Development and utilization of novel SSRs in foxtail millet [Setaria italica (L.) P. Beauv.]

Although the foxtail millet [Setaria italica (L.) P. Beauv.] is recently regarded as a model crop for studying functional genomics of biofuel grasses, its genetic improvement to some extent was limited due to the non‐availability of molecular markers, particularly the microsatellite markers and the saturated genetic linkage map. Considering this, we attempted to generate a significant number of microsatellite markers in cultivar ‘Prasad’. Two hundred and fifty‐six clones were sequenced to generate 41.82‐kb high‐quality sequences retrieved from genomic library enriched with dinucleotide repeat motifs. Microsatellites were identified in 194 (76%) of the 256 positive clones, and 64 primer pairs (pp) were successfully designed from 95 (49%) unique SSR‐containing clones. The 67.4% primer designing ability, 100% PCR amplification efficiency and 45.3% polymorphic potential in the parents of F₂ mapping population established the efficacy of genomic microsatellites. All the 64 microsatellite markers displayed high level of cross‐species amplification (~67%) in 10 millets and non‐millets species. These experimental findings suggest the utility and efficacy of SSRs in diverse genotyping applications, resolving QTLs, phylogenetic relationships and transferability in several important grass species.     

Development of microsatellite markers in sweet passion fruit,and identification of length and conformation polymorphisms within repeat sequences

The aim of this study was to construct and characterize an SSR‐enriched genomic library for Passiflora alata, a fruit species native to the Brazilian plateau and the eastern Amazon region. There is potential to improve this crop, as the fruit is attractive because of its pleasant aroma and flavour characteristics. Of 862 sequences, 391 (45%) were found to have SSRs. We identified 412 microsatellites: 69% were classified as perfect, 23% imperfect, 5% interrupted and 3% were compound microsatellites. The main types of repeat sequences were dinucleotide (62.5%) and trinucleotide (23%) repeats. It was possible to design 312 primer pairs, and 229 of them were synthesized and tested. The amplicons were electrophoresed using denaturing and non‐denaturing gels to screen for divergence between two phenotypically distinct parents of a mapping population of P. alata. Length and conformation polymorphisms within repeat sequences amounted to 35% and 28%, respectively. The importance of the development of SSR markers for sweet passion fruit and the effectiveness of the SSCP approach to increase the available number of polymorphic SSRs are discussed.     

Genetic analysis of forage grasses based on heterologous RFLP markers detected by rice cDNAs

Genetic polymorphism within and between three species of forage grasses, perennial ryegrass (Lolium perenne L), meadow fescue (Festuca pratensis Huds.) and tall fescue (Festuca arundinacea Schreb.), was analyzed using restriction fragment length polymorphism (RFLP) markers detected by rice cDNA probes developed at the Rice Genome Research Programme of Japan (RGP). One hundred and ninety‐seven rice cDNA clones were used for hybridization to genomic DNA of forage grasses. Many of the rice cDNA clones produced no visible band or only a smear with no discrete bands. Twenty‐three clones showed high efficiency cross‐hybridization to the genomic DNA of forage grasses. Genetic variation was evaluated for five varieties and one population of forage grasses using 12 polymorphic rice cDNA RFLP probes. Genetic variability within varieties as measured by Rogers’ genetic distance was considerably lower for the F. pratensis variety ‘Tomosakae’ than for the L. perenne and F. arundinacea varieties. To determine the genetic diversity between varieties of different species, cluster analysis was performed using data from the 12 RFLP probes. The two accessions of Lolium perenne were clustered more closely together than the three varieties of F. arundinacea. Two Japanese varieties of F. arundinacea were grouped in the same cluster. The variety‐specific RFLP markers were seen among six accessions of L. perenne, F. pratensis and F. arundinacea. Such variety‐specific RFLP markers would provide very useful tools for breeding programmes such as the intergeneric hybridization of Lolium and Festuca genera.     

Development, characterization and utilization of microsatellite markers in pigeonpea

Pigeonpea is a major legume of the semi‐arid tropics that has been neglected in terms of molecular breeding. The objectives of this study were to develop microsatellite markers and evaluate their potential for use in pigeonpea genetics and breeding. Two hundred and eight microsatellite loci were isolated by screening a non‐enriched partial genomic library. Primers were designed for 39 microsatellite loci, 20 of which amplified polymerase chain reaction products of the expected size. Nineteen of the primer pairs were polymorphic amongst 15 cultivated and nine wild pigeonpea accessions providing evidence for cross‐species transferability within the genus Cajanus. A total of 98 alleles were detected at the 19 polymorphic loci with an average of 4.9 alleles per locus. The observed heterozygosity ranged from 0.17 to 0.80 with a mean of 0.42 per locus. Less allelic variation (31 alleles) was observed within the cultivated species than across the wild species (92 alleles). The diversity analysis readily distinguished all wild relatives from each other and from the cultivated germplasm. Development of more microsatellites is recommended for future genomic studies in pigeonpea.     

Linkage map construction and mapping QTL for cotton fibre quality using SRAP, SSR and RAPD

Tetraploid cotton is one of the most extensively cultivated species. Two tetraploid species, Gossypium hirsutum L. and G. barbadense L., dominate the world's cotton production. To better understand the genetic basis of cotton fibre traits for the improvement of fibre quality, a genetic linkage map of tetraploid cotton was constructed using sequence‐related amplified polymorphisms (SRAPs), simple sequence repeats (SSRs) and random amplified polymorphic DNAs (RAPDs). A total of 238 SRAP primer combinations, 368 SSR primer pairs and 600 RAPD primers were used to screen polymorphisms between G. hirsutum cv. Handan208 and G. barbadense cv. Pima90 which revealed 749 polymorphic loci in total (205 SSRs, 107 RAPDs and 437 SRAPs). Sixty‐nine F₂ progeny from the interspecific cross of ‘Handan208’בPima90’ were genotyped with the 749 polymorphic markers. A total of 566 loci were assembled into 41 linkage groups with at least three loci in each group. Twenty‐eight linkage groups were assigned to corresponding chromosomes by SSR markers with known chromosome locations. The map covered 5141.8 cM with a mean interlocus space of 9.08 cM. A × test for significance of deviations from the expected ratio (1: 2: 1 or 3: 1) identified 135 loci (18.0%) with skewed segregation, most of which had an excess of maternal parental alleles. In total, 13 QTL associated with fibre traits were detected, among which two QTL were for fibre strength, four for fibre length and seven for micronaire value. These QTL were on nine linkage groups explaining 16.18‐28.92% of the trait variation. Six QTL were located in the A subgenome, six QTL in the D subgenome and one QTL in an unassigned linkage group. There were three QTL for micronaire value clustered on LG1, which would be very useful for improving this trait by molecular marker‐assisted selection.     

Identification of sugarcane microsatellites associated to sugar content in sugarcane and transferability to other cereal genomes

Microsatellites or simple sequence repeats (SSRs) markers are very informative for various applications in genetics and breeding. Information obtained with these markers has contributed to a better understanding of evolution and the complexity of the sugarcane genome. With the objective of identifying a large set of polymorphic microsatellite markers designated as Unigene derived Sugarcane Microsatellite (UGSM) and Sugarcane Enriched Genomic Microsatellite (SEGMS), 351 UGSM and 36 SEGMS were tested to find out informative SSRs marker for sugar content. These markers were screened and validated for their use in genetic diversity, cross transferability and comparative linkage potential in high and low sugar bulk of two segregating progenies and twenty each, cultivated high and low sugar cultivars. 158 (40.83%) of the microsatellite markers (144-UGSM: 14-SEGMS) were found to be highly robust and polymorphic. Cross amplification was estimated among nineteen accessions of six sugarcane cultivars, one inter specific hybrids, five related species, four related genera, and three divergent genera by using 27 UGSM primers. Analysis of 388 alleles, amplified by these markers, indicated the high number of observed allele ranged from 2 to 26, with an average of 14.37 alleles detected per locus. High level of polymorphism detected by these markers among sugarcane species, genera and cultivars was 96.3%, while cross-transferability rate was 98.0% within Saccharum complex and 88.27% to cereals. Wide range of genetic diversity (0.33–0.79 with an average of 0.56) assayed with UGSM markers suggested their importance in various genotypic applications in sugarcane.     

Occurrence of microsatellites in spinach sequences from computer databases and development of polymorphic SSR markers

Microsatellites are valuable tools as molecular markers in plant breeding. To establish genetic linkage maps or for population studies, information about the occurrence and usability of microsatellite markers in different species is necessary. Sequences of spinach Spinacia oleracea from computer databases were therefore searched for the presence of microsatellites. Sixty simple sequence repeats were found in 237 spinach sequences with a total of 349.4 kb DNA. After removing duplicated sequences, 50 different microsatellites with various motifs remained. Differences between nuclear and chloroplast DNA were not in the number of microsatellites but in their type and length. Chloroplast sequences from spinach contain only short strings of A and AT repeats, whereas nuclear sequences show a wider variety of motifs. Flanking primers for polymerase chain reaction (PCR) analysis were designed for 13 of these microsatellites and tested with two different varieties of spinach. Twelve primer pairs gave amplification products and seven of these showed polymorphisms in the variety ‘Wiremona’ but only one in the variety ‘Monatol’. These markers may be used for linkage analysis or population studies in spinach.     

Identification and characterization of microsatellites in eggplant

The potential of microsatellite markers for use in genetic studies in eggplant, Solanum melongena, has been evaluated. A genomic library of eggplant was screened for GA and GT repeat motifs to isolate microsatellite clones. The frequency of each repeat motif in the eggplant genome was found to be every 3200 kb for GA repeats and every 820 kb for GT repeats. Sixty‐one per cent of GT repeats were found to directly flank AT repeats. A total of 37 polymerase chain reaction (PCR) primer pairs were designed, 23 of which amplified a single product or several products. The level of microsatellite polymorphism was evaluated by using S. melongena lines and related Solanum species. Two to six alleles per primer pair were displayed in the S. melongena lines and two to 13 alleles were displayed in the Solanum relatives. Seven microsatellites showed polymorphism between parental lines of the mapping population and segregated in a codominant Mendelian manner. These microsatellite loci were distributed throughout the linkage map.     

Application of microsatellites from maize to teosinte and other relatives of maize

Teosinte comprises different Zea species (Zea mays, Zea diploperennis, Zea perennis, Zea luxurians) that can be crossed with cultivated maize (Z. mays ssp. mays). Nine microsatellites from maize were applied to different teosinte species in order to evaluate their usefulness in markerbased exploitation of these genetic resources. The same microsatellites were tested with rye, barley, and sorghum as potential molecular markers for these species. Almost all microsatellite × teosinte combinations yielded polymerase chain reaction (PCR) fragments in the range of cultivated maize. Using an F₂ population of a cross between maize inbred A188 and an individual of Zea mays ssp. mexicana, amplification products for maize and teosinte originated from the same genomic location for each of nine microsatellites investigated. PCR fragments of reduced intensity were generally obtained by applying maize microsatellites to rye, barley and sorghum. Polymorphisms among accessions within teosinte (sub)species occurred frequently. In contrast, no polymorphisms were obtained within rye, barley, and sorghum. Hence, application of maize microsatellites to teosinte for fingerprinting or marker-assisted introgression of genomic regions from teosinte into cultivated maize appears promising.     

Genetic diversity among elite Bulgarian barley varieties evaluated by RFLP and RAPD markers

Genetic variation among five elite winter barley cultivars (H. vulgare L.) currently grown in Bulgaria was assessed at the molecular level using restriction fragment length polymorphism (RFLP) and randomly amplified polymorphic DNA (RAPD) markers. The present study sampled RFLPs in four well characterized multigene families in barley: the seed storage protein loci; the 18S, 5.8S and 26S ribosomal DNA loci; the loci coding for 5S ribosomal RNA and the loci coding subunit α of ATP-A complex in the mitochondrial genome. RFLPs were detected in three out of five investigated chromosomal loci in the barley cultivars studied. RAPD assay using arbitrary 10-base primers was applied to generate amplified length polymorphic markers in barley. Overall a total of 15 polymorphic phenotypes were found among the studied barley cultivars by using 11 out of 25 tested primers. All RAPDs were considered as dominant genetic markers except for two, where PCR and Southern blot analysis indicated the presence of codominant amplification products. Five RAPD polymorphisms in F₁ and F₂ progenies of the cross between Alpha and Obzor were inherited in Mendelian fashion. The determined values for the genetic variation proved a high genetic similarity among the tested cultivars. Genetic similarity (GS) calculated from RFLP and RAPD data ranged from 0.888 to 0.997 with a mean GS – 0.933. This revised version was published online in July 2006 with corrections to the Cover Date.     

Genetic linkage map construction for white jute (Corchorus capsularis L.) using SRAP,ISSR and RAPD markers

White jute (Corchorus capsularis) and dark jute (Corchorus olitorius) are two important cultivated crops that are used for natural fibre production. Some genetic maps have been developed for dark jute, but the genetic map information for white jute (C. capsularis) is limited. In this study, a linkage map comprising 44 sequence‐related amplified polymorphisms (SRAPs), 57 intersimple sequence repeats (ISSRs) and 18 randomly amplified polymorphic DNA (RAPD) covering 2185.7 cM with a mean density of 18.7 cM per locus was constructed in an F₂ population consisting of 185 individuals derived from a cross between two diverse genotypes of ‘Xinxuan No. 1’ and ‘Qiongyueqing’ in white jute. These markers were evenly distributed in the linkage groups without any clustering. This genetic linkage map construction will facilitate the mapping of agronomic traits and marker‐assisted selection breeding in white jute.     

Genetic maps of RAPD, AFLP and ISSR markers in Ananas bracteatus and A. comosus using the pseudo-testcross strategy

Genetic maps of random amplified polymorphic DNA (RAPD), amplified fragment length polymorphisms (AFLP) and inter simple sequence repeats (ISSR) markers in pineapple (2n = 2x = 50) are reported for the first time. On the basis of a segregating population of 46 F1 individuals from a cross Ananas comosus x A. bracteatus, genetic maps of these two species were constructed using the two‐way pseudo‐testcross approach. The A. bracteatus map consists of 335 markers (60 RAPDs, 264 AFLPs and 11 ISSRs) assembled into 50 linkage groups, 26 of them with at least four markers. The A. comosus map consists of 157 markers (33 RAPDs, 115 AFLPs, eight ISSRs and the ‘piping’ trait locus) organized into 30 linkage groups, 18 of them with at least four markers. These maps cover, respectively, 57.2% of the A. bracteatus genome estimated as 3693 cM long, and 31.6% of the A. comosus genome calculated as 4146 cM. A rough estimate of 120 and 127 kbp/ cM on average was found for the relationship between physical and genetic distance for A. bracteatus and A. comosus, respectively.     

Genetic Relationships in Malus Evaluated by RAPD 'Fingerprinting' of Cultivars and Wild Species

The potential use of RAPD markers for taxonomic studies in Malus was investigated using 18 accessions of wild species and 27 apple cultivars. 29 preselected random decamer primers were applied to three sets of Malus genotypes. Random amplified polymorphic DNA (RAPD) ‘fingerprints’ were analysed for polymorphic amplification fragments, and coefficients estimating genetic similarity were calculated on the basis of about 50 polymorphic RAPD loci in each set of genotypes. Cluster analysis by an unweighted pair-group method with arithmetic averages (UPGMA) revealed that, in the cultivars, the molecular classification was in good agreement with the known lineage. A dendrogram generated for the wild species gave relationships that were, in principle, in accordance with the known phylogenetic information. Closely related species from section I were clearly distinguishable from those of sections III and IV. On the molecular level, a high degree of genetic diversity was found among both different apple cultivars and wild species of the genus Malus. The results gave additional evidence for the hypothesis that M. pumila and M. sylvestris were involved in the origin of the cultivated apples.     

Use of ccSSR markers for the determination of the purity of alloplasmic wheat in different Hordeum cytoplasms

We tested three different consensus chloroplast simple sequence repeat (ccSSR) primers to identify amplified polymorphic products in Hordeum chilense, Hordeum vulgare and other Triticeae species with a double aim. First, to use chloroplast‐specific primers as an indirect method for the assessment of wheat cytoplasmic male sterile purity in seedlings during multiplication and in the development of alloplasmic lines. Second, to analyse cytoplasmic relationships among H. chilense accessions and between H. chilense and other members of the Triticeae tribe. The products from primer ccSSR‐4 were easily discriminated using agarose gel electrophoresis. Based on the lengths of amplification products, three groups were identified: the first included almost exclusively H. chilense accessions, the second contained H. vulgare accessions and the third comprised the wheat and the rest of the analysed accessions. Sequencing of PCR products revealed point mutations and insertions/deletions in addition to the expansion/contraction of the microsatellite repeat length. Data analyses of sequenced fragments revealed six groups of accessions among the material studied. No significant differences were found among H. chilense accessions.     

Microsatellite variability and its use in the characterization of cultivated clones of Hevea brasiliensis

Microsatellite markers were developed and evaluated in Hevea brasiliensis, an important crop species producing natural rubber of commercial utility. Of eight microsatellite markers, four were found to be highly informative, amplifying a total of 19 alleles when evaluated against 27 cultivated Hevea clones/genotypes. Power of discrimination of the microsatellite loci was in the range of 0.62‐0.89, with a mean of 0.76 indicating these microsatellites could be valuable genetic markers for diversity characterization. A combination of four microsatellite markers was successfully used to discriminate uniquely all the 27 Hevea clones and some clone‐specific allelic profiles were generated. Cross‐species amplification of the markers developed in H. brasiliensis had also been demonstrated with two other Hevea species, H. benthamiana and H. spruceana, indicating a high degree of sequence homology at the flanking regions. Sequence analysis of the repeat region at the 3′‐UTR of the hydroxymethylglutaryl‐coenzyme A reductase gene, containing clusters of AG repeats in 15 clones, revealed the existence of two alleles based on the repeat length polymorphisms. Homozygosity as well as heterozygosity for both the alleles had also been detected among the clones. Frequency of homozygotes for the smaller allele (allele‐1) was found to be lower than the larger allele (allele‐2) among the primary clones of H. brasiliensis.     

Development and characterization of a new set of 164 polymorphic EST‐SSR markers for diversity and breeding studies in rubber tree (Hevea brasiliensis Müll. Arg.)

Despite its economic importance and recent genome release, the need for molecular tools for Hevea brasiliensis is high. In the frame of a disease resistance study, EST sequences were retrieved from public database or generated by sequencing SSH libraries. Sequences were trimmed and microsatellite motifs searched using an ad hoc bioinformatic pipeline, and pairs of primers for the amplification of candidate markers were generated. We found a total of 10 499 unigenes from both sources of sequences, and 673 microsatellites motifs were detected using the default parameters of the pipeline. Two hundred sixty‐four primer pairs were tested and 226 (85.6%) successfully amplified. Out of the amplified candidate markers, 164 exhibited polymorphism. Relationships based on dendrograms using simple matching index and diversity statistics based on EST‐SSRs were compared with Genomic SSRs, showing the potentialities of EST‐derived microsatellites for resistance studies but also for population genetics approaches.     

Genome-specific markers of tetraploid wheats and their putative diploid progenitor species

The objective of this study was to isolate genome‐specific markers from the genomes of tetraploid wheats and the putative donor diploid species on the basis of random amplified polymorphic DNA analysis followed by cross‐hybridization. Twenty different Triticum and Aegilops species and accessions were analysed by polymerase chain reaction (PCR) using 30 random primers. The polymorphic PCR fragments were then isolated, labelled and used in cross‐hybridization screenings. The hybridization results established that one marker was specific to the Ae. speltoides S genome, two to the A genome, one to the B genome and five to the G genomes of polyploid species (and to the genomes of the corresponding progenitor species). Four markers were identified that were specific to both the B and G genomes. Analysis of the Triticum and Aegilops species and accessions supported the notion that Ae. speltoides is more closely related to the B and G genomes of polyploid wheat species than were other members of the Sitopsis section. The data also indicated that the B and G genomes had originated from different accessions of Ae. speltoides.     

Identification of cultivars and accessions of Lolium, Festuca and Festulolium hybrids through the detection of simple sequence repeat polymorphism

Molecular diversity and genetic affinity in the Lolium/Festuca grass complex have been assessed using simple sequence repeat (SSR) marker technology. The genotypic set was derived from three accessions of perennial ryegrass, two cultivars of Italian ryegrass, two cultivars of meadow fescue, two cultivars of tall fescue and 10 accessions from different intergeneric hybrid (Festulolium) combinations. The majority of the genomic DNA‐derived SSR primer pairs from perennial ryegrass (LPSSR) and Italian ryegrass (LMSSR) produced clear, simple and distinctive amplification products from the majority of the genotypes. The efficiency of cross‐specific amplification for LPSSR markers varied from 38% in meadow fescue to 93% in two cultivars of Festulolium and from 57% in meadow fescue to 87% in Italian ryegrass for LMSSR markers. Of 40 amplified markers, 14 (35%) produced species‐difference alleles in the relation to cultivars used in the present study. Thirty‐five LPSSR locus‐derived alleles were found to be specific to Lolium species, four to meadow fescue and six to tall fescue. For LMSSR alleles, eight were specific to Lolium species and five were only associated with Italian ryegrass, and null alleles were detected for meadow fescue in all instances. These species‐difference markers could clearly identify different accessions of Festulolium. Cluster analysis separated the individual taxa and showed grouping of intergeneric hybrids based on genomic composition. The data distinguished between the species and reflected the known pedigree of the cultivars and the differences between the species. The dendrogram also distinguished between the Festulolium accessions and clearly demonstrated the relations between Festulolium hybrids and their parent species.     

Genetic relationship of Mediterranean mandarins (Citrus deliciosa Tenore) using RAPD markers

Summary Random amplified polymorphic DNA (RAPD) analysis was carried out to evaluate polymorphism and genetic similarity between 39 Mediterranean mandarin genotypes. One hundred eleven amplification products were identified using 21 random primers. An average of 2.2 RAPD markers was obtained for each primer, corresponding to 42% of the amplification products. Genotype-specific RAPD markers were also found, mainly in known hybrids. UPGMA cluster analysis revealed the low level of genetic variation between accessions of Mediterranean mandarins, whereas their hybrids with other Citrus species showed greater genetic dissimilarity. Twenty accessions yielded very similar patterns, suggesting either that they could be a single clone, or that the technique was not able to detect genomic variation. However, for the other specimens genetic polymorphism can easily be detected by RAPD, although the genetic variation between accessions was quite low. The large number of hybrids and the low polymorphism between accessions support the hypothesis that Mediterranean mandarins are all true hybrid of Common mandarins (Citrus reticulata Blanco).     

Identification of commercial chicory cultivars for hydroponic forcing and their phenetic relationships revealed by random amplified polymorphic DNAs and amplified fragment length polymorphisms

One‐hundred and twenty‐four amplified fragment length polymorphism (AFLP) and 49 random amplified polymorphic DNA (RAPD) markers have been used to distinguish between 20 and 23 commercial chicory cultivars, respectively. These were all Cichorium intybus var. foliosum F₁ hybrids, currently used in hydroponic forcing. Five‐hundred and twenty RAPD primers (OPERON) were tested, of which 156 resulted in reproducible patterns and 26 yielded polymorphisms. Two‐hundred and fifty‐six AFLP primer‐combinations were tested and six combinations were selected for identification purposes. Similarity indices were measured and clustering has been done using pairwise comparison. Both types of marker provide similar conclusions. Two major clusters are formed, representing late and early cultivars. All cultivars were identified using 10 informative RAPD primers or three AFLP primer combinations. A low degree of polymorphism was detected between some early cultivars, suggesting a narrow genetic base in their breeding strategy.