Showing papers in "Theoretical and Applied Genetics in 2002"

Development of microsatellite markers in peach [ Prunus persica (L.) Batsch] and their use in genetic diversity analysis in peach and sweet cherry ( Prunus avium L.).

Abstract: We report the sequence of 41 primer pairs of microsatellites from a CT-enriched genomic library of the peach cultivar 'Merrill O'Henry'. Ten microsatellite-containing clones had sequences similar to plant coding sequences in databases and could be used as markers for known functions. For microsatellites segregating at least in one of the two Prunus F2 progenies analyzed, it was possible to demonstrate Mendelian inheritance. Microsatellite polymorphism was evaluated in 27 peach and 21 sweet cherry cultivars. All primer pairs gave PCR-amplification products on peach and 33 on cherry (80.5%). Six PCR-amplifications revealed several loci (14.6%) in peach and eight (19.5%) in sweet cherry. Among the 33 single-locus microsatellites amplified in peach and sweet cherry, 13 revealed polymorphism both in peach and cherry, 19 were polymorphic only on peach and one was polymorphic only on cherry. The number of alleles per locus ranged from 1 to 9 for peach and from 1 to 6 on sweet cherry with an average of 4.2 and 2.8 in peach and sweet cherry, respectively. Cross-species amplification was tested within the Prunus species: Prunus avium L. (sweet cherry and mazzard), Prunus cerasus L. (sour cherry), Prunus domestica L. (European plum), Prunus amygdalus Batsch. (almond), Prunus armeniaca L. (apricot), Prunus cerasifera Ehrh. (Myrobalan plum). Plants from other genera of the Rosaceae were also tested: Malus (apple) and Fragaria (strawberry), as well as species not belonging to the Rosaceae: Castanea (chestnut tree), Juglans (walnut tree) and Vitis (grapevine). Six microsatellites gave amplification on all the tested species. Among them, one had an amplified region homologous to sequences encoding a MADS-box protein in Malus × domestica. Twelve microsatellites (29.3%) were amplified in all the Rosaceae species tested and 31 (75.6%) were amplified in all the six Prunus species tested. Thirty three (80.5%), 18 (43.9%) and 13 (31.7%) gave amplification on chestnut tree, grapevine and walnut tree, respectively.

Mapping of quantitative trait loci determining agronomic important characters in hexaploid wheat ( Triticum aestivum L.).

Abstract: A set of 114 recombinant inbred lines of the 'International Triticeae Mapping Initiative' mapping population was grown during the seasons 1997, 1998, 1999 and 2000 under several environments. Twenty morphological (glume colour, awn colour, waxiness, leaf erectness, peduncle length), agronomical (ear emergence time, flowering time, grain filling time, ear length, plant height, lodging, grain number, thousand-grain-weight, grain weight per ear, grain protein content, winter hardiness) and disease resistance (powdery mildew, yellow rust, leaf rust, fusarium) traits were studied. Not all traits were scored in each experiment. In total 210 QTLs with a LOD threshold of >2.0 (minor QTLs) were detected of which 64 reached a LOD score of >3.0 (major QTLs). Often QTLs were detected in comparable positions in different experiments. Homologous and homoeologous relationships of the detected QTLs, and already described major genes or QTLs determining the same traits in wheat or other Triticeae members, are discussed.

"Perfect" markers for the Rht-B1b and Rht-D1b dwarfing genes in wheat.

Abstract: PCR-based markers were developed to detect the point mutations responsible for the two major semi-dwarfing genes Rht-B1b (Rht1) and Rht-D1b (Rht2) in wheat. These markers were validated by testing 19 wheat varieties of known Rht genotype. They included Rht-B1b and Rht-D1b dwarfs, double-mutant varieties and tall wheats. These were correctly genotyped with the Rht-B1b and Rht-D1b-specific primers, as well as markers specific for the tall alleles Rht-B1a and Rht-D1a. Using a family of doubled-haploid lines segregating for Rht-B1b and Rht-D1b, the markers were mapped to the expected homoeologous regions of chromosomes 4B and 4D, respectively. Both markers were strongly correlated with a reduction in height, accounting for 23% (Rht-B1b) and 44% (Rht-D1b) of the phenotypic variance in the population. These markers will have utility in marker-assisted selection of the Rht-B1b and Rht-D1b genes in wheat breeding programs.

Molecular mapping of QTLs for Fusarium head blight resistance in spring wheat. I. Resistance to fungal spread (Type II resistance)

Abstract: Fusarium head blight (FHB, scab) is a fungal disease of wheat and other small cereals that is found in both temperate and semi-tropical regions. FHB causes severe yield and quality losses, but the most-serious concern is the possible mycotoxin contamination of cereal food and feed. Breeding for FHB resistance by conventional selection is feasible, but tedious and expensive. This study was conducted to identify and map DNA markers associated with FHB resistance genes in wheat. A population of 364 F1-derived doubled-haploid (DH) lines from the cross ’CM-82036’ (resistant)/’Remus’ (susceptible) was evaluated for Type II resistance (spread within the spike) during 2 years under field conditions. Marker analysis was performed on 239 randomly chosen DH lines. Different marker types were applied, with an emphasis on AFLP and SSR markers. Analysis of variance, as well as simple and composite interval mapping, were applied. Three genomic regions were found significantly associated with FHB resistance. The most-prominent effect was detected on the short arm of chromosome 3B, explaining up to 60% of the phenotypic variance for Type II FHB resistance. A further QTL was located on chromosome 5A and a third one on 1B. The QTL regions on 3B and 5A were tagged with flanking SSR markers, the 1B QTL was found associated with the high-molecular-weight glutenin locus. These results indicate that FHB resistance is under control of a few major QTLs operating together with unknown numbers of minor genes. Marker-assisted selection for these major QTLs involved in FHB resistance appears feasible and should accelerate the development of resistant and agronomically improved wheat cultivars.

Isolation of EST-derived microsatellite markers for genotyping the A and B genomes of wheat.

Abstract: Genetic variation present in 64 durum wheat accessions was investigated by using three sources of microsatellite (SSR) markers: EST-derived SSRs (EST-SSRs) and two sources of SSRs isolated from total genomic DNA. Out of 245 SSR primer pairs screened, 22 EST-SSRs and 20 genomic-derived SSRs were polymorphic and used for genotyping. The EST-SSR primers produced high quality markers, but had the lowest level of polymorphism (25%) compared to the other two sources of genomic SSR markers (53%). The 42 SSR markers detected 189 polymorphic alleles with an average number of 4.5 alleles per locus. The coefficient of similarity ranged from 0.28 to 0.70 and the estimates of similarity varied when different sources of SSR markers were used to genotype the accessions. This study showed that EST-derived SSR markers developed in bread wheat are polymorphic in durum wheat when assaying loci of the A and B genomes. A minumum of ten EST-SSRs generated a very low probability of identity (0.36×10−12) indicating that these SSRs have a very high discriminatory power. EST-SSR markers directly sample variation in transcribed regions of the genome, which may enhance their value in marker-assisted selection, comparative genetic analysis and for exploiting wheat genetic resources by providing a more-direct estimate of functional diversity.

Genetic mapping of 66 new microsatellite (SSR) loci in bread wheat.

Abstract: In hexaploid bread wheat ( Triticum aestivum L. em. Thell), ten members of the IWMMN ( International Wheat Microsatellites Mapping Network) collaborated in extending the microsatellite (SSR = simple sequence repeat) genetic map. Among a much larger number of microsatellite primer pairs developed as a part of the WMC ( Wheat Microsatellite Consortium), 58 out of 176 primer pairs tested were found to be polymorphic between the parents of the ITMI ( International Triticeae Mapping Initiative) mapping population W7984 x Opata 85 (ITMI pop). This population was used earlier for the construction of RFLP ( Restriction Fragment Length Polymorphism) maps in bread wheat (ITMI map). Using the ITMI pop and a framework map (having 266 anchor markers) prepared for this purpose, a total of 66 microsatellite loci were mapped, which were distributed on 20 of the 21 chromosomes (no marker on chromosome 6D). These 66 mapped microsatellite (SSR) loci add to the existing 384 microsatellite loci earlier mapped in bread wheat.

Characterization of the main effects, epistatic effects and their environmental interactions of QTLs on the genetic basis of yield traits in rice

Abstract: Main effects, epistatic effects and their environmental interactions of QTLs are all important genetic components of quantitative traits. In this study, we analyzed the main effects, epistatic effects of the QTLs, and QTL by environment interactions (QEs) underlying four yield traits, using a population of 240 recombinant inbred lines from a cross between two rice varieties tested in replicated field trials. A genetic linkage map with 220 DNA marker loci was constructed. A mixed linear model approach was used to detect QTLs with main effects, QTLs involved in digenic interactions and QEs. In total, 29 QTLs of main effects, and 35 digenic interactions involving 58 loci were detected for the four traits. Thirteen QTLs with main effects showed QEs; no QE was detected for the QTLs involved in epistatic interactions. The amount of variations explained by the QTLs of main effect were larger than the QTLs involved in epistatic interactions, which in turn were larger than QEs for all four traits. This study illustrates the ability of the analysis to assess the genetic components underlying the quantitative traits, and demonstrates the relative importance of the various components as the genetic basis of yield traits in this population.

Mapping of a gene responsible for the difference in amylopectin structure between japonica-type and indica-type rice varieties.

Abstract: The present investigation revealed that the alk and gel(t) genes, which cause the differences between a japonica rice variety Nipponbare and an indica rice variety Kasalath in terms of the disintegration of endosperm starch granules in alkali solution and their gelatinisation in a 4 M urea solution, respectively, cosegregated in backcross inbred lines derived from a cross between the two varieties. The segregation pattern of the profile for amylopectin chain-length, which was distinguished by enrichment in short chains of DP≦11 and depletion in intermediate-size chains of 12≦DP≦24 in japonica as compared with indica, was exactly the same as those of the above physico-chemical properties of starch granules, and the gene was designated as acl(t). Gene-mapping analysis showed that the starch synthase IIa (SSIIa) gene is located at the alk locus on chromosome 6 in the rice genome. These results lead us to the possibility that different alleles of the SSIIa gene are responsible for differences in amylopectin structure between the two varieties, in that SSIIa plays a distinct role in the elongation of short chains within clusters (A+B1 chains) of amylopectin. It is proposed that the activity of SSIIa in japonica rice is reduced in amount or functional capacity relative to the activity of this enzyme in indica rice. This, in turn, would explain why starch from japonica rice has a lower gelatinisation temperature than starch from indica rice and is more susceptible to disintegration in alkali or urea. The evidence for this hypothesis is that the alk(t), gel(t), acl(t) and SSIIa genes all map to the same locus.

Assessing genetic diversity of wheat (Triticum aestivum L.) germplasm using microsatellite markers

Abstract: A set of 24 wheat microsatellite markers, representing at least one marker from each chromosome, was used for the assessment of genetic diversity in 998 accessions of hexaploid bread wheat (Triticum aestivum L.) which originated from 68 countries of five continents. A total of 470 alleles were detected with an average allele number of 18.1 per locus. The highest number of alleles per locus was detected in the B genome with 19.9, compared to 17.4 and 16.5 for genomes A and D, respectively. The lowest allele number per locus among the seven homoeologous groups was observed in group 4. Greater genetic variation exists in the non-centromeric regions than in the centromeric regions of chromosomes. Allele numbers increased with the repeat number of the microsatellites used and their relative distance from the centromere, and was not dependent on the motif of microsatellites. Gene diversity was correlated with the number of alleles. Gene diversity according to Nei for the 26 microsatellite loci varied from 0.43 to 0.94 with an average of 0.77, and was 0.78, 0.81 and 0.73 for three genomes A, B and D, respectively. Alleles for each locus were present in regular two or three base-pair steps, indicating that the genetic variation during the wheat evolution occurred step by step in a continuous manner. In most cases, allele frequencies showed a normal distribution. Comparative analysis of microsatellite diversity among the eight geographical regions revealed that the accessions from the Near East and the Middle East exhibited more genetic diversity than those from the other regions. Greater diversity was found in Southeast Europe than in North and Southwest Europe. The present study also indicates that microsatellite markers permit the fast and high throughput fingerprinting of large numbers of accessions from a germplasm collection in order to assess genetic diversity.

The use of ISSR and RAPD markers for detecting DNA polymorphism, genotype identification and genetic diversity among barley cultivars with known origin

Abstract: The potential of bulk analyses of RAPD and ISSR-PCR markers for fingerprinting purposes was evaluated using ten RAPD and ten ISSR primers. The phylogenetic relationships of 16 barley cultivars from different countries, and all having a known pedigree, were analysed using 353 PCR markers (125 RAPDs and 228 ISSRs). The band profiles generated were reproducible in spite of the different DNA extractions, PCR techniques, electrophoretic methods and gel scorings used. The RAPD primer S10 and four ISSR primers (811, 820, 835 and 881) were both able to distinguish all cultivars. A strong and quite linear relationship was observed between Resolving Power (Rp) of a primer and its ability to distinguish genotypes. The dendrograms obtained using these two molecular markers are in agreement with their known origin, showing clusters that separate very well the spring/winter and six-rows/two-rows cultivars. Thus, bulk analyses of RAPD and ISSR PCR markers provides a quick, reliable and highly informative system for DNA fingerprinting and also permit to establish genetic relationships which agree with, by other means, known origin of the cultivars.

Microsatellite markers isolated in olive ( Olea europaea L.) are suitable for individual fingerprinting and reveal polymorphism within ancient cultivars.

Abstract: We have isolated and sequenced 52 microsatellites or simple sequence repeats (SSRs) from nearly 60 positive clones obtained from two ’Frantoio’ olive genomic libraries enriched in (AC/GT) and (AG/CT) repeats, respectively. The repeat-containing fragments obtained from genomic DNA restricted with Tsp509I were separated using a biotinylated probe bound to streptavidin-coated paramagnetic beads. Fragments were then cloned into lambda ZAPII vector and sequenced. Thirty of the 36 primer pairs which gave correct re-amplification in the source genome were used to assay the polymorphism of 12 olive cultivars, namely four well-known cultivars (’Coratina’, ’Frantoio’, ’Leccino’, ’Pendolino’) and eight ancient cultivars grown locally near Lake Garda (’Casaliva’, ’Favarol’, ’Fort’, ’Grignan’, ’Less’, ’Raza’, ’Rossanel’, ’Trep’). The local cultivars were each re- presented by two to four long-lived individuals. The analysis was carried out using 33P-labelled primers and 6% polyacrylamide sequencing gels. All except two microsatellites showed polymorphism, the number of alleles varying from 1 to 5. The average genetic diversity (H) was 0.55. The power of discrimination (PD) was 0.60. All cultivars, including the local ones, were easily separated from each other. Variations in the SSR pattern were observed among individual plants of the same cultivar in four out of the eight local cultivars analysed. Several primer pairs (17%) amplified more than one locus.

Bay-0 × Shahdara recombinant inbred line population: a powerful tool for the genetic dissection of complex traits in Arabidopsis

Abstract: Natural genetic variation in Arabidopsis is considerable, but has not yet been used extensively as a source of variants to identify new genes of interest. From the cross between two genetically distant ecotypes, Bay-0 and Shahdara, we generated a Recombinant Inbred Line (RIL) population dedicated to Quantitative Trait Locus (QTL) mapping. A set of 38 physically anchored microsatellite markers was created to construct a robust genetic map from the 420 F6 lines. These markers, evenly distributed throughout the five chromosomes, revealed a remarkable equilibrium in the segregation of parental alleles in the genome. As a model character, we have analysed the genetic basis of variation in flowering time in two different environments. The simultaneous mapping of both large- and small-effect QTLs responsible for this variation explained 90% of the total genotypic variance. Two of the detected QTLs colocalize very precisely with FRIGIDA and FLOWERING LOCUS C genes; we provide information on the polymorphism of genes confirming this hypothesis. Another QTL maps in a region where no QTL had been found previously for this trait. This confirms the accuracy of QTL detection using the Bay-0 × Shahdara RIL population, which constitutes the largest in size available so far in Arabidopsis. As an alternative to mutant analysis, this population represents a powerful tool which is currently being used to undertake the genetic dissection of complex metabolic pathways.

Simple sequence repeat map of the sunflower genome

Abstract: Several independent molecular genetic linkage maps of varying density and completeness have been constructed for cultivated sunflower ( Helianthus annuus L.). Because of the dearth of sequence and probe-specific DNA markers in the public domain, the various genetic maps of sunflower have not been integrated and a single reference map has not emerged. Moreover, comparisons between maps have been confounded by multiple linkage group nomenclatures and the lack of common DNA markers. The goal of the present research was to construct a dense molecular genetic linkage map for sunflower using simple sequence repeat (SSR) markers. First, 879 SSR markers were developed by identifying 1,093 unique SSR sequences in the DNA sequences of 2,033 clones isolated from genomic DNA libraries enriched for (AC)(n) or (AG)(n) and screening 1,000 SSR primer pairs; 579 of the newly developed SSR markers (65.9% of the total) were polymorphic among four elite inbred lines (RHA280, RHA801, PHA and PHB). The genetic map was constructed using 94 RHA280 x RHA801 F(7) recombinant inbred lines (RILs) and 408 polymorphic SSR markers (462 SSR marker loci segregated in the mapping population). Of the latter, 459 coalesced into 17 linkage groups presumably corresponding to the 17 chromosomes in the haploid sunflower genome ( x = 17). The map was 1,368.3-cM long and had a mean density of 3.1 cM per locus. The SSR markers described herein supply a critical mass of DNA markers for constructing genetic maps of sunflower and create the basis for unifying and cross-referencing the multitude of genetic maps developed for wild and cultivated sunflowers.

Identification of simple sequence repeats (SSRs) in olive (Olea europaea L.)

Abstract: A small insert genomic library of Olea europaea L., highly enriched in (GA/CT)n repeats, was obtained using the procedure of Kandpal et al. (1994). The sequencing of 103 clones randomly extracted from this library allowed the identification of 56 unique genomic inserts containing simple sequence repeat regions made by at least three single repeats. A sample of 20 primer pairs out of the 42 available were tested for functionality using the six olive varieties whose DNA served for library construction. All primer pairs succeeded in amplifying at least one product from the six DNA samples, and ten pairs detecting more than one allele were used for the genetic characterisation of a panel of 20 olive accessions belonging to 16 distinct varieties. A total of 57 alleles were detected among the 20 genotypes at the ten polymorphic SSR loci. The remaining primer pair allowed the amplification of a single SSR allele for all accessions plus a longer fragment for some genotypes. Considering the simple sequence repeat polymorphism, 5.7 alleles were scored on average for each of the ten SSR loci. A genetic dissimilarity matrix, based on the proportion of shared alleles among all the pair-wise combinations of genotypes, was constructed and used to disentangle the genetic relationships among varieties by means of the UPGMA clustering algorithm. Graphical representation of the results showed the presence of two distinct clusters of varieties. The first cluster grouped the varieties cultivated on the Ionian Sea coasts. The second cluster showed two subdivisions: the first sub-cluster agglomerated the varieties from some inland areas of Calabria; the second grouped the remaining varieties from Basilicata and Apulia cultivated in nearby areas. Results of cluster analysis showed a significant relationship between the multilocus genetic similarities and the geographic origin of the cultivars.

Study of the relationship between pre-harvest sprouting and grain color by quantitative trait loci analysis in a whitexred grain bread-wheat cross.

Abstract: In many wheat (Triticum aestivum L.) growing areas, pre-harvest sprouting (PHS) may cause important damage, and in particular, it has deleterious effects on bread-making quality. The relationship between PHS and grain color is well known and could be due either to the pleiotropic effect of genes controlling red-testa pigmentation (R) or to linkage between these genes and other genes affecting PHS. In the present work, we have studied a population of 194 recombinant inbred lines from the cross between two cultivars, ’Renan’ and ’Recital’, in order to detect QTLs for both PHS resistance and grain color. The variety ’Renan’ has red kernels and is resistant to PHS, while ’Recital’ has white grain and is highly susceptible to PHS. A molecular-marker linkage map of this cross was constructed using SSRs, RFLPs and AFLPs. The population was evaluated over 2 years at Clermont-Ferrand (France). PHS was evaluated on mature spikes under controlled conditions and red-grain color was measured using a chromameter. Over the 2 years, we detected four QTLs for PHS, all of them being co-localized with QTLs for grain color. Three of them were located on the long arm of chromosomes 3 A, 3B and 3D, close to the loci where the genes R and taVp1 were previously mapped. For these three QTLs, the resistance to PHS is due to the allele of the variety ’Renan’. Another co-located QTL for PHS and grain color was detected on the short arm of chromosome 5 A. The resistance for PHS for this QTL is due to the allele of ’Recital’.

QTL clusters reflect character associations in wild and cultivated rice.

Abstract: The genetic basis of character association related to differentiation found in the primary gene pool of rice was investigated based on the genomic distribution of quantitative trait loci (QTLs). Major evolutionary trends in cultivated rice of Asiatic origin (Oryza sativa) and its wild progenitor (O. rufipogon) are: (1) differentiation from wild to domesticated types (domestication), (2) ecotype differentiation between the perennial and annual types in wild races, and (3) the Indica versus Japonica type differentiation in cultivated races. Using 125 recombinant inbred lines (RILs) derived from a cross between an Indica cultivar of O. sativa and a strain of O. rufipogon carrying some Japonica-like characteristics, we mapped 147 markers, mostly RFLPs, on 12 chromosomes. Thirty-seven morphological and physiological quantitative traits were evaluated, and QTLs for 24 traits were detected. The mapped loci showed a tendency to form clusters that are composed of QTLs of the domestication-related traits as well as Indica/Japonica diagnostic traits. QTLs for perennial/annual type differences did not cluster. This cluster phenomenon could be considered "multifactorial linkages" followed by natural selection favoring co-adapted traits. Further, it is possible that the clustering phenomenon is partly due to pleiotropy of some unknown key factor(s) controlling various traits through diverse metabolic pathways. Chromosomal regions where QTL clusters were found coincided with the regions harboring genes or gene blocks where the frequency of cultivar-derived alleles in RILs is higher than expected. This distortion may be partly due to unconscious selection favoring cultivated plant type during the establishment of RILs.

PCR-mediated recombination in amplification products derived from polyploid cotton.

Abstract: PCR recombination describes a process of in vitro chimera formation from non-identical templates. The key requirement of this process is the inclusion of two partially homologous templates in one reaction, a condition met when amplifying any locus from polyploid organisms and members of multigene families from diploid organisms. Because polyploids possess two or more divergent genomes ("homoeologues") in a common nucleus, intergenic chimeras can form during the PCR amplification of any gene. Here we report a high frequency of PCR-induced recombination for four low-copy genes from allotetraploid cotton ( Gossypium hirsutum). Amplification products from these genes ( Myb3, Myb5, G1262 and CesA1) range in length from 860 to 4,050 bp. Intergenomic recombinants were formed frequently, accounting for 23 of the 74 (31.1%) amplicons evaluated, with the frequency of recombination in individual reactions ranging from 0% to approximately 89%. Inspection of the putative recombination zones failed to reveal sequence-specific attributes that promote recombination. The high levels of observed in vitro recombination indicate that the tacit assumption of exclusive amplification of target templates may often be violated, particularly from polyploid genomes. This conclusion has profound implications for population and evolutionary genetic studies, where unrecognized artifactually recombinant molecules may bias results or alter interpretations.

Chromosomal regions associated with segregation distortion in maize.

Abstract: Segregation distortion skews the genotypic frequencies from their Mendelian expectations. Our objectives in this study were to assess the frequency of occurrence of segregation distortion in maize, identify chromosomal regions consistently associated with segregation distortion, and examine the effects of gametophytic factors on linkage mapping. We constructed a simple sequence repeat (SSR) linkage map for a LH200/LH216 F(2)Syn3 (i.e., random-mated three times) population, and compared the segregation distortion in this map with the segregation distortion in three published linkage maps. Among 1,820 codominant markers across the four mapping populations, 301 (17%) showed segregation distortion ( P < 0.05). The frequency of markers showing segregation distortion ranged from 19% in the Tx303/CO159 mapping population to 36% in the B73/Mo17 mapping population. A positive relationship was found between the number of meioses and the frequency of segregation distortion detected in a population. On a given chromosome, nearly all of the markers showing segregation distortion favored the allele from the same parent. A total of 18 chromosomal regions on the ten maize chromosomes were associated with segregation distortion. The consistent location of these chromosomal regions in four populations suggested the presence of segregation distortion regions (SDRs). Three known gametophytic factors are possible genetic causes of these SDRs. As shown in previous research, segregation distortion does not affect the estimate of map distance when only one gametophytic factor is present in an SDR.

QTL mapping and introgression of yield-related traits from Oryza glumaepatula to cultivated rice (Oryza sativa) using microsatellite markers

Abstract: Rice ( Oryza sativa) cultivar development currently faces the task of overcoming yield plateaus, which is difficult due to the narrow genetic base of breeding programs. Oryza glumaepatula is a diploid wild relative of cultivated rice, native to Central and South America, and is therefore a potential source of alleles of agronomic importance to rice breeding programs. We studied 11 agronomic traits in BC(2)F(2) families of the interspecific cross Oryza sativa x O. glumaepatula. Transgressive lines which are almost isogenic to the elite recurrent O. sativa parent were identified for most of these traits. Quantitative trait locus (QTL) analysis was performed by single-point and interval mapping using a molecular map based on 157 microsatellite and STS markers. Marker regions accounting for 14.5 to 72.9% of a phenotypic variation trait were identified in 9 of the 12 rice chromosomes. Positive QTL effects from O. glumaepatula were observed in chromosomal regions associated with tillering and panicle-number traits.

The origin of cultivated Coffea arabica L. varieties revealed by AFLP and SSR markers

Abstract: Molecular markers were used to assess polymorphism between and within the genetic bases of coffee (i.e. Typica and Bourbon) spread from Yemen since the early 18th century that have given rise to most arabica cultivars grown world-wide. Eleven Coffea arabica accessions derived from the disseminated bases were evaluated by amplified fragment length polymorphism (AFLP) using 37 primer combinations and simple-sequence repeats (SSRs) produced by six microsatellites. Four cultivars growing in Yemen and 11 subspontaneous accessions collected in the primary centre of diversity of the species were included in the study in order to define their relationship with the accessions derived from the genetic bases of cultivars. One hundred and seven AFLP markers were used to calculate genetic distances and construct a dendrogram. The accessions derived from the disseminated bases were grouped separately, according to their genetic origin, and were distinguished from the subspontaneous accessions. The Yemen cultivars were classified with the Typica-derived accessions. Except for one AFLP marker, all AFLP and SSR markers present in the cultivated accessions were also detected in the subspontaneous accessions. Polymorphism among the subspontaneous accessions was much higher than among the cultivated accessions. It was very low within the genetic bases, confirming the historical documentation on their dissemination. The results enabled a discussion of the genetic diversity reductions that successively occurred during the dissemination of C. arabica from its primary centre of diversity.

Substitution mapping of Pup1: a major QTL increasing phosphorus uptake of rice from a phosphorus-deficient soil.

Abstract: A major QTL for P uptake had previously been mapped to a 13-cM marker interval on the long arm of chromosome 12. To map that major QTL with higher precision and certainty, a secondary mapping population was developed by backcrossing a near-isogenic line containing the QTL from the donor parent to the recurrent parent of low P uptake. Two different mapping strategies have been followed in this study. A conventional QTL mapping approach was based on individual F2 RFLP data and the phenotypic evaluation of family means in the F3. The second strategy employed a substitution-mapping approach. Phenotypic and marker data were obtained for 160 F3 individuals of six highly informative families that differed in the size of donor chromosomal segments in the region of the putative QTL. QTL mapping showed that close to 80% of the variation between families was due to a single QTL, hereafter referred to as Pup1 (Phosphorus uptake 1). Pup1 was placed in a 3-cM interval flanked by markers S14025 and S13126, which is within 1 cM of the position identified in the original QTL mapping experiment. Other chromosomal regions and epistatic effects were not significant. Substitution mapping revealed that Pup1 co-segregated with marker S13126 and that the flanking markers, S14025 and S13752, were outside the interval containing Pup1. The two mapping strategies therefore yielded almost identical results and, in combining the advantages of both, Pup1 could be mapped with high certainty. The QTL mapping appoach showed that the phenotypic variation between families was due to only one QTL without any additional epistacic interactions, whereas the advantage of substitution mapping was to place clearly defined borders around the QTL.

Characterisation of polymorphic microsatellite markers from Aegilops tauschii and transferability to the D-genome of bread wheat.

Abstract: Microsatellites were isolated from a Aegilops tauschii (the D-genome donor of bread wheat) library enriched for various motifs. Primers generated from the flanking region of the microsatellites were used successfully to amplify the corresponding loci in the D genome of bread wheat. Additional amplification sometimes also occurred from the A and B genomes. The majority of the microsatellites contained (GA)n and (GT)n motifs. GA and GT repeats appeared to be both more abundant in this library and more polymorphic than other types of repeats. The allele number for both types of dinucleotide repeats fitted a Poisson distribution. Deviance analysis showed that GA and GT were more polymorphic than other motifs in bread wheat. Within each motif type (di-, tri- and tetra-nucleotide repeats), repeat number has no influence on polymorphism. The microsatellites were mapped using the Triticum aestivum Courtot × Chinese Spring mapping population. A total of 100 markers was developed on this intraspecific map, mainly on the D genome. For polyploid species, isolation of microsatellites from an ancestral diploid donor seems to be an efficient way of developing markers for the corresponding genome in the polyploid plant.

Microsatellites in Zea - variability, patterns of mutations, and use for evolutionary studies.

Abstract: To evaluate the performance of microsatellites or simple sequence repeats (SSRs) for evolutionary studies in Zea, 46 microsatellite loci originally derived from maize were applied to diverse arrays of populations that represent all the diploid species of Zea and 101 maize inbreds. Although null phenotypes and amplification of more than two alleles per plant were observed at modest rates, no practical obstacle was encountered for applying maize microsatellites to other Zea species. Sequencing of microsatellite alleles revealed complex patterns of mutation including frequent indels in the regions flanking microsatellite repeats. In one case, all variation at a microsatellite locus came from indels in the flanking region rather than in the repeat motif. Maize microsatellites show great variability within populations and provide a reliable means to measure intraspecific variation. Phylogeographic relationships of Zea populations were successfully reconstructed with good resolution using a genetic distance based on the infinite allele model, indicating that microsatellite loci are useful in evolutionary studies in Zea. Microsatellite loci show a principal division between tropical and temperate inbred lines, and group inbreds within these two broad germplasm groups in a manner that is largely consistent with their known pedigrees.

Molecular linkage map of allotetraploid cotton (Gossypium hirsutum L. × Gossypium barbadense L.) with a haploid population

Abstract: In the present study, a haploid population from the cross of the two cultivated allotetraploid cottons, Gossypium hirsutum L. and Gossypium barbadense L., was developed by means of Vsg, a virescently marked semigamous line of Sea island cotton, and some target haploids were successfully doubled with colchicine. A molecular linkage map was constructed with 58 doubled and haploid plants. Among the total of 624 marker loci (510 SSRs and 114 RAPDs), 489 loci were assembled into 43 linkage groups and covered 3,314.5 centi-Morgans (cM). Using the monosomic and telodisomic genetic stocks, the linkage groups of the present map were associated with chromosomes of the allotetraploid genome, and some of the unassociated groups were connected to corresponding A or D subgenomes. Through the analysis of the assignment of the duplicated SSR loci in the chromosomes or the linkage groups, ten pairs of possible homoeologous chromosome (or linkage group) regions were identified. Among them, the pairs of Chrs. 1 and 15, Chrs. 4 and 22, and Chrs. 10 and 20 had already been determined as homoeologous by classical genetic and cytogenetic research, and the pair of Chrs. 9 and 23 had also been identified by the ISH method of molecular cytogenetics. But, from present research, it was assumed that Chrs. 5 and 18 might be a new pair of homoeologous chromosomes of the allotetraploid cotton genome detected by molecular mapping of the cotton genome.

Identification and mapping of molecular markers linked to rust resistance genes located on chromosome 1RS of rye using wheat-rye translocation lines

Abstract: The short arm of rye (Secale cereale) chromosome 1 has been widely used in breeding programs to incorporate new disease resistance genes into wheat. Using wheat-rye translocation and recombinant lines, molecular markers were isolated and mapped within chromosomal regions of 1RS carrying rust resistance genes Lr26, Sr31, Yr9 from 'Petkus' and SrR from 'Imperial' rye. RFLP markers previously mapped to chromosome 1HS of barley – flanking the complex Mla powdery mildew resistance gene locus – and chromosome 1DS of Aegilops tauschii – flanking the Sr33 stem rust resistance gene – were shown to map on either side of rust resistance genes on 1RS. Three non cross-hybridising Resistance Gene Analog markers, one of them being derived from the Mla gene family, were mapped within same region of 1RS. PCR-based markers were developed which were tightly linked to the rust resistance genes in 'Imperial' and 'Petkus' rye and which have potential for use in marker-assisted breeding.

Transgenics of an elite indica rice variety Pusa Basmati 1 harbouring the codA gene are highly tolerant to salt stress.

Abstract: Transgenic lines of indica rice were generated by Agrobacterium-mediated transformation with the choline oxidase (codA) gene from Arthrobacter globiformis. Choline oxidase catalyses conversion of choline to glycine betaine. Glycine betaine is known to provide tolerance against a variety of stresses. Molecular analyses of seven independent transgenic lines as performed by Southern, Northern and Western hybridization revealed integration and expression of the transgene as well as inheritance in the progeny plants. A good correlation was observed between levels of mRNA and protein accumulation, and a significant amount of choline oxidase product, i.e. glycine betaine, accumulated in R0 as well as R1 plants. Mendelian as well as non-Mendelian segregation patterns were obtained in the progeny plants. Challenge studies performed with R1 plants by exposure to salt stress (0.15 M NaCl) for 1 week, followed by a recovery period, revealed that in some cases more than 50% of the transgenic plants could survive salt stress and set seed whereas wild-type plants failed to recover.

QTL mapping of stay-green in two sorghum recombinant inbred populations

Abstract: The stay-green trait is a reported component of tolerance to terminal drought stress in sorghum. To map quantitative trait loci (QTLs) for stay-green, two sorghum recombinant inbred populations (RIPs) of 226 F3:5 lines each were developed from crosses (1) IS9830 × E36-1 and (2) N13 × E36-1. The common parental line, E36-1 of Ethiopian origin, was the stay-green trait source. The genetic map of RIP 1 had a total length of 1,291 cM, with 128 markers (AFLPs, RFLPs, SSRs and RAPDs) distributed over ten linkage groups. The map of RIP 2 spanned 1,438 cM and contained 146 markers in 12 linkage groups. The two RIPs were evaluated during post-rainy seasons at Patancheru, India, in 1999/2000 (RIP 2) and 2000/2001 (RIP 1). The measures of stay-green mapped were the green leaf area percentages at 15, 30 and 45 days after flowering (% GL15, % GL30 and % GL45, respectively). Estimated repeatabilities for % GL15, % GL30 and % GL45 amounted to 0.89, 0.81 and 0.78 in RIP 1, and 0.91, 0.88 and 0.85 in RIP 2, respectively. The number of QTLs for the three traits detected by composite interval mapping ranged from 5 to 8, explaining 31% to 42% of the genetic variance. In both RIPs, both parent lines contributed stay-green alleles. Across the three measures of the stay-green trait, three QTLs on linkage groups A, E and G were common to both RIPs, with the stay-green alleles originating from E36-1. These QTLs were therefore consistent across the tested genetic backgrounds and years. After QTL validation across sites and verification of the general benefit of the stay-green trait for grain yield performance and stability in the target areas, the corresponding chromosomal regions could be candidates for marker-assisted transfer of stay-green into elite materials.

Comparative analysis of genetic diversity in the mangrove species Avicennia marina (Forsk.) Vierh. (Avicenniaceae) detected by AFLPs and SSRs.

Abstract: Avicennia marina is an important mangrove species with a wide geographical and climatic distribution which suggests that large amounts of genetic diversity are available for conservation and breeding programs. In this study we compare the informativeness of AFLPs and SSRs for assessing genetic diversity within and among individuals, populations and subspecies of A. marina in Australia. Our comparison utilized three SSR loci and three AFLP primer sets that were known to be polymorphic, and could be run in a single analysis on a capillary electrophoresis system, using different- colored fluorescent dyes. A total of 120 individuals representing six populations and three subspecies were sampled. At the locus level, SSRs were considerably more variable than AFLPs, with a total of 52 alleles and an average heterozygosity of 0.78. Average heterozygosity for AFLPs was 0.193, but all of the 918 bands scored were polymorphic. Thus, AFLPs were considerably more efficient at revealing polymorphic loci than SSRs despite lower average heterozygosities. SSRs detected more genetic differentiation between populations (19 vs 9%) and subspecies (35 vs 11%) than AFLPs. Principal co-ordinate analysis revealed congruent patterns of genetic relationships at the individual, population and subspecific levels for both data sets. Mantel testing confirmed congruence between AFLP and SSR genetic distances among, but not within, population comparisons, indicating that the markers were segregating independently but that evolutionary groups (populations and subspecies) were similar. Three genetic criteria of importance for defining priorities for ex situ collections or in situ conservation programs (number of alleles, number of locally common alleles and number of private alleles) were correlated between the AFLP and SSR data sets. The congruence between AFLP and SSR data sets suggest that either method, or a combination, is applicable to expanded genetic studies of mangroves. The codominant nature of SSRs makes them ideal for further population-based investigations, such as mating-system analyses, for which the dominant AFLP markers are less well suited. AFLPs may be particularly useful for monitoring propagation programs and identifying duplicates within collections, since a single PCR assay can reveal many loci at once.

Isolation and characterization of microsatellites in Brassica rapa L.

Abstract: We report here the isolation and characterization of microsatellites, or simple sequence repeats (SSRs), in Brassica rapa. The size-fractionated genomic library was screened with (GA)15 and (GT)15 oligonucleotide probes. A total of 58 clones were identified as having the microsatellite repeats, and specific primer pairs were designed for 38 microsatellite loci. All primer pairs, except two, amplified fragments having the sizes expected from the sequences. Of the 36 primer pairs, 35 amplified polymorphic loci in 19 cultivars of B. rapa, while monomorphism was observed in only one primer pair. A total of 232 alleles was identified by the 36 primer pairs in 19 cultivars of B. rapa, and these primer pairs were examined also in nine Brassicaceae species. Most of the 36 primer pairs amplified the loci in the Brassicaceae species. Segregation of the microsatellites was studied in an F2 population from a cross of doubled-haploid lines DH27 × G309. The microsatellites segregated in a co-dominant manner. These results indicate that the microsatellites isolated in this study were highly informative and could be useful tools for genetic analysis in B. rapa and other related species.

Molecular characterization and similarity relationships among apricot ( Prunus armeniaca L.) genotypes using simple sequence repeats.

Abstract: A collection of 48 apricot genotypes, originated from diverse geographic areas, have been screened with 37 SSR primer pairs developed in different species of Prunus in order to identify and characterize the genotypes and establish their genetic relations. Thirty one of those primer pairs resulted in correct amplifications and 20 produced polymorphic repeatable amplification patterns with the 48 genotypes studied. A total of 82 alleles were detected for the 20 loci. All the genotypes studied could be unequivocally distinguished with the combination of SSRs used. The results obtained evidence for the cross-species transportability of microsatellite sequences, allowing the discrimination among different genotypes of a given fruit-tree species with sequences developed in other species. UPGMA cluster analysis of the similarity data grouped the genotypes studied according to their geographic origin and/or their pedigree information.