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PubMed2001

Soybean genomic survey: BAC-end sequences near RFLP and SSR markers.

We are building a framework physical infrastructure across the soybean genome by using SSR (simple sequence repeat) and RFLP (restriction fragment length polymorphism) markers to identify BACs (bacterial artificial chromosomes) from two soybean BAC libraries. The libraries were prepared from two genotypes, each digested with a different restriction enzyme. The BACs identified by each marker were grouped into contigs. We have obtained BAC- end sequence from BACs within each contig. The sequences were analyzed by the University of Minnesota Center for Computational Genomics and Bioinformatics using BLAST algorithms to search nucleotide and protein databases. The SSR-identified BACs had a higher percentage of significant BLAST hits than did the RFLP-identified BACs. This difference was due to a higher percentage of hits to repetitive-type sequences for the SSR-identified BACs that was offset in part, however, by a somewhat larger proportion of RFLP-identified significant hits with similarity to experimentally defined genes and soybean ESTs (expressed sequence tags). These genes represented a wide range of metabolic functions. In these analyses, only repetitive sequences from SSR-identified contigs appeared to be clustered. The BAC-end sequences also allowed us to identify microsynteny between soybean and the model plants Arabidopsis thaliana and Medicago truncatula. This map-based approach to genome sampling provides a means of assaying soybean genome structure and organization.

Algorithms↗

PubMed2002

[Genetic diversity revealed by ISSR molecular marker in common wheat, spelt, compactum and progeny of recurrent selection].

It is important to estimate the genetic diversity between the parents for improving the heterosis of hybrid wheat. In this study, ISSR(inter-simple sequence repeat) marker was used to measure the genetic diversity within and among common wheat (Triticum aestivum L.), spelt (Triticum spelta L.), compactum (Triticum compactum Host.) and progeny of foreign wheat-based recurrent selection, and the possibility of establishing the new heterotic group was also assessed. Forty seven genotypes were used for ISSR analysis, which included 14 common wheat, 10 spelt wheat, 11 compactum and 12 progeny of recurrent selection. Eleven of 33 ISSR primers that can produce distinguishable bands were selected for PCR amplification. A total of 238 bands were amplified, among which 207 (87%) bands were polymorphic. The polymorphic bands amplified by each primer ranged from 11 to 38, with an averaged of 18.8. The percentage of polymorphic band (80.3%) in common wheat was higher than that in progeny of recurrent selection (78.7%), spelt (75.0%) and compactum (74.9%). The 238 polymorphic products were used to calculate Nei's similarity index (GS) and the genetic distance (GD). It was found that the mean genetic distance between different wheat types (0.3115-0.3442) was obviously higher than that within common wheat (0.2743), spelt (0.2351), compactum (0.2622). In addition, progeny of recurrent selection also showed much higher genetic distance with other three wheat types (0.3217, 0.3256, 0.3198). The cluster analysis was performed based on the genetic distance (GD) matrix by using UPGMA method. Common wheat, spelt, compactum and progeny of recurrent selection were classified into four different groups. In this study, ISSR marker was firstly used to assess genetic diversity among common wheat, spelt, compactum and progeny of recurrent selection, and can differentiate the wheat cultivars (lines) that selected from the same cross combination. It was concluded that spelt, compactum and progeny of recurrent selection can be used to diversify the genetic basis for hybrid wheat breeding and improve heterosis. It is possible to establish the wheat heterotic group by ISSR marker.

Genetic Markers↗

PubMed2002

[Genetic diversity of D-genome revealed by SSR markers in synthesized hexaploid wheat introduced from CIMMYT].

Simple sequence repeat (SSR) molecular marker was used to measure the genetic diversity of D-genome in 26 synthesized hexaploid wheat (AABBDD) introduced from CIMMYT. Twenty-three D-genome specific SSR primers were selected for PCR amplification, among which 22 primers can detect polymorphism. A total of 92 alleles were identified at 23 loci using the above SSR primers, with an average of 4 alleles per locus. The 92 alleles were used to calculate Nei's similarity index (GS) and the genetic distance (GD). It was also found that the mean genetic distance between 26 synthesized hexaploid wheat was 0.4955, which was obviously high. From the above results, it can be indicated that the genetic variation of D-genome in synthesized hexaploid wheat was abundant and could be used to improve the genetic diversity in wheat breeding. Interestingly, synthesized hexaploid wheat 17 and 18 shared the same D-genome donor, but three of 23 detected SSR loci were polymorphic between the two materials. Therefore, during the period of allopolyploidization, there was genetic differentiation in repeat region of donor genome.

Alleles↗

PubMed2002

[Genetic variation of main restorer lines of hybrid rice in China was revealed by microsatellite markers].

A total of thirty-five restorer lines of hybrid rice (Oryza sativa L.) were analyzed by twenty-five SSR (simple sequence repeats) primer pairs, which disperse on 12 chromosomes in rice. Those primers detected 65 alleles among 35 restorer lines of hybrid rice. Per primer pair detected 2.6 alleles on the average. PIC (polymorphism index content) values ranged from 0.206 to 0.682. PIC value is 0.414 on the average. The result from cluster analysis shows that hybrid rice restorer lines have abundant resource in China, but the genetic diversity is small and the genetic background is vulnerable among them. The utilization of rice heterosis was limited seriously.

Genetic Variation↗

PubMed2002

[Construction, characterization and screening of a transformation-competent artificial chromosome (TAC) library of wheat-Thinopyrum intermedium translocation line with resistance to barley yellow dwarf virus].

A transformation-competent artificial chromosome (TAC) library was constructed from the genomic DNA of wheat-Th. intermidium translocation line HW642 that harbor the barley yellow dwarf virus (BYDV) resistance gene derived from Th. intermidium. The library consists of 2.3 x 10(6) clones with an average insert size of 22kb, representing approximately 2.5 haploid genome equivalents and is able to give a greater than 95.77% probability of isolating single-copy DNA sequences from this library. The library was stored as frozen cultures in 24 96-well formats, each well containing approximately 1000 different clones. TAC clones containing interest gene could be identified by the pooled PCR technique. A sequence characterized amplified region (SCAR) marker cosegregated with BYDV resistance gene, derived from a simple sequence repeat (SSR) or microsatellite marker wms37 of wheat, was applied to screen the TAC library. Twelve clones were successfully selected by the pooled PCR method. PCR products were identified by hybridizing with the SCAR marker band of Th. intermidium. Out of 12 clones, 10 positive clones restricted by Hind III were shown to hybridize with genomic DNA of Th. intermidium. These results showed evidences that the 10 clones could be used as candidate clones for isolation of BYDV resistance and its related genes, and the TAC library is a useful resource for isolating genes.

Base Sequence↗

PubMed2002

Use of molecular markers in cereal breeding.

Great advances have been made in recent years in marker detection systems and in the techniques used to identify markers linked to useful traits. While RFLP markers have been the basis for most work in crop plants, useful markers have been generated using RAPD and AFLP methods. More recently, microsatellite or simple sequence repeat (SSR) markers have been developed for major crop plants and this marker system is predicted to lead to even more rapid advances in both marker development and implementation in breeding programs. Identification of markers linked to useful traits has been based on complete linkage maps and bulked segregant analysis. However, alternative methods, such as the construction of partial maps and combination of pedigree and marker information, have also proved useful in identifying marker/trait associations. The value of markers in analysing the inheritance of traits in crop plants and understanding genome structure and organization is now well established. The different properties of markers systems and their applications in genome analysis and molecular breeding of cereals species are discussed.

Breeding↗

PubMed2002

In silico analysis on frequency and distribution of microsatellites in ESTs of some cereal species.

During the last decade microsatellites or SSRs (simple sequence repeats) have been proven to be the markers of choice in plant genetics research and for breeding purposes because of their hypervariability and ease of detection. However, development of these markers is expensive, labour intensive and time consuming, in particular, if they are being developed from genomic libraries. In the context of large-scale sequencing and genomics programmes in various cereal species at different laboratories, a large set of expressed sequence tags (ESTs) is being generated, which can be used to search for microsatellites. Keeping in view the importance of such type of SSRs, available ESTs of some cereal species like barley, maize, oats, rice, rye and wheat were investigated for a study of abundance, frequency and distribution of various types of microsatellites. SSRs were present in about 7% to 10% of the total ESTs in the investigated cereal genomes. On the basis of surveying EST sequences amounting to 75.2 Mb in barley, 54.7 Mb in maize, 43.9 Mb in rice, 3.7 Mb in rye, 41.6 Mb in sorghum and 37.5 Mb in wheat, the frequency of SSRs was 1/7.5 kb in barley, 1/7.5 kb in maize, 1/6.2 kb in wheat, 1/5.5 kb in rye and sorghum and 1/3.9 kb in rice. The overall average SSR frequency for these species is 1/6.0 kb. Trimeric repeats are the most abundant (54% to 78%) class of microsatellites followed by dimeric repeats (17% to 40%). Among the trimeric repeats the motifs CCG are the most common in all the cases ranging from 32% in wheat to 49% in sorghum. When all these SSRs were analysed for assessing their potential to develop new markers, unique primer pairs could be designed for 30% to 70% of the total non-redundant microsatellites which are up to 3% of total ESTs in the studied species.

Crops, Agricultural↗

PubMed2002

Generation and mapping of AFLP, SSRs and SNPs in Lycopersicon esculentum.

Amplified Fragment Length Polymorphism (AFLP), Simple Sequence Repeat (SSR) and Single Nucleotide Polymorphism (SNP), were applied to the tomato genome for assessment of polymorphism and for mapping. The polymorphism of AFLP was studied in twenty-one commercial tomato (L. esculentum) varieties. Four AFLP primer combinations produced 298 clear bands; an average of 75 bands per combination. SSR markers were generated from two sources: (1) size-selected genomic libraries screened with (AT)n, (CT)n, (GT)n, (ATT)n and (CTT)n probes. (2) GeneBank database. Primers were designed for 114 loci and used for genotyping 13 tomato varieties and three Lycopersicon species. Eighteen markers were used to evaluate the polymorphism among the commercial cultivars and were found to be a useful tool for cultivar identification. In-silico comparison of DNA sequences (ESTs and genes) of L. pennellii and L. esculentum, yielded 312 SNPs. Ten L. pennelli genomic fragments were sequenced and the comparison with L. esculentum yielded 22 SNPs. Another 19 SNPs were discovered by sequencing and comparing L. pennellii genomic DNA to L. esculentum DNA fragments containing SSRs. The average SNP frequency was found to be one in a few tens of base pairs. A total of 52 microsatellites, 159 polymorphic AFLP markers and six SNPs were mapped using the Introgression Lines generated by [1]. Map location and markers' distribution are presented.

Alleles↗

PubMed2002

A microsatellite marker based study of chromosomal regions and gene effects on yield and yield components in maize.

The genetic basis of different traits and the effect of chromosomal regions on the expression of such traits is the primary interest of this study. In the present investigation, the effect of chromosomal regions on yield and its two primary components was studied, and two genetic parameters, additive effect and degree of dominance, were estimated based on a single-locus model using markers with significant effects on the traits (informative markers). Eight inbred lines from diverse geographical regions of India were crossed in all possible pairs, and F1s were evaluated for the yield and yield components in a replicated trial in two environments. Sequential path analysis was employed to find those yield components showing significant direct effects on yield with negligible multicollinearity. The parental lines were profiled using 56 polymorphic SSR (Simple Sequence Repeat) markers covering 10 chromosomes of maize. Stepwise multiple regression analysis was used to determine the informative markers on yield and its primary components detected through path analysis. Fourteen markers were found to have association with chromosomal regions showing significant effects on the total grain yield, 100-grain weight and total number of kernels per ear. Chromosome 1 with four informative markers revealed the highest genic effects on yield and its components. Markers bnlg594 and bnlg1360 on chromosome 10, and bnlg147 on chromosome 1 revealed the highest additive effects on the total grain yield, 100-grain weight and total number of kernels per ear, respectively. For the analyzed traits, overdominance occurred in all the loci and d/a values, varying from 8.60 for 100-grain weight to 1.40 for total grain yield.

Breeding↗

PubMedDate not supplied

Exclusion of melanocortin-1 receptor (mc1r) and agouti as candidates for dominant black in dogs.

The domestic dog exhibits a variety of coat colors that encompass a wide range of variation among different breeds. Very little is known about the molecular biology of dog pigmentation; current understanding is based mostly on traditional breeding experiments, which in some cases have suggested genetic interactions that are different from those reported in other mammals. We have examined the molecular genetics of dominant black, a uniform coat color characteristic of black Labrador retrievers or Newfoundlands that has been proposed to be caused by either variation in the melanocortin-1 receptor gene (Mc1r) or by variation in the Agouti gene (A). We identified several coding polymorphisms within Mc1r and several simple sequence repeat polymorphisms closely linked to A, and examined their inheritance in a Labrador retriever x greyhound cross that segregates dominant black. No single Mc1r allele was found consistently in animals carrying dominant black, and neither Mc1r nor A cosegregated with dominant black. These results refine our understanding of mammalian coat color inheritance and suggest that dominant black coat color in dogs is caused by a gene not previously implicated in pigment type switching.

Agouti Signaling Protein↗

PubMed2003

Widespread bimodal intrachromosomal genomic instability in sporadic breast cancers associated with 13q allelic imbalance.

Genomic instability is thought to underlie tumor progression in solid tumors, such as breast cancer. Although evidence that the hereditary breast cancer genes, BRCA1 and BRCA2, are involved in DNA repair suggests that genomic instability plays an important role in hereditary breast tumorigenesis, genomic instability remains poorly characterized in sporadic breast cancers. Using a DNA fingerprinting technique, inter-(simple sequence repeat) PCR (inter-SSR PCR), the degree of genomic instability was quantified in 47 sporadic breast cancers compared with matched adjacent normal breast tissues. Almost all sporadic breast cancers show significant genomic instability by inter-SSR PCR. The distribution of this instability is bimodal; 57% of the tumors show fewer changes, whereas 43% show striking genomic alterations. Further analysis of two inter-SSR PCR tumor-normal differences revealed a genomic amplification and probable deletion. Thus, inter-SSR PCR can detect chromosomal breakage-related genomic alterations in most sporadic breast cancers. Genomic instability as detected by inter-SSR PCR is not correlated with aneuploidy, suggesting that this technique preferentially detects intrachromosomal alterations. Chromosomal instability in breast cancer can therefore be subdivided into at least two groups: (a) intrachromosomal and (b) gross chromosomal. Allelic imbalance at markers at the 13q13 and retinoblastoma loci (13q) and not at 17q loci was significantly associated with high levels of intrachromosomal instability, suggesting genes at 13q13 and retinoblastoma loci are either selectively targeted or involved in the genesis of genomic instability in sporadic breast cancers.

Adult↗

PubMed2003

Insertion of a reamplification round into the ISSR-PCR protocol gives new flax fingerprinting patterns.

We expanded the basic ISSR-PCR protocol by an additional PCR reamplification round in order to detect whether increased PCR productivity would give new bands in ISSR patterns. We found that the reamplification step had a prominent impact on the quality of the inter-simple-sequence repeat (ISSR) PCR patterns of flax, depending on the particular primer used for PCR amplification. We could clearly distinguish between two types of reamplification effect. Most ISSR primers (16 out of 21) gave no reamplification effect as usual, but five primers (23.8%) provided a new ISSR fingerprinting pattern after the 2nd reamplification round, leaving the previous 1st round pattern completely blank. Therefore, we recommend the expansion of a basic ISSR-PCR protocol for another reamplification round in order to mine out full the fingerprinting potential from ISSR-PCR method.

DNA Fingerprinting↗

PubMed1992

Isolation of YAC clones from the pericentromeric region of chromosome 10 and development of new genetic markers linked to the multiple endocrine neoplasia type 2A gene.

Genetic linkage mapping and contig assembly using yeast artificial chromosome (YAC) technology form the basis of our strategy to clone and define the genomic structure of the pericentromeric region of chromosome 10 containing the multiple endocrine neoplasia type 2A gene. Thus far YAC walks have been initiated from five chromosome 10 pericentromeric loci including RBP3, D10S94, RET, D10Z1, and FNRB. Long range pulsed-field gel electrophoresis maps are constructed from the YACs isolated to define clone overlaps and to identify putative CpG islands. Bidirectional YAC walks are continued by rescreening the YAC library with sequence-tagged site assays developed from end-clones. Several new restriction fragment length polymorphisms and simple sequence repeat polymorphism markers have been identified from the YAC clones. In particular, two highly informative (CA)n dinucleotide repeat markers, sTCL-1 from proximal chromosome 10p (16 alleles, PIC = 0.68) and sJRH-1 from the RBP3 locus (18 alleles, PIC = 0.88), provide useful reagents for a polymerase chain reaction-based predictive genetic test that can be performed rapidly from small amounts of DNA.

Chromosomes, Fungal↗

PubMed2001

Molecular markers for leaf rust resistance genes in wheat.

Over 100 genes of resistance to rust fungi: Puccinia recondita f. sp. tritici, (47 Lr - leaf rust genes), P. striiformis (18 Yr - yellow rust genes) and P. graminis f. sp. tritici (41 Sr - stripe rust genes) have been identified in wheat (Triticum aestivum L.) and its wild relatives according to recent papers. Sixteen Lr resistance genes have been mapped using restriction fragments length polymorphism (RFLP) markers on wheat chromosomes. More than ten Lr genes can be identified in breeding materials by sequence tagged site (STS) specific markers. Gene Lrk 10, closely linked to gene Lr 10, has been cloned and its function recognized. Available markers are presented in this review. The STS, cleaved amplified polymorphic sequence (CAPS) and sequence characterized amplified regions (SCAR) markers found in the literature should be verified using Triticum spp. with different genetic background. Simple sequence repeats (SSR) markers for Lr resistance genes are now also available.

Journal Article↗

PubMed2003

[Towards the positional cloning of a spikelet identity gene frizzle panicle (FZP) in rice (Oryza sativa L.)].

FZP is a key gene for spikelet differentiation in rice. Mutation of the gene blocks the differentiation of spikelets and makes rachis branches develop unlimitedly. A mutant of the gene named frizzle panicle (fzp) was previously found from the high-generation progeny of a cross between two Oryza sativa ssp. indica rice varieties, V20B and Hua1B. With the mutant, FZP had been mapped to a chromosomal region of about 26.4 cM in width between two SSR (Simple Sequence Repeat) markers, RM172 and RM18, on chromosome 7. In this study, high-resolution mapping of the gene was carried out for the positional cloning of the gene. Two flanking SSR markers, NRM6 and NRM8, were identified, which are 0.2 cM and 1.0 cM apart from the target gene, respectively, bracketing the target gene within an interval of 1.2 cM or 144 kb. An APETALA2 (AP2)-domain like gene was found at the expected position of FZP. As AP2 is known to play an important role in the floral development, we took it as the most possible candidate of FZP. PCR analysis showed that the mutant allele of the AP2-domain like gene contains an insert of about 4 kb in length, suggesting that the gene is very likely FZP.

Chromosome Mapping↗

PubMed2003

MICAS: a fully automated web server for microsatellite extraction and analysis from prokaryote and viral genomic sequences.

MICAS is a web server for extracting microsatellite information from completely sequenced prokaryote and viral genomes, or user-submitted sequences. This server provides an integrated platform for MICdb (database of prokaryote and viral microsatellites), W-SSRF (simple sequence repeat finding program) and Autoprimer (primer design software). MICAS, through dynamic HTML page generation, helps in the systematic extraction of microsatellite information from selected genomes hosted on MICdb or from user-submitted sequences. Further, it assists in the design of primers with the help of Autoprimer, for sequences containing selected microsatellite tracts.

Algorithms↗

PubMedDate not supplied

Zygotic and nucellar embryo survival following dehydration and cryopreservation of citrus intact seeds.

A cryopreservation procedure by dehydration and direct immersion in liquid nitrogen was developed for seeds of four polyembryonic Citrus species, and the sexual or nucellar origin of the recovered seedlings was investigated. Seeds of three species could be desiccated in a sterile air flow to 16 percent (C. sinensis) or 10 percent (C. aurantium and C. limon) moisture content with a negligible reduction in germination levels. Differently, the germinability of C. deliciosa seeds dropped to 50 percent after drying to 15 percent moisture content. Following dehydration treatments, a reduction in the average number of seedlings per germinated seed was always observed. However, all four species benefited from desiccation in terms of protection during immersion in liquid nitrogen, with C. sinensis and C. aurantium showing the greatest survival (93 percent germination) after cryopreservation. The Inter-Simple Sequence Repeat analysis of seedlings recovered from cryopreserved seeds showed that the dehydration/cryopreservation procedure promotes the germination of zygotic embryos and reduces the number of apomictic seedlings per seed.

Citrus↗

PubMed2004

Extension of the Messapia x dicoccoides linkage map of Triticum turgidum (L.) Thell.

A set of recombinant inbred lines (RIL) derived from a cross between the cultivar Messapia of durum wheat (Triticum turgidum var. durum) and the accession MG4343 of T. turgidum var. dicoccoides was analysed to increase the number of assigned markers and the resolution of the previously constructed genetic linkage map. An updated map of the durum wheat genome consisting of 458 loci was constructed. These loci include 261 Restriction Fragment Length Polymorphisms (RFLPs), 91 microsatellites (Simple Sequence Repeats, SSRs), 87 Amplified Fragment Length Polymorphisms (AFLPs), two ribosomal genes, and nine biochemical (seven seed storage proteins and two isozymes) and eight morphological markers. The loci were mapped on all 14 chromosomes of the A and B genomes, and covered a total distance of 3038.4 cM with an average distance of 6.7 cM between adjacent markers. The molecular markers were evenly distributed between the A and the B genomes (240 and 218 markers, respectively). An additional forty loci (8.8%) could not be assigned to a specific linkage group. A fraction (16.4%) of the markers significantly deviated from the expected Mendelian ratios; clusters of loci showing distorted segregation were found on the 1B, 2A, 2B, 3A, 4A, 7A and 7B chromosomes. The genetic lengths of the chromosomes range from 148.8 cM (chromosome 6B) to 318.0 cM (chromosome 2B) and approximately concur with their physical lengths. Chromosome 2B has the largest number of markers (47), while the chromosomes with the fewest markers are 3A and 6B (23). There are two gaps larger than 40 cM on chromosomes 2A and 3B. The durum wheat map was compared with the published maps of bread and durum wheats; the order of most common RFLP and SSR markers on the 14 chromosomes of the A and B genomes were nearly identical. A core-map can be extracted from the high-density Messapia x dicoccoides map and a subset of uniformly distributed markers can be used to detect and map quantitative trait loci.

Chromosome Mapping↗