Development and mapping of Oryza glumaepatula-derived microsatellite markers in the interspecific cross Oryza glumaepatula x O. sativa

Wild germplasm of domesticated crops is a source of genetic variation little utilized in breeding programs. Interspecific crosses can potentially uncover novel gene combinations that can be important for quantitative trait analysis. The combined use of wide crosses and genetic maps of chromosomal regions associated with quantitative traits can be used to broaden the genetic basis of rice breeding programs. Oryza glumaepatula is a diploid (AA genome) wild rice species native from South and Central America. A genetic map was constructed with 162 PCR-based markers (155 microsatellite and 7 STS markers) using a backcross population derived from the cross O. glumaepatula, accession RS-16 from the Brazilian Amazon Region x O. sativa BG-90-2, an elite rice inbred line. The map included 47 new SSR markers developed from an O. glumaepatula genomic library enriched for AG/TC sequences. All SSR markers were able to amplify the O. sativa genome, indicating a high degree of SSR flanking region conservation between O. glumaepatula and O. sativa species. The map covered 1500.4 cM, with an average of one marker every 10 cM. Despite some chromosomes being more densely mapped, the overall coverage was similar to other maps developed for rice. The advantage to construct a SSR-based map is to permit the combination of the speed of the PCR reaction, and the codominant nature of the SSR marker, facilitating the QTL analysis and marker assisted selection for rice breeding programs.     

Analysis of genetic variability of South American wild rice populations (Oryza glumaepatula) with isozymes and RAPD markers

The knowledge of population structure and genetic diversity of wild relatives of rice is needed to investigate their evolutionary history and potential use in breeding programs. Very little is known about the wild rice species ( Oryza spp.), particularly those that are native to South America. A study using isozyme and RAPD markers was conducted to estimate the level of genetic diversity of four South American wild rice populations ( Oryza glumaepatula ) recently collected in the Amazon forest and western Brazil rivers. F -statistics and genetic diversity parameters calculated from isozyme and RAPD markers indicated high values for inbreeding coefficients and differentiation among the four populations. In agreement with this, a pattern of greater variation between than within populations was observed with both types of markers. These findings were corroborated by an AMOVA analysis, which indicated that a large portion of the total genetic variation was attributed to regional divergence. The partition of the AMOVA analysis among populations showed that most of the genetic diversity was due to differences among populations. This distribution pattern of genetic variation of O. glumaepatula populations is in agreement with the expectation for an autogamous species and provides important baseline data for conservation and collection strategies for this species.     

Development of microsatellite markers for identifying Brazilian Coffea arabica varieties

Microsatellite markers, also known as SSRs (Simple Sequence Repeats), have proved to be excellent tools for identifying variety and determining genetic relationships. A set of 127 SSR markers was used to analyze genetic similarity in twenty five Coffea arabica varieties. These were composed of nineteen commercially important Brazilians and six interspecific hybrids of Coffea arabica, Coffea canephora and Coffealiberica. The set used comprised 52 newly developed SSR markers derived from microsatellite enriched libraries, 56 designed on the basis of coffee SSR sequences available from public databases, 6 already published, and 13 universal chloroplast microsatellite markers. Only 22 were polymorphic, these detecting 2-7 alleles per marker, an average of 2.5. Based on the banding patterns generated by polymorphic SSR loci, the set of twenty-five coffee varieties were clustered into two main groups, one composed of only Brazilian varieties, and the other of interspecific hybrids, with a few Brazilians. Color mutants could not be separated. Clustering was in accordance with material genealogy thereby revealing high similarity.     

Transferability of microsatellite and sequence tagged site markers in Oryza species

The genus Oryza comprises 22 species which are potentially useful as a source of genetic variability that can be introgressed into the worldwide cultivated rice, Oryza sativa. Molecular markers are useful tools for monitoring gene introgressions and for detecting polymorphism among species. In this study, cross-amplification was estimated among 28 accessions of 16 Oryza species, representing the genomes AA, BB, CC, BBCC and CCDD, using 59 microsatellite (OG, OS and RM series) and 15 STS (Sequence Tagged Sites) markers. All markers amplified at least one Oryza species, indicating different levels of transferability across species. Markers based on microsatellite sequences amplified 37 % of the accessions, with an average of 6.58 alleles per locus and an average polymorphism information content (PIC) of 70 %. For STS markers, the amplification level was 53.3 %, and the average number of alleles and PIC values were 1.6 and 10 %, respectively. These Results showed that although the STS markers detected a reduced level of genetic diversity, the transferability was higher, indicating that they can be used for genetic analysis when evaluating less genetically related species of Oryza. Among the microsatellite markers, an analysis of species with an AA genome showed that the OG markers produced the highest level of polymorphic loci (54.6 %), followed by RM markers (48 %). Highly polymorphic and transferable molecular markers in Oryza can be useful for exploiting the genetic resources of this genus, for detecting allelic variants in loci associated with important agronomic traits, and for monitoring alleles introgressed from wild relatives to cultivated rice.     

Interspecific microsatellite markers for the study of pinniped populations

Microsatellites have rapidly become the marker of choice for a wide variety of population genetic studies. Here we describe 20 pinniped microsatellite markers which have been tested across 18 pinniped species. The majority of these markers have broad utility in all pinnipeds and provide a strong base for detailed population genetic studies in the Pinnipedia.     

Genetic variability in Brazilian wheat cultivars assessed by microsatellite markers

Wheat (Triticum aestivum) is one of the most important food staples in the south of Brazil. Understanding genetic variability among the assortment of Brazilian wheat is important for breeding. The aim of this work was to molecularly characterize the thirty-six wheat cultivars recommended for various regions of Brazil, and to assess mutual genetic distances, through the use of microsatellite markers. Twenty three polymorphic microsatellite markers (PMM) delineated all 36 of the samples, revealing a total of 74 simple sequence repeat (SSR) alleles, i.e. an average of 3.2 alleles per locus. Polymorphic information content (PIC value) calculated to assess the informativeness of each marker ranged from 0.20 to 0.79, with a mean of 0.49. Genetic distances among the 36 cultivars ranged from 0.10 (between cultivars Ocepar 18 and BRS 207) to 0.88 (between cultivars CD 101 and Fudancep 46), the mean distance being 0.48. Twelve groups were obtained by using the unweighted pair-group method with arithmetic means analysis (UPGMA), and thirteen through the Tocher method. Both methods produced similar clusters, with one to thirteen cultivars per group. The results indicate that these tools may be used to protect intellectual property and for breeding and selection programs.     

Genetic variability in local Brazilian horse lines using microsatellite markers

Genetic variability at 11 microsatellite markers was analyzed in five naturalized/local Brazilian horse breeds or genetic groups. Blood samples were collected from 328 animals of the breeds Campeira (Santa Catarina State), Lavradeira (Roraima State), Pantaneira (Pantanal Mato-Grossense), Mangalarga Marchador (Minas Gerais State), as well as the genetic group Baixadeiro (Maranh?o State), and the exotic breeds English Thoroughbred and Arab. We found significant genetic variability within evaluated microsatellite loci, with observed heterozygosis varying between 0.426 and 0.768 and polymorphism information content values of 0.751 to 0.914. All breeds showed high inbreeding coefficients and were not in Hardy-Weinberg equilibrium. The smallest genetic distance was seen between the Pantaneira and Arab breeds. The principal component analyzes and Bayesian approach demonstrated that the exotic breeds have had a significant influence on the genetic formation of the local breeds, with introgression of English Throroughbred in Pantaneira and Lavradeira, as well as genetic proximity between the Arab, Pantaneira and Mangalarga Marchador populations. This study shows the need to conserve traits acquired by naturalized horse breeds over centuries of natural selection in Brazil due to the genetic uniqueness of each group, suggesting a reduced gene flow between them. These results reinforce the need to include these herds in animal genetic resource conservation programs to maximize the genetic variability and conserve useful allele combinations.     

Genetic variability of dromedary camel populations based on microsatellite markers

Understanding existing levels of genetic variability of camel populations is capital for conservation activities. This study aims to provide information on the genetic diversity of four dromedary populations, including Guerzni, Harcha, Khouari and Marmouri. Blood samples from 227 individuals belonging to the aforementioned populations were obtained and genotyped by 16 microsatellite markers. A total of 215 alleles were observed, with the mean number of alleles per locus being 13.4 ± 6.26. All loci were polymorphic in the studied populations. The average expected heterozygosity varied from a maximum of 0.748 ± 0.122 in Guerzni population to a minimum of 0.702 ± 0.128 in Harcha population; Guerzni population showed the highest value of observed heterozygosity (0.699 ± 0.088), whereas Harcha population the lowest (0.646 ± 0.130). Mean estimates of F-statistics obtained over loci were F_IS = 0.0726, F_IT = 0.0876 and F_ST = 0.0162. The lowest genetic distance was obtained between Guerzni and Khouari (0.023), and the highest genetic distance between Harcha and Marmouri (0.251). The neighbour-joining phylogenetic tree showed two groups of populations indicating a cluster of Guerzni, Khouari and Marmouri, and a clear isolation of Harcha. The genetic distances, the factorial correspondence analysis, the analysis of genetic structure and the phylogenetic tree between populations revealed significant differences between Harcha and other populations, and a high similarity between Guerzni, Khouari and Marmouri. It is concluded from this study that the camel genetic resources studied are well diversified. However, the herd management, especially the random selection of breeding animals, can increase the level of genetic mixing between different populations, mainly among Guerzni, Khouari and Marmouri, that live in the same habitat and grazing area.     

Analysis of genetic variation in 28 dog breed populations with 100 microsatellite markers

Dog breeds were created by man choosing for select phenotypic traits such as size, shape, coat color, conformation, and behavior. Rigorous phenotypic selection likely resulted in a loss of genetic information. The present study extends previous dog population observations by assessing the genotypic variation within and across 28 breeds representing the seven recognized breed groups of the American Kennel Club (AKC). One hundred autosomal microsatellite markers distributed across the canine genome were used to examine variation within breeds. Resulting breed-specific allele frequencies were then used in an attempt to elucidate phylogeny and genetic distances between breeds. While the set of autosomal microsatellites was useful in describing genetic variation within breeds, establishing the genetic relatedness between breeds was less conclusive. A more accurate determination of breed phylogeny will likely require the use of single-nucleotide polymorphisms (SNPs).     

Genetic diversity of Brazilian and introduced olive germplasms based on microsatellite markers

Olive trees have been grown since the beginning of civilization, and the consumption of olives and olive products is increasing worldwide, due to their health benefits and organoleptic qualities. To meet the growing market for olives, commercial cultivation of this species is expanding from traditional areas to new regions. Although the Brazilian olive industry has just begun to be established, breeding programs are already developing cultivars that are more adapted to local conditions. We used 12 microsatellite markers to evaluate 60 olive accessions, including several cultivars that were developed in Brazil. The analyses identified 72 distinct alleles; the largest number of alleles per locus were at the markers GAPU 101 and GAPU 71B, which contained 10 and 9 alleles, respectively. The largest allelic diversity and polymorphic information contents were also found at the GAPU 101 and GAPU 71B markers, with values of 0.8399/0.8203 and 0.8117/0.7863, respectively. Additionally, the 12 microsatellite markers generated a cumulative identity probability of 1.51 x 10(-10), indicating a high level of accuracy of accession identification. The set of markers that we used allowed the identification of 52 of the 60 olive genotypes, in addition to the recognition of several varietal synonyms. The components of a two-dimensional principal coordinate analysis explained 48.6% of the total genetic variation. The results obtained from the microsatellite markers showed a substantial degree of genetic diversity in the olive tree accessions used in Brazil.     

水稻微卫星标记的发展和应用

微卫星又称简单序列重复。它是由几个核苷酸（一般２～４个）为重复单位组成的串联重复序列。相同座位上的重复序列由于重复次数的不同而造成序列长度的多态性。微卫星标记是一种共显性标记,具有等位基因丰富、检测技术简单等优点。微卫星标记在基因组作图、品种鉴定、种质保存、分子标记辅助选择等方面有着广泛的应用。目前水稻中已发展了３００多个微卫星标记。     

微卫星标记分析罗非鱼群体的遗传潜力

利用25个微卫星标记,对奥利亚罗非鱼2个群体["夏奥1号"(ZA)、广西群体(GA)]和尼罗罗非鱼4个群体[埃及品系(ZN)、88品系(XN)、广西群体(GN)、美国品系(MN)]进行检测。共检测到7 775个扩增片段,长度在100~400 bp;等位基因数3~8个不等,共计143个等位基因;平均每个基因座扩增得到5.72个等位基因。各群体平均观测杂合度(H o)在0.7253~0.8160之间,平均期望杂合度(He)在0.5146~0.6834之间,平均多态信息含量(PIC)在0.4212~0.6105之间,平均有效等位基因数(A e)在2.20~3.23之间。ZA与GA遗传相似系数最高(0.9130),ZA与ZN遗传相似系数最低(0.4352)。总的说来,4个尼罗罗非鱼群体的遗传潜力较高,2个奥利亚罗非鱼群体的遗传潜力适中。     

Stock composition in North Atlantic populations of whiting using microsatellite markers

A size-selected library constructed from DNA of the whiting Merlangius merlangus was screened. From about 3200 recombinant clones, 43 microsatellite loci were detected. Thirteen were sequenced in full. Primers were designed from the sequence of the flanking regions for six loci and used to test the allelic variability at these loci using the polymerase chain reaction (PCR). In addition, five primer pairs developed for the stickleback and another seven for cod were tested. Only six primer pairs revealed at least three alleles per locus. The three useful loci Gmo2, Mmer- UEAW01 and Mmer- UEAW02, had 14–23 alleles per locus in 370 samples. Estimates of genetic structure (φ) were not statistically significant. However, estimates of genetic differentiation ( F _st) were significantly different from zero. Heterogeneity χ²-analysis of allele frequencies among populations suggested relatively low levels of differentiation among samples. Significantly different allele frequency distributions were found for Borgensfjord and northern and southern North Sea samples for at least one locus, and between the latter samples for Mmer -UEAW02 and Gmo2 . There were significant excesses of homozygotes in all samples, over expectation for randomly mating populations in Hardy-Weinberg equilibrium. The estimated frequencies of null alleles were 14.3%, for Mmer -UEAW01, 10.2% for Mmer- UEAW02 and 11.6% for Gmo2 . This result calls for a careful interpretation of the significance of these microsatellite data.     

Ultrastructure of Oryza glumaepatula, a wild rice species endemic of tropical America

Orv'za gluniaepatula is a perennial wild rice species, endemic to tropical America, previously known as the Latin American race of Orrza rufipogon. In Costa Rica, it is found in the northern region of the country, mainly in the wetland of the Medio Queso River, Los Chiles, Alajuela. It is diploid, of AA type genome and because of its genetic relatedness to cultivated rice it is included in the O. saliva complex. We describe the ultrastructure of leaf blade, spikelet, ligule and auricles. Special emphasis is given to those traits of major taxonomic value for O. glumaepatula and to those characters that distinguish this species from O. rufipogon and O. sativa. O. glumaepatula has a leaf blade covered with tombstone-shaped, oblong and spheroid epicuticular wax papillae. It has diamond-shaped stomata surrounded by spherical papillae, rows of zipper-like silica cells, bulky prickle trichomes of ca. 40 microm in length and small hirsute trichomes of ca. 32 tpm in length. The central vein is covered with large, globular papillae of ca. 146 microm in length, a characteristic that distinguishes this species from O. rufipogon and O. sativa. The border of the leaf blade exhibits a row of even-sized bulky prickle trichomes of ca. 42.5 microm in length. Auricles have attenuated trichomes of ca. 5.5 mm in length on the edges and small bicellular trichomes of 120 microm in length on the surface. The ligule has a large number of short attenuated trichomes on its surface of 100 microm in length. These latter two traits have important taxonomic value since they were found in O. glumaepatula but not found in O. sativa or in O. rufipogon. The spikelet has the typical morphology of the Oryza genus. Fertile lemmas have abundant spines, a trait shared with O. rufipogon but not with O. sativa. The sterile lemmas are wing-shaped with serrated borders, a characteristic that distinguishes this species from O. rufipogon and O. sativa. All the ultrastructure characters observed in O. glumaepatula from Costa Rica are also common to the specimens from Brazil.     

Sequence-tagged microsatellite sites as markers in chicken reference and resource populations

Two chicken genomic libraries were screened for the presence of poly(TG/AC) microsatellite tracts. The number of positive clones was low, confirming the low frequency of such micro-satellites in the chicken genome relative to mammalian genomes. Polymorphism of 29 microsatellite tracts, comprising 11 from the library screening and 18 obtained from GenBank, was examined in the East Lansing and Compton reference families, in a resource population formed by a cross between a single White Rock broiler and inbred Leghorn females, and in a panel of birds from five layer stocks. Twenty microsatellites, primarily of the poly(TG/AC) type, were polymorphic in at least one of the populations. Thirteen of the microsatellites were polymorphic in the East Lansing reference family and 13 were also polymorphic in the resource population, confirming that the genetic distance between White Rock and White Leghorn is about as great as between Jungle fowl and White Leghorn. Only six microsatellites were polymorphic in the Compton reference family, formed by a cross between two White Leghorn strains. Twelve of the microsatellites were mapped in the East Lansing and/or Compton reference families. These were well dispersed among the various linkage groups and did not show any indications of terminal clustering.     

Population structures of Brazilian tall coconut (Cocos nucifera L.) by microsatellite markers

Coconut palms of the Tall group were introduced to Brazil from the Cape Verde Islands in 1553. The present study sought to evaluate the genetic diversity among and within Brazilian Tall coconut populations. Samples were collected of 195 trees from 10 populations. Genetic diversity was accessed by investigating 13 simple sequence repeats (SSR) loci. This provided a total of 68 alleles, ranging from 2 to 13 alleles per locus, with an average of 5.23. The mean values of gene diversity (H_e ) and observed heterozygosity (H_o ) were 0.459 and 0.443, respectively. The genetic differentiation among populations was estimated at θ^P=0.1600and the estimated apparent outcrossing rate was t_a = 0.92. Estimates of genetic distances between the populations varied from 0.034 to 0.390. Genetic distance and the corresponding clustering analysis indicate the formation of two groups. The first consists of the Baía Formosa, Georgino Avelino, and São José do Mipibu populations and the second consists of the Japoatã, Pacatuba, and Praia do Forte populations. The correlation matrix between genetic and geographic distances was positive and significant at a 1% probability. Taken together, our results suggest a spatial structuring of the genetic variability among the populations. Geographically closer populations exhibited greater similarities.     

Efficiency of semi-automated fluorescent multiplex PCRs with 11 microsatellite markers for genetic studies of deer populations

Thirty bovine and eight ovine microsatellite primer pairs were tested on four tropical deer species: Eld's and Swamp deer (highly threatened) and Rusa and Vietnamese Sika deer (economically important). Thirty markers gave an amplified product in all four species (78.9%). The number of polymorphic microsatellite markers varied among the species from 14 in Eld's deer (47%) to 20 in Swamp deer (67%). Among them, 11 microsatellite loci were multiplexed in three polymerase chain reactions (PCRs) and labelled with three different fluorochromes that can be loaded in one gel-lane. To test the efficiency of the multiplex, primary genetic studies (mean number of alleles, expected heterozygosities and Fis values) were carried out on four deer populations. Parentage exclusion probability and probability of identity were computed and discussed on a Swamp deer population. These multiplexes PCRs were also tested on several other deer species and subspecies. The aim of this study is to establish a tool useful for genetic studies of population structure and diversity in four tropical deer species which with few modifications can be applied to other species of the genus Cervus.