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     

Diversity of microsatellites derived from genomic libraries and GenBank sequences in rice (Oryza sativa L.)

The growing number of rice microsatellite markers warrants a comprehensive comparison of allelic variability between the markers developed using different methods, with various sequence repeat motifs, and from coding and non-coding portions of the genome. We have performed such a comparison over a set of 323 microsatellite markers; 194 were derived from genomic library screening and 129 were derived from the analysis of rice-expressed sequence tags (ESTs) available in public DNA databases. We have evaluated the frequency of polymorphism between parental pairs of six inter- subspecific crosses and one inter-specific cross widely used for mapping in rice. Microsatellites derived from genomic libraries detected a higher level of polymorphism than those derived from ESTs contained in the GenBank database (83.8% versus 54.0%). Similarly, the other measures of genetic variability [the number of alleles per locus, polymorphism information content (PIC), and allele size ranges] were all higher in genomic library-derived microsatellites than in their EST-database counterparts. The highest overall degree of genetic diversity was seen in GA-containing microsatellites of genomic library origin, while the most conserved markers contained CCG- or CAG-trinucleotide motifs and were developed from GenBank sequences. Preferential location of specific motifs in coding versus non-coding regions of known genes was related to observed levels of microsatellite diversity. A strong positive correlation was observed between the maximum length of a microsatellite motif and the standard deviation of the molecular-weight of amplified fragments. The reliability of molecular weight standard deviation (SDmw) as an indicator of genetic variability of microsatellite loci is discussed. Received: 5 May 1999 / Accepted: 16 August 1999     

Development and mapping of 2240 new SSR markers for rice (Oryza sativa L.).

A total of 2414 new di-, tri- and tetra-nucleotide non-redundant SSR primer pairs, representing 2240 unique marker loci, have been developed and experimentally validated for rice (Oryza sativa L.). Duplicate primer pairs are reported for 7% (174) of the loci. The majority (92%) of primer pairs were developed in regions flanking perfect repeats > or = 24 bp in length. Using electronic PCR (e-PCR) to align primer pairs against 3284 publicly sequenced rice BAC and PAC clones (representing about 83% of the total rice genome), 65% of the SSR markers hit a BAC or PAC clone containing at least one genetically mapped marker and could be mapped by proxy. Additional information based on genetic mapping and "nearest marker" information provided the basis for locating a total of 1825 (81%) of the newly designed markers along rice chromosomes. Fifty-six SSR markers (2.8%) hit BAC clones on two or more different chromosomes and appeared to be multiple copy. The largest proportion of SSRs in this data set correspond to poly(GA) motifs (36%), followed by poly(AT) (15%) and poly(CCG) (8%) motifs. AT-rich microsatellites had the longest average repeat tracts, while GC-rich motifs were the shortest. In combination with the pool of 500 previously mapped SSR markers, this release makes available a total of 2740 experimentally confirmed SSR markers for rice, or approximately one SSR every 157 kb.     

Efficiency of microsatellite enrichment in<Emphasis Type="Italic">Prosopis chilensis</Emphasis> using magnetic capture

Microsatellites (i.e., simple sequence repeats [SSRs]) are highly variable genetic markers that are widely used at an intraspecific level in population genetic studies. Here we employed an enrichment strategy for microsatellite isolation by using microsatellite oligoprobes and magnetic capture of the fragments (Fischer and Bachmann, 1998) inProsopis chilensis (Mol.) Stuntz (Fabaceae). We analyzed the obtained level of enrichment by sequencing 120 enriched genomic fragments. A total of 521 SSR motives were detected. According to specific search criteria (SSR motifs ≥3 repeat units and ≥6 bp length), 95.8% of the clones contained SSR motifs. Of these, 7.8% showed homology to chloroplast sequences and 92.2% to nuclear sequences. When regarding only nuclear SSRs with 5 or more repeat units and a minimum length of 10 bp, the level of enrichment was 30.8%. A FASTA search against the European Molecular Biology Laboratory (EMBL) database univocally revealed 4 clones in transcribed regions, 102 clones in genomic regions with unknown function, and 9 clones in chloroplast regions. Among the loci with longer repeat units (≥10 bp, ≥5 repeat units), 3 were in transcribed regions and 65 were in other genomic regions. We discuss the applicability of these markers for population genetic studies.     

Abundance and polymorphism of microsatellite markers in the tea tree (Melaleuca alternifolia, Myrtaceae)

 The sequencing of 831 clones from an enriched microsatellite library of Melaleuca alternifolia (Myrtaceae) yielded 715 inserts containing repeat motifs. The majority of these (98%) were dinucleotide repeats or trinucleotide repeats averaging 22 and 8 repeat motifs respectively. The AG/GA motif was the most common, accounting for 43% of all microsatellites. From a total of 139 primer pairs designed, 102 produced markers within the expected size range. The majority of these (93) were polymorphic. Primer pairs were tested on five selected M. alternifolia genotypes. Loci based on dinucleotide repeats detected on average a greater number of alleles (4.2) than those based on trinucleotide repeats (2.9). The loci described will provide a large pool of polymorphisms useful for population studies, genetic mapping, and possibly application in other Myrtaceae. Received: 28 July 1998 / Accepted: 8 October 1998     

Development of microsatellite markers and characterization of simple sequence length polymorphism (SSLP) in rice (Oryza sativa L.)

Microsatellite markers containing simple sequence repeats (SSR) are a valuable tool for genetic analysis. Our objective is to augment the existing RFLP map of rice with simple sequence length polymorphisms (SSLP). In this study, we describe 20 new microsatellite markers that have been assigned to positions along the rice chromosomes, characterized for their allelic diversity in cultivated and wild rice, and tested for amplification in distantly related species. Our results indicate that the genomic distribution of microsatellites in rice appears to be random, with no obvious bias for, or clustering in particular regions, that mapping results are identical in intersubspecific and interspecific populations, and that amplification in wild relatives ofOryza sativa is reliable in species most closely related to cultivated rice but becomes less successful as the genetic distance increases. Sequence analysis of SSLP alleles in three relatedindica varieties demonstrated the clustering of complex arrays of SSR motifs in a single 300-bp region with independent variation in each. Two microsatellite markers amplified multiple loci that were mapped onto independent rice chromosomes, suggesting the presence of duplicated regions within the rice genome. The availability of increasing numbers of mapped SSLP markers can be expected to increase the power and resolution of genome analysis in rice.     

Abundance, polymorphism and genetic mapping of microsatellites in rice

Dinucleotide microsatellites have been characterized and used as genetic markers in rice. Screening of a rice genomic library with poly(dG-dA)·(dC-dT) and poly(dG-dT)·(dC-dA) probes indicated that (GA)_n repeats occurred, on average, once every 225 kb and (GT)_n repeats once every 480 kb. DNA sequencing of ten randomly selected microsatellites indicated that the numbers of repeats ranged from 12 to 34 and that the patterns of microsatellites in rice were similar to those of humans and other mammals. Primers to these microsatellite loci as well as to four published microsatellite-containing sequences have been designed and degrees of polymorphism has been examined with 20 rice accessions. Multiple alleles, ranging from 5 to 11, have been observed at all the microsatellite loci in 20 rice accessions. Alleles specific to two cultivated subspecies, indica and japonica, were found in some microsatellite loci. Heterozygosity values of all the microsatellite markers were significantly higher than those of RFLP markers, based upon a parallel comparison. Ten microsatellite loci have been genetically mapped to four rice chromosomes. The genomic distribution of microsatellites appears to be random in rice.     

An integrated genetic linkage map of avocado

 An avocado genomic library was screened with various microsatellite repeats. (A/T)_n and (TC/AG)_n sequences were found to be the most frequent repeats. One hundred and seventy-two positive clones were sequenced successfully of which 113 were found to contain simple sequence repeats (SSR). Polymerase chain reaction primers were designed to the regions flanking the SSR in 62 clones. A GenBank search of avocado DNA sequences revealed 1 sequence containing a (CT)₁₀ repeat. A total of 92 avocado-specific SSR markers were screened for polymorphism using 50 offspring of a cross between the avocado cultivars ‘Pinkerton’ and ‘Ettinger’. Both are standard avocado cultivars which are normally outcrossed and highly heterozygous. Fifty polymorphic SSR loci, 17 random amplified polymorphic DNA (RAPD) and 23 minisatellite DNA Fingerprint (DFP) bands were used to construct the avocado genetic map. The resulting data were analyzed with various mapping programs in order to assess which program best accommodated data from progeny of heterozygous parents. The analyses resulted in 12 linkage groups with 34 markers (25 SSRs, 3 RAPDs and 6 DFP bands) covering 352.6 cM. This initial map can serve as a basis for developing a detailed genomic map and for detection of linkage between markers and quantitative trait loci. Received: 2 April 1996 / Accepted: 28 February 1997     

Characterization and analysis of microsatellite loci in Elymus caninus (Triticeae: Poaceae)

 Microsatellites are highly variable DNA sequences that can be used as markers for the genetic analysis of plants. The potential of microsatellite markers for use in a genetic diversity study in Elymus species was evaluated. Genomic libraries of Elymus caninus were constructed. The libraries were screened with two dinucleotide, (GA)n and (GT)n, and two trinucleotide repeats, (TCT)n and (CAC)n. A total of 19 positive clones were found for the two dinucleotide repeats; no positive clone was found for the trinucleotide repeats. Positive clones were sequenced to confirm the presence of microsatellites and to generate polymerase chain reaction (PCR) primers based on the sequences flanking the microsatellite. All sequenced (GA)n clones have repeats of n>10; over half of the (GT)n microsatellites have n<10 repeats. Primer pairs were designed and evaluated for 8 selected microsatellites. PCR products were amplified from 15 Elymus caninus accessions. The number of alleles found for the eight loci varied from 1 for ECGA89 and ECGT35 to 13 for ECGA22, as determined by non-denaturing polyacrylamide electrophoresis. Six microsatellite loci were found to be polymorphic in E. caninus. The eight primer pairs were tested on three other species; seven were successful in amplifying DNA from Elymus alaskanus and E. mutabilis, and four amplified DNA from E. caucasicus. Based on these results, microsatellites appear to be useful markers in detecting variation in E. caninus. Received: 8 September 1997/Accepted: 6 October 1997     

Mapping and genome organization of microsatellite sequences in rice (Oryza sativa L.)

In order to enhance the resolution of an existing genetic map of rice, and to obtain a comprehensive picture of marker utility and genomic distribution of microsatellites in this important grain species, rice DNA sequences containing simple sequence repeats (SSRs) were extracted from several small-insert genomic libraries and from the database. One hundred and eighty eight new microsatellite markers were developed and evaluated for allelic diversity. The new simple sequence length polymorphisms (SSLPs) were incorporated into the existing map previously containing 124 SSR loci. The 312 microsatellite markers reported here provide whole-genome coverage with an average density of one SSLP per 6 cM. In this study, 26 SSLP markers were identified in published sequences of known genes, 65 were developed based on partial cDNA sequences available in GenBank, and 97 were isolated from genomic libraries. Microsatellite markers with different SSR motifs are relatively uniformly distributed along rice chromosomes regardless of whether they were derived from genomic clones or cDNA sequences. However, the distribution of polymorphism detected by these markers varies between different regions of the genome. Received: 5 May 1999 / Accepted: 16 August 1999