Physical molecular maps of wheat chromosomes

In bread wheat, a set of 527 simple sequence repeats (SSRs) were tried on 164 deletion lines, leading to a successful mapping of 270 SSRs on 313 loci covering all 21 chromosomes. A maximum of 119 loci (38%) were located on B subgenome, and a minimum of 90 loci (29%) mapped on D subgenome. Similarly, homoeologous group 7 carried a maximum of 61 loci (19%), and group 4 carried a minimum of 22 loci (7%). Of the cited 270 SSRs, 39 had multiple loci, but only eight of these detected homoeologous loci. Linear order of loci in physical maps largely corresponded with those in the genetic maps. Apparently, distances between each of only 26 pairs of loci significantly differed from the corresponding distances on genetic maps. Some loci, which were genetically mapped close to the centromere, were physically located distally, while other loci that were mapped distally in the genetic maps were located in the proximal bins in the physical maps. This suggested that although the linear order of the loci was largely conserved, variation does exist between genetic and physical distances.Electronic Supplementary Material Supplementary material is available for this article at .     

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.     

海参微卫星DNA的多态性

微卫星DNA(microsatellite DNA)广泛存在于真核生物的基因组中。由于其具有突变频率快、多态性丰富、呈共显性遗传、通用性等特点,已成为近年来被广泛应用的分子遗传标记。本研究对10个海参微卫星DNA进行了克隆与测序。结果表明:90%的微卫星DNA序列存在长度多态性,这为进一步研究海参的分子标记辅助育种奠定了基础。     

Microsatellite markers developed from Theobroma cacao L. expressed sequence tags

Theobroma cacao L. expressed sequence tags (ESTs) were converted into useful genetic markers for fingerprinting individuals and genetic linkage mapping. Primers were designed to microsatellite‐containing ESTs. Twenty‐two T. cacao accessions, parents of various mapping populations segregating for disease resistance and crop yield characteristics, were tested. Twenty‐seven informative loci were discovered with 26 primer pairs. The number of detected alleles ranged from two to 11 and averaged 4.4 per locus. All 27 markers could be mapped into at least one of the existing F₁ or F₂ populations segregating for agronomically important traits.     

Microsatellite marker development,mapping and applications in rice genetics and breeding

Microsatellites are simple, tandemly repeated di- to tetra-nucleotide sequence motifs flanked by unique sequences. They are valuable as genetic markers because they are co-dominant, detect high levels of allelic diversity, and are easily and economically assayed by the polymerase chain reaction (PCR). Results from screening a rice genomic library suggest that there are an estimated 5700-10 000 microsatellites in rice, with the relative frequency of different repeats decreasing with increasing size of the motif. A map consisting of 120 microsatellite markers demonstrates that they are well distributed throughout the 12 chromosomes of rice. Five multiple copy primer sequences have been identified that could be mapped to independent chromosomal locations. The current level of genome coverage provided by these simple sequence length polymorphisms (SSLPs) in rice is sufficient to be useful for genotype identification, gene and quantitative trait locus (QTL) analysis, screening of large insert libraries, and marker-assisted selection in breeding. Studies of allelic diversity have documented up to 25 alleles at a single locus in cultivated rice germplasm and provide evidence that amplification in wild relatives of Oryza sativa is generally reliable. The availability of increasing numbers of mapped SSLP markers can be expected to complement existing RFLP and AFLP maps, increasing the power and resolution of genome analysis in rice.     

Microsatellite markers from sugarcane (Saccharum spp.) ESTs cross transferable to erianthus and sorghum

Analysis of a sugarcane (Saccharum spp.) EST (expressed sequence tag) library of 8678 sequences revealed approximately 250 microsatellite or simple sequence repeats (SSRs) sequences. A diversity of dinucleotide and trinucleotide SSR repeat motifs were present although most were of the (CGG)_n trinucleotide motif. Primer sets were designed for 35 sequences and tested on five sugarcane genotypes. Twenty-one primer pairs produced a PCR product and 17 pairs were polymorphic. Primer pairs that produced polymorphisms were mainly located in the coding sequence with only a single pair located within the 5′ untranslated region. No primer pairs producing a polymorphic product were found in the 3′ untranslated region. The level of polymorphism (PIC value) in cultivars detected by these SSRs was low in sugarcane (0.23). However, a subset of these markers showed a significantly higher level of polymorphism when applied to progenitor and related genera (Erianthus sp. and Sorghum sp.). By contrast, SSRs isolated from sugarcane genomic libraries amplify more readily, show high levels of polymorphism within sugarcane with a higher PIC value (0.72) but do not transfer to related species or genera well.     

Assessment of ISSR based molecular genetic diversity of Hassawi rice in Saudi Arabia

Inter simple sequence repeat (ISSR) analysis, using 14 primers was performed to estimate genetic diversity among 27 landraces of Hassawi rice growing in Al-Ahsa region of Saudi Arabia and deposited at King Abdulaziz City for Science and Technology with KACST IDs. The average polymorphism produced by 11 selected primers was more than 75%. The analysis of ISSR polymorphism divided the examined rice landraces into two groups; In one group (A), one accession (KACST 191) was clearly delimited as a distant landrace from other 12 landraces grouped in two clusters; cluster I of seven landraces of close geographic distributions; four of them grow at close geographic locations (KACST IDs 32, 183, 184, 185, 186, 187 and 188) and cluster II is comprised of five landraces KACST IDs (190, 308, 352, 353 and 355). In group B, the landraces were more closely related to each other as compared to the landraces of group A. In this group a small cluster of two landraces (KACST 305 & KACST 333) was clearly distant from a large group of three clusters comprised of landraces having KACST IDs 189 & 192, landraces 302, 306, 307, 308 & 310 and landraces with KACST IDs 334, 351, 354, 356 & 357 respectively. These results indicate that ISSR fingerprints are efficient in the identification and resolution of genetic diversity between the landraces of the Hassawi rice and will be an efficient method in the authentication of the rice germplasm in the gene bank of Saudi Arabia.     

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.     

Use of DNA markers to study bird migration

The molecular methods that are presently being used for studying phylogenetics, phylogeography and population genetics can also be applied to study bird migration. They are powerful and can supplement the information obtained from ringing, telemetry, morphometrics, ringing, radar tracking and isotope analysis. This short review describes the principles, scopes and limitations DNA methods and DNA markers that are relevant for migration research, such as DNA sequences, short tandem repeats (microsatellites), single nucleotide polymorphisms, amplified fragment length polymorphism, inter simple sequence repeats and molecular sexing.     

Development and characterization of a large set of microsatellite markers in grapevine (Vitis vinifera L.) suitable for multiplex PCR

Despite their numerous advantages, the use of microsatellites as genetic markers could be limited because of the low number of loci that can be simultaneously analysed per experiment. To increase the information per simple sequence repeat (SSR) assay in the grapevine, we developed a large set of new markers suitable for multiplexing and multi-loading. We produced microsatellite motif-enriched genomic libraries containing preferentially large size inserts which allowed us to design primers generating a wide range of allele sizes in a very standard and unique PCR condition. Three hundred and fifty clones were sequenced and 190 of them (54%) contained microsatellite motifs with suitable flanking regions for primer design. We developed 169 new SSR markers giving suitable signal with fluorescent-based DNA detection. The total number of alleles detected varied from 1 to 8 per locus with an average of 3.5 and the mean expected heterozygosity was 0.544 (range: 0 0.86). Sixty-eight loci (40%) were perfect types, 73 (43%) were imperfect and 28 (17%) were compound or imperfect-compound. The number of alleles generated by perfect and imperfect type loci was positively correlated to the length of the microsatellite motif. Forty-six multiplex sets based on 125 selected loci were developed. Considering their allele size range, up to four PCR multiplex were pooled together for multi-loading. The 169 SSR loci developed in this study represent a new and informative set of markers easy to combine for multiplexing and multi-loading according to the needs of any user and suitable for large scale genetic analyses in grapevine.     

用微卫星标记评估红尾鲃的种群结构

本文检测了三种鲤科鱼的 1 6对微卫星引物在红尾中的适用性 ,其中 6对引物可以成功扩增 ,且 5个位点具有多态性。对采自两条不同河流的标本 ,通过检测这些多态微卫星位点的遗传变异情况 ,评估了它们在红尾种群结构分析的适合性。结果显示这 5个多态位点在上述两个样本中的平均表观杂合度分别是 0 2 93和0 4 71。这两个样本显著的基因异质性表明我们所确定的微卫星标记可用于红尾的种内遗传分化研究     

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     

Microsatellite and sequence-tagged site markers diagnostic for the rice bacterial leaf blight resistance gene xa-5

 Microsatellite and sequence-tagged site (STS) markers tightly linked to the bacterial leaf blight (BLB) resistance gene xa-5 were identified in this study. A survey was conducted to find molecular markers that detected polymorphisms between the resistant (IRBB5) and susceptible (‘IR24’) nearly isogenic lines for xa-5, and between Chinsurah Boro II (CBII), an alternative source of xa-5, and a widely planted variety (‘IR64’) that lacks xa-5. Two F₂ populations, from the crosses ‘IR24’×IRBB5 and CBIIבIR64’, were used to estimate linkage based on marker genotype and reaction to disease inoculation with Xanthomonas oryzae pv. oryzae. Two RFLP clones, RZ390 and RG556, were found to co-segregate with xa-5 and were converted into STS markers. A microsatellite marker, RM390, was developed based on a simple sequence repeat in the 5′ untranslated region of the cDNA probe, RZ390, and found to co-segregate with resistance. Two other microsatellites, RM122 and RM13, were located 0.4 cM and 14.1 cM away from xa-5. A germplasm survey of diverse lines containing BLB resistance genes using automated fluorescent detection indicated the range of allelic diversity for each of the microsatellite loci linked to xa-5 and confirmed their usefulness in following genes through the narrow crosses typical of a breeding program. The limited number of alleles observed at the microsatellite loci linked to the resistance gene in 35 xa-5-containing accessions suggested either a single ancestral origin or a few independent origins of the xa-5 gene. PCR-based markers, like the ones developed in this study, are economical and easy to use, and have applicability in efforts to pyramid the recessive xa-5 gene with other BLB resistance genes. Received: 27 September 1996/Accepted: 7 February 1997     

Exact tandem repeats analyzer (E-TRA): A new program for DNA sequence mining

Exact Tandem Repeats Analyzer 1.0 (E-TRA) combines sequence motif searches with keywords such as ‘organs’, ‘tissues’, ‘cell lines’ and ‘development stages’ for finding simple exact tandem repeats as well as non-simple repeats. E-TRA has several advanced repeat search parameters/options compared to other repeat finder programs as it not only accepts GenBank, FASTA and expressed sequence tags (EST) sequence files, but also does analysis of multiple files with multiple sequences. The minimum and maximum tandem repeat motif lengths that E-TRA finds vary from one to one thousand. Advanced user defined parameters/options let the researchers use different minimum motif repeats search criteria for varying motif lengths simultaneously. One of the most interesting features of genomes is the presence of relatively short tandem repeats (TRs). These repeated DNA sequences are found in both prokaryotes and eukaryotes, distributed almost at random throughout the genome. Some of the tandem repeats play important roles in the regulation of gene expression whereas others do not have any known biological function as yet. Nevertheless, they have proven to be very beneficial in DNA profiling and genetic linkage analysis studies. To demonstrate the use of E-TRA, we used 5,465,605 human EST sequences derived from 18,814,550 GenBank EST sequences. Our results indicated that 12.44% (679,800) of the human EST sequences contained simple and non-simple repeat string patterns varying from one to 126 nucleotides in length. The results also revealed that human organs, tissues, cell lines and different developmental stages differed in number of repeats as well as repeat composition, indicating that the distribution of expressed tandem repeats among tissues or organs are not random, thus differing from the un-transcribed repeats found in genomes.     

Development of a microsatellite framework map providing genome-wide coverage in rice (Oryza sativa L.)

 Ninety-four newly developed microsatellite markers were integrated into existing RFLP framework maps of four rice populations, including two doubled haploid, a recombinant inbred, and an interspecific backcross population. These simple sequence repeats (SSR) were predominantly poly(GA) motifs, targetted because of their abundance in rice. They were isolated from a previously described sheared library and a newly constructed enzyme-digested library. Differences in the average length of poly(GA) tracts were observed for clones isolated from the two libraries. The length of GA motifs averaged 21 repeat units for clones isolated from the Tsp-509-digested library, while motifs averaged 17 units for clones from the sheared library. There was no evidence of clustering of microsatellite markers near centromeres or telomeres. Mapping of the 94 newly developed markers as well as of 27 previously reported microsatellites provided genome-wide coverage of the 12 chromosomes, with an average distance of 1 SSLP (simple sequence repeat polymorphism) per 16–20 cM. Received: 13 February 1997/Accepted: 28 February 1997     

Development and mapping of SSR markers for maize

Microsatellite or simple sequence repeat (SSR) markers have wide applicability for genetic analysis in crop plant improvement strategies. The objectives of this project were to isolate, characterize, and map a comprehensive set of SSR markers for maize (Zea mays L.). We developed 1051 novel SSR markers for maize from microsatellite-enriched libraries and by identification of microsatellite-containing sequences in public and private databases. Three mapping populations were used to derive map positions for 978 of these markers. The main mapping population was the intermated B73 × Mo17 (IBM) population. In mapping this intermated recombinant inbred line population, we have contributed to development of a new high-resolution map resource for maize. The primer sequences, original sequence sources, data on polymorphisms across 11 inbred lines, and map positions have been integrated with information on other public SSR markers and released through MaizeDB at URL:www.agron.missouri.edu. The maize research community now has the most detailed and comprehensive SSR marker set of any plant species.     

Development of multiplex sets of simple sequence repeat DNA markers covering the soybean genome

Multiplexing involves the analysis of several markers in a single gel lane that is based on the allele size range of marker loci. Multiplex SSR marker analysis is conducted with primers that are labeled with one of three dyes. The development of an SSR multiplex system requires estimates of the allele size range of markers to strategize primer labeling and for grouping markers into multiplex sets. A method is presented that describes the development of multiplex sets of SSR markers in soybean (Glycine max (L.) Merr.) by the selective placement of primer sites and by the analysis of diverse germplasm. Primer sites were placed at specific distances from the SSR to adjust the allele size range of marker loci. The analysis of pooled DNA samples comprising diverse soybean genotypes provided robust estimates of the allele size range of marker loci that enabled the development of multiplex sets. Eleven multiplex sets comprising 74 SSR markers distributed across the 20 linkage groups of soybean were developed. Multiplex sets constructed from the analysis of diverse soybean germplasm should have a wide range of genotyping applications. The procedures used in this study were systematic and rapid and should be applicable for multiplex development in any species with SSR marker technology.     

Trinucleotide Repeats as Bait for Vectorette PCR: A Tool for Developing Genetic Mapping Markers

Trinucleotide repeats are common within gene coding regions and could serve as beacons to locate genes. Five of the most common trinucleotide repeats in an Actinidia (kiwifruit) expressed sequence tag (EST) database were found to be (ACC)₄, (CAC)₄, (CCA)₄, (CTC)₄, and (TGG)₄. These repeats, with or without an artificial 5′-end tail, were tested by vectorette PCR against genomic DNA from Actinidia chinensis. Eighty-nine randomly selected clones showed an average insert size of 383 bp, with a maximum of 1,151 bp and a minimum of 78 bp. Two-thirds of the clones contained the artificial tail attached to the trinucleotide, showing a slight advantage of possessing such a tail during annealing and amplification. The sequences were searched against the Actinidia EST database and GenBank. Of the 89 clones, 33 had a significant hit (expect value < e⁻¹⁵). Twenty-four of those clones matched an Actinidia EST. Twenty-one clones contained one or more simple sequence repeats. This methodology can be applied by conventional cloning and sequencing methods or by high throughput pyrosequencing technologies to develop genetic markers and also for gene mining in species with little or no genetic/genomic resources.