A Strategy to Investigate the Intravarietal Genetic Variability in Vitis vinifera L. for Clones and Biotypes Identification and to Correlate Molecular Profiles with Morphological Traits or Geographic Origins

Grapevine is the most economically important and widely cultivated fruit crop in the world. Molecular markers have been used on Vitis vinifera to distinguish among both varieties and clones. Microsatellites are used to fingerprint varieties and several other techniques, reported in many papers, are used to analyze the differences among clones, but it is not available in the literature as a well defined strategy to screen a large number of Vitis cultivars. In fact, it is often necessary to use different techniques to investigate the genetic variability in different grapevine varieties and a proposed technique is used to study a cultivar, which is often not suitable for either the study of another cultivar or compare the genetic relationship among various cultivars. We describe here a strategy used for the analysis of several grapevine cultivars to describe a universal method to obtain DNA polymorphisms of Vitis vinifera genotypes from the same cultivar by using amplified fragment length polymorphism (AFLP), selective amplification of microsatellite polymorphic loci (SAMPL), microsatellites AFLP (M-AFLP), and ISSR molecular markers. The strategy here adopted permitted both to identify different biotypes (i.e., Primitivo), accessions (i.e., Garnacha tinta), and clones (i.e., Callet, Manto Negro, Moll) among the variability of same variety and to correlate the genetic differences to their geographical origins (i.e., Garnacha tinta; Malvasia nera di Brindisi/Lecce) or morphological traits (i.e., Malvasia of Candia). Here is also described the application of the protocol that allows to highlight the genetic variability accumulated during centuries of cultivations and selections of the same variety in different environments by vine growers.     

Comparison of the effects of different virus infections on performance of three Majorcan grapevine cultivars in field conditions

The effects of viruses on grape production and must quality are not fully understood. In this study, the evaluation of the impact of different virus infections on performance of the main autochthonous grapevine varieties of Mallorca (Callet, Manto Negro and Moll) was pursued. Therefore, a large number of vines were observed in field conditions over 4 years and tested by enzyme-linked immunosorbent assay for viruses listed by the international certification programmes. In each variety, some specific virus infections resulted to be more effective than the others in inducing losses in production. In Callet, yield (Y) reduction was over 20% in plants infected by Grapevine fanleaf virus (GFLV). In Moll, plants subject to more than one infection showed over 40% Y decrease. In Manto Negro, the most surprising results were obtained, because plants showed almost 40% Y reduction because of Grapevine leafroll-associated virus-1 (GLRaV-1) infection. In addition, virus infections were linked to some must quality parameter increase in Manto Negro and Moll, but in the majority of cases it was an indirect effect, because the decrease in production parameters played a predominant role by producing an important concentration effect. However, in Manto Negro, anthocyanin content decrease was directly related to GFLV infection.     

Isolation and characterization of microsatellite markers from Hibiscus rosa-sinensis (Malvaceae) and cross-species amplifications

We report on the development of 10 microsatellite markers in Hibiscus rosa-sinensis (Hrs). Three markers were obtained from sequences available in GenBank and seven were isolated using a two-step ‘primer extension’ procedure, based on the microsatellite-AFLP (M-AFLP) technique. Polymorphism was explored in 21 Hrs genotypes representing the genetic variation within commercial varieties. Inter-specific amplification was assessed on 12 Hibiscus wild species. A total of 45 and 56 alleles (ranging from 1 to 10 for each locus) was amplified respectively from the 21 Hrs varieties and among the full Hibiscus spp. genotype set. Primers and conditions for polymerase chain reaction (PCR) amplification of the detected loci are reported.     

Inter-simple sequence repeat (ISSR) amplification for analysis of microsatellite motif frequency and fingerprinting in rice (Oryza sativa L.)

 Inter-simple sequence repeat (ISSR) amplification was used to analyze microsatellite motif frequency in the rice genome and to evaluate genetic diversity among rice cultivars. A total of 32 primers, containing different simple sequence repeat (SSR) motifs, were tested for amplification on a panel of 59 varieties, representative of the diversity of cultivated rice (Oryza sativa L.). The ISSR analysis provided insights into the organization, frequency and levels of polymorphism of different simple sequence repeats in rice. The more common dinucleotide motifs were more amenable to ISSR analysis than the more infrequent tri-, tetra- and penta-nucleotide motifs. The ISSR results suggested that within the dinucleotide class, the poly(GA) motif was more common than the poly(GT) motif and that the frequency and clustering of specific tri- and tetra-nucleotide simple sequence repeats was variable and motif-specific. Furthermore, trinucleotide ISSR markers were found to be less polymorphic than either dinucleotide or certain tetranucleotide ISSR markers, suggesting which motifs would be better targets for microsatellite marker development. The ISSR amplification pattern was used to group the rice genotypes by cluster analysis. These results were compared to surveys of the same varieties for amplified fragment length polymorphism (AFLP), restriction fragment length polymorphism (RFLP) and isozyme markers. The ISSR fingerprint could be used to differentiate the genotypes belonging to either Japonica or Indica sub species of cultivated rice and to dissect finer levels of diversity within each subspecies. A higher percentage of polymorphic bands was produced with the ISSR technique than the AFLP method, based on a similar PCR reaction. Therefore, ISSR amplification proved to be a valuable method for determining genetic variability among rice varieties and for rapidly identifying cultivars. This efficient genetic fingerprinting technique would be useful for characterizing the large numbers of rice accessions held in national and international germplasm centers. Received: 25 May 1998 / Accepted: 17 September 1998     

Novel polymorphic microsatellite markers in section Caulorrhizae (Arachis,Fabaceae)

This work reports the characterization of 11 polymorphic microsatellite loci in section Caulorrhizae. The primer pairs were designed from Arachis pintoi and showed full transferability to Arachis repens species. These new markers were used to evaluate the genetic diversity in germplasm (accessions and cultivars) of section Caulorrhizae. This new set of markers detected greater gene diversity than morphological and molecular markers such as AFLP (amplified fragment length polymorphism) and RAPD (rapid analysis of polymorphic DNA) previously used in this germplasm.     

Use of Three Different Marker Systems to Estimate Genetic Diversity of Indian Elite Rice Varieties

Genetic diversity among 42 Indian elite rice varieties, which is important for selection of parents for conventional breeding and hybrid program, was evaluated using three different types of DNA markers and parentage analysis. Random amplified polymorphic DNA (RAPD), inter-simple sequence repeat (ISSR) and sequence tagged microsatellite site (STMS) markers resulted in mean heterozygosity values of 0.429, 0.675 and 0.882 over all loci, respectively, and marker index values of 2.21, 4.05 and 5.49, respectively. The three molecular marker systems together provide wider genome coverage and, therefore, would be a better indicator of the genetic relationships among the 42 elite rice cultivars than those revealed using individual molecular markers. A total of 153 bands (91%) were polymorphic out of 168 bands amplified, considering all the markers together. The average genetic similarity coefficient across all the 861 cultivar pairs was 0.70 while the average coefficient of parentage was 0.10. Cluster analysis revealed that there was a very poor correlation (correlation coefficient <0.1) between dendrograms generated using coefficients of parentage and molecular marker generated genetic similarities, which can be attributed to selection pressure, genetic drift, sampling of loci and unknown relationships among supposedly unrelated ancestors. This revised version was published online in July 2006 with corrections to the Cover Date.     

A genetic linkage map of the model legume Lotus japonicus and strategies for fast mapping of new loci

A genetic map for the model legume Lotus japonicus has been developed. The F(2) mapping population was established from an interspecific cross between L. japonicus and L. filicaulis. A high level of DNA polymorphism between these parents was the source of markers for linkage analysis and the map is based on a framework of amplified fragment length polymorphism (AFLP) markers. Additional markers were generated by restriction fragment length polymorphism (RFLP) and sequence-specific PCR. A total of 524 AFLP markers, 3 RAPD markers, 39 gene-specific markers, 33 microsatellite markers, and six recessive symbiotic mutant loci were mapped. This genetic map consists of six linkage groups corresponding to the six chromosomes in L. japonicus. Fluorescent in situ hybridization (FISH) with selected markers aligned the linkage groups to chromosomes as described in the accompanying article by Pedrosa et al. 2002(this issue). The length of the linkage map is 367 cM and the average marker distance is 0.6 cM. Distorted segregation of markers was found in certain sections of the map and linkage group I could be assembled only by combining colormapping and cytogenetics (FISH). A fast method to position genetic loci employing three AFLP primer combinations yielding 89 markers was developed and evaluated by mapping three symbiotic loci, Ljsym1, Ljsym5, and Ljhar1-3.     

Isolation and characterization of polymorphic microsatellite loci in the field vole,Microtus agrestis,and their cross‐utility in the common vole,Microtus arvalis

We developed nine polymorphic microsatellite loci for the field vole, Microtus agrestis. The number of alleles ranged from five to 15 and observed heterozygosities ranged from 0.40 to 1.00. We also tested the microsatellite loci for amplification and polymorphism in the congeneric species Microtus arvalis. Five of the nine loci were successfully analysed in this species. The microsatellite markers will be employed in studies of reproductive success and fine‐scale spatial genetic structure.     

Characterization of microsatellite loci in silver carp (Hypophthalmichthys molitrix), and cross‐amplification in other cyprinid species

Captive populations of silver carp (Hypophthalmichthys molitrix), a major aquaculture species in Asia, would undoubtedly benefit from genetic monitoring and improvement programs. We report the isolation and preliminary characterization of 16 microsatellite loci derived from both conventional and microsatellite‐enriched libraries. Inheritance studies confirmed the allelic nature of observed polymorphisms at all loci, while identifying null alleles at two loci. These loci, having varying degrees of polymorphism, should provide useful markers for applied genetic studies. A high degree of cross‐amplification among 10 other cyprinid species suggests that these loci may have more widespread utility.     

Characterization of microsatellite markers in cassava based on microsatellite-AFLP technique

We developed molecular markers for cassava based on the microsatellite-amplified fragment length polymorphism (M-AFLP) technique. Twenty primer pairs were developed and used for the analysis of 48 samples of Manihot species, consisting of M. esculenta (33), M. esculenta ssp flabellifolia (3), M. chlorosticta (3), M. carthaginensis (3), M. filamentosa (3), and M. tristis (3). Nine microsatellite loci that were polymorphic among these Manihot species were identified, giving 32 polymorphic alleles and from two to seven alleles per locus. Unbiased and direct count heterozygosity varied from 0.0233 to 0.7924 and 0.0000 to 0.7083, respectively. There was significant deviation (P < 0.05) from Hardy-Weinberg equilibrium at five loci. Genotypic data from the Manihot species were subjected to genetic diversity analysis. We found that M. chlorosticta and M. esculenta ssp flabellifolia were the closest populations, while M. filamentosa and M. esculenta ssp flabellifolia were the most divergent. Considering within M. esculenta, the samples from Nigeria and Fiji were the most closely related, while those from Venezuela and of unknown origin were the most divergent. We conclude that the M-AFLP technique is an effective method for generating microsatellite markers that are useful for genetic diversity analysis in Manihot species.     

Molecular mapping in tropical maize (Zea mays L.) using microsatellite markers. 1. Map construction and localization of loci showing distorted segregation

Microsatellites have become the most important class of markers for mapping procedures. Primarily based on restriction fragment length polymorphism (RFLP) markers, several molecular genetic maps of maize have been developed, mainly using temperate inbred maize lines. To characterize the level of polymorphism of microsatellite loci and construct a genetic map in tropical maize, two elite inbred lines, L-08-05F and L-14-4B, were crossed to produce 400 F(2) individuals that were used as a mapping population. A survey of 859 primer pair sequences of microsatellites was used. The polymorphism screens of each microsatellite and genotype assignment were performed using high-resolution agarose gels. About 54 % of the primer sets gave clearly scorable amplification products, 13 % did not amplify and 33 % could not be scored on agarose gels. A total of 213 polymorphic markers were identified and used to genotype the mapping population. Among the polymorphic markers, 40 showed loci deviating from expected Mendelian ratios and clusters of deviating markers were located in three chromosome regions. Non-Mendelian scoring was present in 19 markers. The final genetic map with 117 markers spanned 1634 cM in length with an average interval of 14 cM between adjacent markers.     

Isolation and identification of eight microsatellite loci in the Cherokee darter (Etheostoma scotti) and their variability in other members of the genera Etheostoma, Ammocrypta, and Percina

The Cherokee darter Etheostoma scotti is a federally threatened fish endemic to the Etowah River system of northwest Georgia. In order to analyse the population structure and genetic diversity of this fish, eight tetranucleotide microsatellite genetic markers were developed. The marker set was applied to 13 additional darter species to test cross-species amplification and polymorphism. Successful amplification was obtained for all eight loci in each of the 13 other species of darters, with between seven and eight polymorphic loci per species.     

Turkey microsatellite DNA loci amplified by chicken-specific primers

Forty-eight primer-pairs complementary to unique DNA sequences flanking chicken (genus Gallus ) genomic (TG)_n microsatellite repeats were previously designed. These primer-pairs were used in the polymerase chain reaction to amplify turkey (genus Meleagris ) genomic DNA loci. Results indicated that the majority (92%) of these primer-pairs generated amplification products in turkey genomic DNA. Hybridization using end-labelled (TG)₈ as a probe showed that, out of 41 primer-pairs tested, only 14 generated an amplification product that also contained a detectable (TG)_n microsatellite repeat when turkey DNA was the template. Among 18 primerpairs tested for polymorphism, using three commercial turkey lines, five were found to exhibit length polymorphism, three of which did not contain a detectable TG repeat. Therefore, a significant portion of chicken microsatellite markers can be useful for genomic mapping and linkage analysis in the turkey, reducing the costs involved in producing turkey-specific microsatellite markers.     

Genetic Variation and Population Structure of Oriental Migratory Locust, Locusta migratoria manilensis, in China by Allozyme, SSRP-PCR, and AFLP Markers

Allozyme analysis, microsatellite primer PCR (SSRP-PCR), and amplified fragment length polymorphism (AFLP) techniques were used to assess genetic diversity and population structure of the Chinese oriental migratory locust, Locusta migratoria manilensis. A total of 299 PCR markers (67 SSRPs and 232 AFLPs) were detected in eight populations, of which 98.7% were polymorphic markers. The proportion of polymorphic loci (95.5–98.8%) by SSRP+AFLP markers indicated no significant differences between populations, and all populations exhibited a similar level of variability; results of the allozyme analysis demonstrated that 19 loci gave rise to a lower level of polymorphism (55.6–66.7%). The genetic distances between the populations were relatively low. Shannon’s index and Nei’s gene diversity showed low differentiation among the populations. Allozyme analysis, however, reflected greater similarity and smaller differentiation between the populations than those shown by SSRP and AFLP markers. Neighbor-joining dendrograms derived from both the allozyme and SSRP+AFLP markers showed that the genetic distances among Chinese oriental migratory locust populations were not greatly influenced by geographic distance and breeding habitats.     

Development and characterization of 43 microsatellite markers for the critically endangered primrose Primula reinii using MiSeq sequencing

Primula reinii (Primulaceae), a perennial herb belonging to the Primula section Reinii, occurs on wet, shaded rocky cliffs in the mountains of Japan. This threatened species comprises four varieties; these plants are very localized and rare in the wild. In this study, 43 microsatellite markers were developed using MiSeq sequencing to facilitate conservation genetics of these critically endangered primroses. We developed novel microsatellite markers for three varieties of P. reinii, and tested its polymorphism and genetic diversity using natural populations. These novel markers displayed relatively high polymorphism; the number of alleles and expected heterozygosities ranged from 2 to 6 (mean=3.2) and 0.13 to 0.82 (mean=0.45), respectively. All loci were in HardyeWeinberg equilibrium. These microsatellite markers will be powerful tools to assess P. reinii genetic diversity and develop effective conservation and management strategies.     

Inheritance and usefulness of AFLP markers in channel catfish (Ictalurus punctatus ), blue catfish ( I. furcatus) , and their F1, F2, and backcross hybrids

Eight primer combinations were used to investigate the application of amplified fragment length polymorphism (AFLP) markers in catfish for genetic analysis. Intraspecific polymorphism was low among channel catfish or blue catfish strains. Interspecific AFLP polymorphism was high between the channel catfish and blue catfish. Each primer combination generated from 70 to more than 200 bands, of which 38.6–75.7% were polymorphic between channel catfish and blue catfish. On average, more than 20 polymorphic bands per primer combination were produced as quality markers suitable for genetic analysis. All AFLP markers were transmitted into channel catfish × blue catfish F1 hybrids, except rare markers that were heterozygous in the parents and therefore were segregating in F1 hybrids. The two reciprocal channel catfish × blue catfish F1 hybrids (channel catfish female × blue catfish male; blue catfish female × channel catfish male) produced identical AFLP profiles. The AFLP markers were inherited and segregated in expected Mendelian ratios. At two loci, E8-b9 and E8-b2, markers were found at significantly lower frequencies than expected with F2 and backcross hybrids which had been selected for increased growth rates. The reproducibility of AFLP was excellent. These characteristics of the catfish AFLP markers make them highly useful for genetic analysis of catfish, especially for construction of genetic linkage and quantitative trait loci maps, and for marker-assisted selection. Received: 10 September 1997 / Accepted: 10 December 1997     

Clones Identification and Genetic Characterization of Garnacha Grapevine by Means of Different PCR-Derived Marker Systems

This study uses PCR-derived marker systems to investigate the extent and distribution of genetic variability of 53 Garnacha accessions coming from Italy, France and Spain. The samples studied include 28 Italian accessions (named Tocai rosso in Vicenza area; Alicante in Sicily and Elba island; Gamay perugino in Perugia province; Cannonau in Sardinia), 19 Spanish accessions of different types (named Garnacha tinta, Garnacha blanca, Garnacha peluda, Garnacha roja, Garnacha erguida, Garnacha roya) and 6 French accessions (named Grenache and Grenache noir). In order to verify the varietal identity of the samples, analyses based on 14 simple sequence repeat (SSR) loci were performed. The presence of an additional allele at ISV3 locus (151 bp) was found in four Tocai rosso accessions and in a Sardinian Cannonau clone, that are, incidentally, chimeras. In addition to microsatellite analysis, intravarietal variability study was performed using AFLP, SAMPL and M-AFLP molecular markers. AFLPs could discriminate among several Garnacha samples; SAMPLs allowed distinguishing few genotypes on the basis of their geographic origin, whereas M-AFLPs revealed plant-specific markers, differentiating all accessions. Italian samples showed the greatest variability among themselves, especially on the basis of their different provenance, while Spanish samples were the most similar, in spite of their morphological diversity.     

AFLP and SAMPL markers for characterization of genetic diversity in Terminalia arjuna: a backbone tree of Tasar silk industry

Overexploitation of Terminalia arjuna by the Tasar silk, pharmaceutical, leather, and other industries has led to gradual depletion of its germplasm resource and genetic diversity. Information on its existing gene pool is currently lacking. We report the first evaluation of genetic variation in this tree, using amplified fragment length polymorphism (AFLP) and selective amplification of microsatellite polymorphic loci (SAMPL) markers. The study consisted of (1) characterization of genetic diversity at interzonal and intrazonal levels, (2) comparison of diversity within and among populations, and (3) comparison of efficiency of the two marker systems. The germplasm used in the present study had a wide genetic base, which is evident through the high level of genetic diversity observed, as expected for a widespread, long-lived tropical species. The clustering obtained after interzonal genetic diversity analysis of T.?arjuna is not region specific, a reflection of the lack of any defined population structure in this genus in India. The high value of r obtained through Mantel??s test for evaluation of cophenetic correlation in cluster analysis indicated the high fitness of the accessions to a group. The region-specific bands obtained during the present investigation can be used as diagnostic markers for authentication and to identify raw materials for herbal drug preparations.     

Comparative analysis of microsatellite loci in chicken and turkey.

Cross-species amplification of 520 chicken microsatellite markers was tested by polymerase chain reaction with genomic DNA of the turkey (Meleagris gallopavo). Each primer pair was tested at six different combinations of annealing temperature and MgCl2 concentration. A total of 280 (54%) of the primer pairs produced amplification products. The majority of these products were similar, if not identical in size to those expected based on the fragment sizes of the corresponding chicken loci. Structure of the dinucleotide repeat and flanking sequences was examined for 13 turkey fragments (amplified with chicken primers) and 5 chicken fragments (amplified with turkey primers). Sequence analysis found a wide array of mutations between species in addition to differences in repeat length. To estimate the usefulness of the amplified loci for genetic mapping in the turkey, allelic polymorphism was determined for 57 of the 280 amplified loci. A total of 20 of 57 markers (35%) were polymorphic with an average of 1.4 alleles per locus. The results of this study suggest that approximately 20% of the chicken microsatellite markers will be useful for mapping the turkey genome.     

A Preliminary Genetic Linkage Map of the Pacific Abalone Haliotis discus hannai Ino

Preliminary genetic linkage maps were constructed for the Pacific abalone (Haliotis discus hannai Ino) using amplified fragment length polymorphism (AFLP), randomly amplified polymorphic DNA (RAPD), and microsatellite markers segregating in a F₁ family. Nine microsatellite loci, 41 RAPD, and 2688 AFLP markers were genotyped in the parents and 86 progeny of the mapping family. Among the 2738 markers, 384 (including 365 AFLP markers, 10 RAPD markers, and 9 microsatellite loci) were polymorphic and segregated in one or both parents: 241 in the female and 146 in the male. The majority of these markers, 232 in the female and 134 in the male, segregated according to the expected 1:1 Mendelian ratio (α = 0.05). Two genetic linkage maps were constructed using markers segregating in the female or the male parent. The female framework map consisted of 119 markers in 22 linkage groups, covering 1773.6 cM with an average intermarker space of 18.3 cM. The male framework map contained 94 markers in 19 linkage groups, spanning 1365.9 cM with an average intermarker space of 18.2 cM. The sex determination locus was mapped to the male map but not to the female map, suggesting a XY-male determination mechanism. Distorted markers showing excess of homozygotes were mapped in clusters, probably because of their linkage to a gene that is incompatible between two parental populations.