genome. Ribosomal DNA, interspersed repeated sequences, and cDNA probes were tested for detection of polymorphism within and between single egg mass lines of each population. Cloned cDNAs and ribosomal intergenic spacer sequences detect polymorphism both within and between populations, demonstrating the usefulness of these sequence classes for molecular genetic analysis of population structure and genome evolution.

Genetic linkage mapping in peach using morphological, RFLP and RAPD markers.

We have constructed a genetic linkage map of peach [Prunus persica (L.) Batsch] consisting of RFLP, RAPD and morphological markers, based on 71 F2 individuals derived from the self-fertilization of four F1 individuals of a cross between 'New Jersey Pillar' and KV 77119. This progeny, designated as the West Virginia (WV) family, segregates for genes controlling canopy shape, fruit flesh color, and flower petal color, size and number. The segregation of 65 markers, comprising 46 RFLP loci, 12 RAPD loci and seven morphological loci, was analyzed. Low-copy genomic and cDNA probes were used in the RFLP analysis. The current genetic map for the WV family contains 47 markers assigned to eight linkage groups covering 332 centi Morgans (cM) of the peach nuclear genome. The average distance between two adjacent markers is 8 cM. Linkage was detected between Pillar (Pi) and double flowers (Dl) RFLP markers linked to Pi and flesh color (γ) loci were also found. Eighteen markers remain unassigned. The individuals analyzed for linkage were not a random sample of all F2 trees, as an excess of pillar trees were chosen for analysis. Because of this, Pi and eight other markers that deviated significantly from the expected Mendelian ratios (e.g., 1∶2∶1 or 3∶1) were not eliminated from the linkage analysis. Genomic clones that detect RFLPs in the WV family also detect significant levels of polymorphism among the 34 peach cultivars examined. Unique fingerprint patterns were created for all the cultivars using only six clones detecting nine RFLP fragments. This suggests that RFLP markers from the WV family have a high probability of being polymorphic in crosses generated with other peach cultivars, making them ideal for anchor loci. This possibility was examined by testing RFLP markers developed with the WV family in three other unrelated peach families. In each of these three peach families respectively 43%, 54% and 36% of RFLP loci detected in the WV family were also polymorphic. This finding supports the possibility that these RFLP markers may serve as anchor loci in many other peach crosses.

Trans-splicing of a Meloidogyne incognita mRNA encoding a putative esophageal gland protein.

A monoclonal antibody, 7A9, specific for antigens in the subventral esophageal glands of adult female Meloidogyne incognita and for antigens in the longitudinal muscles of second-stage juveniles, was used to isolate a clone from a M. incognita cDNA expression library. The corresponding genomic DNA was isolated by hybridization and the gene designated sec-1. DNA sequence analysis of sec-1 revealed the presence of 9 introns having structural similarities to introns from the free-living nematode Caenorhabditis elegans. The sec-1 message was also trans-spliced and the leader sequence differed in one position from the sequence of the C. elegans trans-spliced leader SL1. Sequences analogous to sec-1 were specific to the genus Meloidogyne, and regulation of sec-1 expression did not involve differences in overall rates of transcript accumulation. The deduced amino acid sequence of the protein encoded by sec-1 had some similarities to the rod portions of several myosin heavy chains.

Genomic RFLP Analysis of Meloidogyne arenaria Race 2 Populations.

Traditional morphological methods of Meloidogyne identification have been unsuccessful in distinguishing three South Carolina, USA Meloidogyne arenaria race 2 populations-Govan, Pelion, and Florence. These populations differ greatly in reproductive rate and aggressiveness on soybean hosts. Total genomic DNA from eggs of each population was digested with the restriction endonuclease Eco RI and Southern hybridization analyses were performed with single-copy and interspersed multi-copy cloned probes. Probes were isolated from a genomic library of Eco RI, M. arenaria DNA fragments cloned into pUC8. One probe, designated pE1.6A, when hybridized to Southern blots of M. arenaria genomic DNAs, displayed an interspersed repetitive pattern, and the RFLPs distinguished the Govan population from the Pelion and Florence populations. Another clone, pE6.0A, carrying moderately repeated sequences, distinguished the Pelion and Florence isolates. This communication demonstrates the utility of genomic RFLP analysis for distinguishing populations of the same race within the same species. To test the possible utility of these moderately repeated sequence probes for detecting the presence of nematode DNA in DNA samples from roots inoculated with varying numbers of nematodes, dot blot hybridization analyses were performed. It is possible to detect as few as 30 nematodes per root sample with these cloned probes.

Higher plant mitochondrial DNA expression : 2. Influence of nuclear background on the transcription of a mitochondrial open reading frame, ORF25.

Mitochondria are semi autonomous organelles, with their own genome and transcription/translation systems. Although the regulation of mitochondrial gene expression is fairly well characterized in the animal system, little is known about these processes in plants. We have been studying the expression of ORF25, a mitochondrial open reading frame, in normal male-fertile maize. In all the N lines that we have examined, the ORF25 transcript pattern is similar, except for that in B37N. We have compared ORF25 transcription patterns between B73N and B37N: B73N has one major transcript of 2,300 nucleotides and two minor transcripts of 3,400 and 1,600 nucleotides, while B37N has a single transcript, 3,400 bases long. The ORF25 reading frame and 5' flanking regions have been analyzed by restriction mapping and found to be identical in these lines. Interestingly, the F1 progeny from reciprocal crosses between B73N and B37N have ORF25 transcript patterns identical to B73N. This suggests that the process of mitochondrial transcription is influenced by nuclear factors in normal cytoplasm. This factor(s) appears to be dominant in B73N and the F1 progeny. S1 nuclease analyses have revealed that identical fragments are protected in B73N and the F1 hybrids, indicating that the ORF25 transcripts in the F1 progeny are identical on the 5' ends to those of the parent B73N. This nuclear regulation may be at the level of initiation of transcription or processing of the mtRNA.

Higher plant mitochondrial DNA expression : 1. Variant expression of the plant mitochondrial open reading frame, ORF25, in B37N and B73N maize lines.

In studying the process of mitochondrial transcription, mutants that show altered gene expression as evidenced from transcript pattern differences are a valuable resource. However, such mutants are difficult to find since changes in mitochondrial gene expression will most likely be lethal. Several laboratories have been investigating cytoplasmic male-sterile mutants in maize and have reported changes in transcription patterns due to nuclear background influences on the complex chimeric gene region TURF-2H3 in T-cms. There have been no reports of altered transcription patterns for N cytoplasm that can be attributed to nuclear background differences. Through a Northern hybridization analysis of ORF25 transcription in a number of N lines, we reported invariant expression of this region. Subsequently, we have discovered a line B37N, which shows the presence of a single ORF25-specific transcript of 3,400 nucleotides, in contrast to the transcript sizes of 3,400, 2,300 and 1,600 displayed by most of the cytoplasms we have examined. Experiments presented in this communication demonstrate that the differences in the B37N, ORF25 transcript pattern map to the 5' flanking sequences of the reading frame. Using restriction enzyme mapping and Southern hybridization analysis, no detectable differences were found in the transcription unit structure for this reading frame in B37N and B73N, which shows the standard, three-transcript pattern. Analysis of nuclear background influences indicates that the transcript patterns for this open reading frame are dependent on nuclear background. These data are presented in part 2 of this study.

Toxin resistance and/or male fertility reversion is correlated with defined transcription changes in the 1.5 kb AvaI region of cmsT.

Reversion of T type cytoplasmic male sterility (cmsT) to fertility is correlated with sequence changes in a 1.5 kb AvaI fragment. This 1.5 kb AvaI fragment is composed of 5' flanking sequences of ATPase subunit 6, one complete open reading frame (ORF 13) and part of the another (ORF 25). The sequence of the 1.5 kb AvaI fragment was compared to the sequences of homologous regions in the N (male fertile) and T revertant V3 mitochondrial DNA. Sequences were found to diverge between ORF 13 and ORF 25 coding regions. To further characterize the transcription of these rearranged sequences, specific probes for ORF 13, ORF 25 and 5' flanking sequences of ATPase 6 were hybridized to Northern blots of N, cmsT and the T revertant V3 and V18 mtRNAs. Each revertant has a single ORF 25 homologous transcript in contrast to the multitranscript pattern in cmsT. ORF 13 homologous transcripts were not detected in either revertant cytoplasm. The loss of ORF 13 and/or altered ORF 25 transcription in the fertile revertants may be responsible for the male fertility and/or toxin resistance in these plants.

Northern hybridization analysis of mitochondria gene expression in maize cytoplasm with varied nuclear backgrounds.

Type T cytoplasmic male sterility in Zea mays is associated with mitochondrial RNA (mtRNA) coding sequences found on a 1.5 kb AvaI mitochondrial DNA (mtDNA) fragment not found in other cytoplasms (N, C, or S) (Abbott and Fauron 1986). Three probes (pH3.2N, pH2.7N, and ORF 13) specific to different parts of the 1.5 AvaI T region were used in a Northern blot analysis of N mtDNAs from lines with diverse nuclear backgrounds (Rf1, Rf2 included). The N mtDNA clone pH 3.2N shows homology with the right-hand boundary of the 1.5 AvaI T region and includes a portion of an open reading frame (ORF 25). Southern blots of AvaI and BamH1 digestions of N, T, S, and C mtDNA, probed with pH3.2N demonstrate that sequences in or adjacent to this region are highly active in recombination. The clone pH 2.7N is homologous to an untranscribed region of ATPase 6 and the structural gene; and ORF 13 is a portion of the 1.5 AvaI region derived predominantly from 26S 3' flanking sequences. pH3.2N and 1.5 AvaI sequences showed identical hybridization patterns on Northern blots of N, T, Trev and Tres mtRNAs. Transcript sizes of mtRNAs homologous to the pH 3.2N probe in all of these lines were different, however, there was no variation in transcript sizes when pH 2.7N was used as a probe. Northern blots of mtRNA from N cytoplasms with various nuclear restored backgrounds showed no difference in expression when probed with pH 3.2N or pH 2.7N; however, transcripts homologous to an ORF 13 specific probe can be detected in N cytoplasm with a particular nuclear background. This may suggest activity of nuclear restorer alleles in N cytoplasm.

Structural alterations in a transcribed region of the T type cytoplasmic male sterile maize mitochondrial genome.

H. maydis toxin resistance, and cytoplasmic reversion from sterility to fertility in Zea mays T type cytoplasm are correlated with sequence changes in a 6.6 kb XhoI fragment of T mitochondrial DNA. Comparative Northern blot analysis of N (normal male fertile), and cmsT (cytoplasmic male sterile) mitochondrial RNAs using subclones of the 6.6 kb Xho I region of cmsT as probes, reveals different sized transcripts. In cmsT these RNA coding sequences have been mapped onto a 1.5 kb AvaI fragment completely internal to the 6.6 kb XhoI region. Comparative Southern analysis of AvaI digested mitochondrial DNA from cmsT, N and a T-type cytoplasm which has reverted to fertility (designated V3) (Brettel et al. 1979), positions the RNA coding sequences on a 1.5 kb, 2.1 kb and 2.1 kb fragment respectively. These sequence rearrangements in the fertile T revertants create a novel mtRNA transcript. Southern hybridization experiments of other higher plant mitochondrial DNAs using the 1.5 kb Ava I fragment from cmsT mtDNA as a probe, indicate the presence of homologous sequences.

Quantitative variation in components of the maize mitochondrial genome between tissues and between plants with different male-sterile cytoplasms.

The amounts of a 1.9 kb mitochondrial plasmid relative to sequences in another mitochondrial DNA replicon and also to nuclear ribosomal DNA sequences have been compared in maize leaves and anthers. Similar comparisons have been made between plants with the same nuclear genotype but containing normal, S, or T cytoplasms. The ratio of 1.9 kb plasmid to nuclear rDNA is lower in plants with normal cytoplasm than in plants with S or T cytoplasm. It also differs between leaves and anthers. Furthermore, the relative concentration of the mitochondrial DNA sequences belonging to different replicons differs between leaves and anthers. It is concluded that components of different mitochondrial replicons are not maintained in fixed ratios during development and that the concentration of the 1.9 kb plasmid is regulated, in part, by cytoplasmically-inherited determinants. The 1.9 kb plasmid is absent from lines with the Vg cytoplasm, but related sequences are found in the maize nuclear genome.