Nonrandom distribution and frequencies of genomic and EST-derived microsatellite markers in rice, wheat, and barley

BACKGROUND: Earlier comparative maps between the genomes of rice (Oryza sativa L.), barley (Hordeum vulgare L.) and wheat (Triticum aestivum L.) were linkage maps based on cDNA-RFLP markers. The low number of polymorphic RFLP markers has limited the development of dense genetic maps in wheat and the number of available anchor points in comparative maps. Higher density comparative maps using PCR-based anchor markers are necessary to better estimate the conservation of colinearity among cereal genomes. The purposes of this study were to characterize the proportion of transcribed DNA sequences containing simple sequence repeats (SSR or microsatellites) by length and motif for wheat, barley and rice and to determine in-silico rice genome locations for primer sets developed for wheat and barley Expressed Sequence Tags. RESULTS: The proportions of SSR types (di-, tri-, tetra-, and penta-nucleotide repeats) and motifs varied with the length of the SSRs within and among the three species, with trinucleotide SSRs being the most frequent. Distributions of genomic microsatellites (gSSRs), EST-derived microsatellites (EST-SSRs), and transcribed regions in the contiguous sequence of rice chromosome 1 were highly correlated. More than 13,000 primer pairs were developed for use by the cereal research community as potential markers in wheat, barley and rice. CONCLUSION: Trinucleotide SSRs were the most common type in each of the species; however, the relative proportions of SSR types and motifs differed among rice, wheat, and barley. Genomic microsatellites were found to be primarily located in gene-rich regions of the rice genome. Microsatellite markers derived from the use of non-redundant EST-SSRs are an economic and efficient alternative to RFLP for comparative mapping in cereals

RAPD markers associated with resistance to blackleg disease in Brassica species

Blackleg, caused by Leptosphaeria maculans, is a serious disease of Brassica species. Genetic analysis of resistance to L. maculans was carried out with 15 accessions from the USDA Brassica germplasm collections, representing diploids (A, C), and tetraploid (AC) genomes, respectively; and 9 cultivars from the National Winter Canola Variety Trials (NWCVT) all carrying AC genomes. All genotypes were screened for blackleg disease at the cotyledonary stage. The results indicated that 46% of the 24 genotypes were resistant, while 54% were susceptible. On the other hand, adult plant screening revealed that all the public genotypes were resistant. In an effort to identify molecularmarkers associated with resistance to blackleg disease, all genotypes were screened with 13 RAPD and 8 SSR markers producing 169 amplified products. Six RAPD markers (OPB01, OPE03, OPE16, OPF10, OPE12, and OPI01) were polymorphic, while the SSR markers were monomorphic. Chi-square analysis indicated that 5 amplified fragments (OPE03-4000, OPE16-1100, OPE16-1300, OPE16-1900, and OPI01- 280) from RAPD primers were significantly associated with blackleg resistance. Thus this study demonstrated that RAPD primers could be effectively used to identify DNA markers that are associatedwith blackleg disease resistance, and that resistance to L. maculans might also exist in the A and C genomes

Phylogenetic relationships within and among Brassica species from RAPD loci associated with blackleg resistance

The genus Brassica comprises economically important oilseed and vegetable crops. Their susceptibility to fungal diseases such as blackleg causes yield loss. In this study, thirty accessionsfrom USDA germplasm collection representing two diploid Brassica species (Brassica rapa and Brassica oleracea var. virids) and fifteen tetraploid cultivars (Brassica napus) from the national wintercanola variety trials (NWCVT) were evaluated using 13 sets of random amplified polymorphic DNA (RAPD) associated with blackleg resistance in Brassica nigra. 126 highly polymorphic bands with an average of 10 per primer were detected. A UPGMA dendrogram showed B. rapa as highly diverse and was supported from three different basal branches, while B. napus accessions were generally monophyletic. Similarly, all of B. oleraceae accessions were supported from the same basal node.Generally, the three species were reciprocally paraphyletic, suggesting that the RAPD markers showed both functional relationships as well as homology, possibly due to selection at the RAPD loci associated with blackleg resistance. Consequently, two potentially susceptible B. napus accessions were identified. The high polymorphic information content (PIC) and number of phylogenetically informative bands established RAPD as a useful tool for phylogenetic reconstruction, quantification ofgenetic diversity for conservation, cultivar classification and molecular breeding in Brassica

DNA-based identification of Lentinula edodes strains with species-specific primers

Lentinula edodes is among the five globally cultivated edible mushrooms, which are wood decaying spore bearing Basidiomycetes possessing separate hyphae. Specific identification of this fungus from others in the division Basidiomycota using specific primers enables a fast and accurate detection through polymerase chain reaction (PCR). As a prelude to additional nutritional and sequence characterization research, we have developed a species specific PCR assay for this fungus after screening four primer-pairs and two universal primer pairs. The primer-pair LE1F/R was specific in amplifications of ATCC-defined L. edodes strains and did not amplify DNA from six medicinally and nutritionally important fungal reference strains, Oyster (Pleurotus ostreatus), Maitake (Grifola frondosa), Enoki (Flammulina velutipes), Baby bella (Agaricus bisporus), Porcini (Boletus edulis), and Chanterelle (Cantharellus cibarius). However, amplifications using the universal primers were positive for all six strains. This assay will therefore serve to validate morphology-based identifications of L. edodes strains.Keywords: Lentinula edodes, LE1F/R, species-specific primer

Identification of genes related to germination in aged maize seed by screening natural variability

Ageing reduces vigour and viability in maize inbred lines due to non-heritable degenerative changes. Besides non-heritable genetic changes due to chromosome aberrations and damage in the DNA sequence, heritable changes during maize conservation have been reported. Genetic variability among aged seeds of inbred lines could be used for association studies with seed germination. The objective of this study was to identify genes related to germination in aged seeds. The sweet corn inbred line P39 and the field corn inbred line EP44 were used as plant material. Bulks of living and dead seeds after 20 and 22 years of storage were compared by using simple sequence repeats (SSRs) and, when the bulks differed for a marker, the individual grains were genotyped. Differences between dead and living seeds could be explained by residual variability, spontaneous mutation, or ageing. Variability was larger for chromosome 7 than for other chromosomes, and for distal than for proximal markers, suggesting some relationships between position in the genome and viability in aged seed. Polymorphic SSRs between living and dead seeds were found in six known genes, including pathogenesis-related protein 2, superoxide dismutase 4, catalase 3, opaque endosperm 2, and metallothionein1 that were related to germination, along with golden plant 2. In addition, five novel candidate genes have been identified; three of them could be involved in resistance to diseases, one in detoxification of electrophillic compounds, and another in transcription regulation. Therefore, genetic variability among aged seeds of inbreds was useful for preliminary association analysis to identify candidate genes

Development of Genome-wide Simple Sequence Repeat Markers Using Whole-genome Shotgun Sequences of Sorghum (Sorghum bicolor (L.) Moench)

Simple sequence repeat (SSR) markers with a high degree of polymorphism contribute to the molecular dissection of agriculturally important traits in sorghum (Sorghum bicolor (L.) Moench). We designed 5599 non-redundant SSR markers, including regions flanking the SSRs, in whole-genome shotgun sequences of sorghum line ATx623. (AT/TA)n repeats constituted 26.1% of all SSRs, followed by (AG/TC)n at 20.5%, (AC/TG)n at 13.7% and (CG/GC)n at 11.8%. The chromosomal locations of 5012 SSR markers were determined by comparing the locations identified by means of electronic PCR with the predicted positions of 34 008 gene loci. Most SSR markers had a similar distribution to the gene loci. Among 970 markers validated by fragment analysis, 67.8% (658 of 970) markers successfully provided PCR amplification in sorghum line BTx623, with a mean polymorphism rate of 45.1% (297 of 658) for all SSR loci in combinations of 11 sorghum lines and one sudangrass (Sorghum sudanense (Piper) Stapf) line. The product of 5012 and 0.678 suggests that ∼3400 SSR markers could be used to detect SSR polymorphisms and that more than 1500 (45.1% of 3400) markers could reveal SSR polymorphisms in combinations of Sorghum lines

Development of Simple Sequence Repeats (SSR) Markers in Setaria italica (Poaceae) and Cross-Amplification in Related Species

Foxtail millet is one of the world’s oldest cultivated crops. It has been adopted as a model organism for providing a deeper understanding of plant biology. In this study, 45 simple sequence repeats (SSR) markers of Setaria italica were developed. These markers showing polymorphism were screened in 223 samples from 12 foxtail millet populations around Taiwan. The most common dinucleotide and trinucleotide repeat motifs are AC/TG (84.21%) and CAT (46.15%). The average number of alleles (Na), the average heterozygosities observed (Ho) and expected (He) are 3.73, 0.714, 0.587, respectively. In addition, 24 SSR markers had shown transferability to six related Poaceae species. These new markers provide tools for examining genetic relatedness among foxtail millet populations and other related species. It is suitable for germplasm management and protection in Poaceae

Optimization of sequence alignment for simple sequence repeat regions

Abstract Background Microsatellites, or simple sequence repeats (SSRs), are tandemly repeated DNA sequences, including tandem copies of specific sequences no longer than six bases, that are distributed in the genome. SSR has been used as a molecular marker because it is easy to detect and is used in a range of applications, including genetic diversity, genome mapping, and marker assisted selection. It is also very mutable because of slipping in the DNA polymerase during DNA replication. This unique mutation increases the insertion/deletion (INDELs) mutation frequency to a high ratio - more than other types of molecular markers such as single nucleotide polymorphism (SNPs). SNPs are more frequent than INDELs. Therefore, all designed algorithms for sequence alignment fit the vast majority of the genomic sequence without considering microsatellite regions, as unique sequences that require special consideration. The old algorithm is limited in its application because there are many overlaps between different repeat units which result in false evolutionary relationships. Findings To overcome the limitation of the aligning algorithm when dealing with SSR loci, a new algorithm was developed using PERL script with a Tk graphical interface. This program is based on aligning sequences after determining the repeated units first, and the last SSR nucleotides positions. This results in a shifting process according to the inserted repeated unit type. When studying the phylogenic relations before and after applying the new algorithm, many differences in the trees were obtained by increasing the SSR length and complexity. However, less distance between different linage had been observed after applying the new algorithm. Conclusions The new algorithm produces better estimates for aligning SSR loci because it reflects more reliable evolutionary relations between different linages. It reduces overlapping during SSR alignment, which results in a more realistic phylogenic relationship.</p

Effect of Ppd-1 photoperiod sensitivity genes on dry matter production and allocation in durum wheat

Understanding the effect of genetic factors controlling flowering time is essential to fine-tune phenological development and to maximize yield. Thirty-four spring durum wheat genotypes classified in five allelic combinations for Ppd-A1/Ppd-B1 loci were grown for two years at three contrasting latitudes: Mexico-North, Spain- South and Spain-North. In all them, a delay in flowering date due to the presence of photoperiod sensitivity alleles Ppd-A1b and Ppd-B1b resulted in lower yields. The number of days to flowering, determined by an increasing number of photoperiod sensitivity alleles, accounted in all sites for more than 80% of the variation in the contribution of translocation of pre-flowering assimilates to grain yield. In Mexico and Spain-North lateflowering resulted in decreased harvest index as influenced by high temperatures during grain filling. In Mexico, where grain filling occurred under high temperatures and solar radiation, translocation of pre-flowering assimilates accounted from 55 to 63% of yield, independently of the flowering date of the genotype. In Spain- North, where water was available during grain filling, current photosynthesis was the main contributor to yield (57–73%), with independence of the allelic combination at Ppd loci. In Spain-South, the relative contribution of photosynthesis and translocation depended on the allelic composition at Ppd loci, with translocation increasing by 24% in the latest-flowering genotypes compared with the earliest ones. In all sites the limiting factor for attaining high yields was the capacity of the plant canopy to photosynthesize after anthesis. This study suggests that the expression of genes Ppd-A1 and Ppd-B1 regulating the response to photoperiod modulates the physiological strategy adopted by durum wheat to fill its grains, underlining the importance of phenology fitting in maximizing grain yield.This study was partially supported by INIA (Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria) Spain [RTA2009- 00085, RTA2015-00038, AGL2012-37217]

Exploiting a wheat EST database to assess genetic diversity

Expressed sequence tag (EST) markers have been used to assess variety and genetic diversity in wheat (Triticum aestivum). In this study, 1549 ESTs from wheat infested with yellow rust were used to examine the genetic diversity of six susceptible and resistant wheat cultivars. The aim of using these cultivars was to improve the competitiveness of public wheat breeding programs through the intensive use of modern, particularly marker-assisted, selection technologies. The F2 individuals derived from cultivar crosses were screened for resistance to yellow rust at the seedling stage in greenhouses and adult stage in the field to identify DNA markers genetically linked to resistance. Five hundred and sixty ESTs were assembled into 136 contigs and 989 singletons. BlastX search results showed that 39 (29%) contigs and 96 (10%) singletons were homologous to wheat genes. The database-matched contigs and singletons were assigned to eight functional groups related to protein synthesis, photosynthesis, metabolism and energy, stress proteins, transporter proteins, protein breakdown and recycling, cell growth and division and reactive oxygen scavengers. PCR analyses with primers based on the contigs and singletons showed that the most polymorphic functional categories were photosynthesis (contigs) and metabolism and energy (singletons). EST analysis revealed considerable genetic variability among the Turkish wheat cultivars resistant and susceptible to yellow rust disease and allowed calculation of the mean genetic distance between cultivars, with the greatest similarity (0.725) being between Harmankaya99 and Sönmez2001, and the lowest (0.622) between Aytin98 and Izgi01