A Fast Silver Staining Protocol Enabling Simple and Efficient Detection of SSR Markers using a Non-denaturing Polyacrylamide Gel

Simple Sequence Repeat (SSR) is one of the most effective markers used in plant and animal genetic research and molecular breeding programs. Silver ...

United States Department of Agriculture - Agricultural Research Service

Light-harvesting chlorophyll a/b-binding protein (LHCP) is one of the most abundant chloroplast proteins in plants. Its main function is to collect and transfer light energy to photosynthetic reaction centers. However, the roles of different LHCPs in light-harvesting antenna systems remain obscure. Exploration of nucleotide variation in the genes encoding LHCP can facilitate a better understanding of the functions of LHCP. In this study, nucleotide variations in Lhcb1, a LHCP gene in barley, were investigated across 292 barley accessions collected from 35 different countries using EcoTILLING technology, a variation of the Targeting Induced Local Lesions In Genomes (TILLING). A total of 23 nucleotide variations were detected including three insert/deletions (indels) and 20 single nucleotide polymorphisms (SNPs). Among them, 17 SNPs were in the coding region with nine missense changes. Two SNPs with missense changes are predicted to be deleterious to protein function. Seventeen SNP formed 31 distinguishable haplotypes in the barley collection. The levels of nucleotide diversity in the Lhcb1 locus differed markedly with geographic origins and species of accessions. The accessions from Middle East Asia exhibited the highest nucleotide and haplotype diversity. H. spontaneum showed greater nucleotide diversity than H. vulgare. Five SNPs in Lhcb1 were significantly associated with at least one of the six agronomic traits evaluated, namely plant height, spike length, number of grains per spike, thousand grain weight, flag leaf area and leaf color, and these SNPs may be used as potential markers for improvement of these barley traits.

Allelic variations of a light harvesting chlorophyll a/b-binding protein gene (Lhcb1) associated with agronomic traits in barley.

Small heat shock protein 17.8 (HSP17.8) is produced abundantly in plant cells under heat and other stress conditions and may play an important role in plant tolerance to stress environments. However, HSP17.8 may be differentially expressed in different accessions of a crop species exposed to identical stress conditions. The ability of different genotypes to adapt to various stress conditions resides in their genetic diversity. Allelic variations are the most common forms of genetic variation in natural populations. In this study, single nucleotide polymorphisms (SNPs) of the HSP17.8 gene were investigated across 210 barley accessions collected from 30 countries using EcoTILLING technology. Eleven SNPs including 10 from the coding region of HSP17.8 were detected, which form nine distinguishable haplotypes in the barley collection. Among the 10 SNPs in the coding region, six are missense mutations and four are synonymous nucleotide changes. Five of the six missense changes are predicted to be deleterious to HSP17.8 function. The accessions from Middle East Asia showed the higher nucleotide diversity of HSP17.8 than those from other regions and wild barley (H. spontaneum) accessions exhibited greater diversity than the cultivated barley (H. vulgare) accessions. Four SNPs in HSP17.8 were found associated with at least one of the agronomic traits evaluated except for spike length, namely number of grains per spike, thousand kernel weight, plant height, flag leaf area and leaf color. The association between SNP and these agronomic traits may provide new insight for study of the gene's potential contribution to drought tolerance of barley.

Single nucleotide polymorphisms in HSP17.8 and their association with agronomic traits in barley.

Silver staining is one of the widely used methods for DNA fragment detection in biological research. Silver staining protocols have been steadily optimized to improve detection efficiency. This research reports a continuous effort to simplify the existing silver staining protocols, lower experiment cost, and improve DNA detection sensitivity and image clarity. The new method only requires three reagents (silver nitrate, sodium hydroxide, and formaldehyde) and 6-7 min with high detection sensitivity to visualize as low as 14.6 pg (3.3 pg/mm ) of DNA in a non-denaturing polyacrylamide gel. In comparison to previous reported protocols, the new one has the highest resolution, is the easiest to operate, takes the shortest time, and uses the fewest chemical reagents. Therefore, the new method can be used for quick generation of high quality molecular marker data in genetic analysis.

Development of a simple and effective silver staining protocol for detection of DNA fragments.

CI13227 is a U.S. winter wheat line with adult-plant slow-rusting resistance that has been the subject of several studies on the characteristics and components of slow rusting. Previous genetic studies used different populations and approaches and came to different conclusions about the genetic basis of resistance in CI13227. To clarify the situation, a new doubled-haploid (DH) population of CI13227 × Lakin was produced and a linkage map was constructed using 5,570 single-nucleotide polymorphism (SNP) markers derived from wheat 90K SNP assays and 84 simple sequence repeat markers. Three quantitative trait loci (QTL) were identified for three slow-rusting traits on chromosome arms 2DS, 7AL, and 7BL from CI13227. A fourth QTL mapped on chromosome 3BS was from Lakin. The QTL on 2DS, designated QLr.hwwg-2DS, explained 11.2 to 25.6% of the phenotypic variation. It was found in the same position as a slow-rusting QTL in the CI13227 × Suwon 92 population in a previous study and, thus, verified the 2DS QTL. The QTL on chromosome 7BL explained 8.1 and 19.3% of the phenotypic variation and is likely to be Lr68. The other two QTL showed a minor effect on some of the traits evaluated in a single experiment. Flanking SNP closely linked to all QTL were converted to Kompetitive allele-specific polymerase chain reaction markers that can be used in marker-assisted selection to transfer these QTL into adapted wheat cultivars.

Quantitative Trait Loci for Slow-Rusting Resistance to Leaf Rust in Doubled-Haploid Wheat Population CI13227 × Lakin.

Flowering Chinese cabbage is one of the most important vegetable crops in southern China. Genetic improvement of various agronomic traits in this crop is underway to meet high market demand in the region, but the progress is hampered by limited number of molecular markers available in this crop. This study aimed to develop EST-SSR markers from transcriptome sequences generated by next-generation sequencing. RNA-seq of eight cabbage samples identified 48,975 unigenes. Of these unigenes, 23,267 were annotated in 56 gene ontology (GO) categories, 6,033 were mapped to 131 KEGG pathways, and 7,825 were assigned to clusters of orthologous groups (COGs). From the unigenes, 8,165 EST-SSR loci were identified and 98.57% of them were 1-3 nucleotide repeats with 14.32%, 41.08% and 43.17% of mono-, di- and tri-nucleotide repeats, respectively. Fifty-eight types of motifs were identified with A/T, AG/CT, AT/AT, AC/GT, AAG/CTT and AGG/CCT the most abundant. The lengths of repeated nucleotide sequences in all SSR loci ranged from 12 to 60 bp, with most (88.51%) under 20 bp. Among 170 primer pairs were randomly selected from a total of 4,912 SSR primers we designed, 48 yielded unambiguously polymorphic bands with high reproducibility. Cluster analysis using 48 SSRs classified 34 flowering Chinese cabbage cultivars into three groups. A large number of EST-SSR markers identified in this study will facilitate marker-assisted selection in the breeding programs of flowering Chinese cabbage.

Development of EST-SSR markers in flowering Chinese cabbage (Brassica campestris L. ssp. chinensis var. utilis Tsen et Lee) based on de novo transcriptomic assemblies.

 Genetic diversity is an essential resource for breeders to improve new cultivars with desirable characteristics. Recently, genotyping-by-sequencing (GBS), a next-generation sequencing (NGS) technology that can simplify complex genomes, has now be used as a high-throughput and cost-effective molecular tool for routine breeding and screening in many crop species, including the species with a large genome.  We genotyped a diversity panel of 369 Iranian hexaploid wheat accessions including 270 landraces collected between 1931 and 1968 in different climate zones and 99 cultivars released between 1942 to 2014 using 16,506 GBS-based single nucleotide polymorphism (GBS-SNP) markers. The B genome had the highest number of mapped SNPs while the D genome had the lowest on both the Chinese Spring and W7984 references. Structure and cluster analyses divided the panel into three groups with two landrace groups and one cultivar group, suggesting a high differentiation between landraces and cultivars and between landraces. The cultivar group can be further divided into four subgroups with one subgroup was mostly derived from Iranian ancestor(s). Similarly, landrace groups can be further divided based on years of collection and climate zones where the accessions were collected. Molecular analysis of variance indicated that the genetic variation was larger between groups than within group.  Obvious genetic diversity in Iranian wheat was revealed by analysis of GBS-SNPs and thus breeders can select genetically distant parents for crossing in breeding. The diverse Iranian landraces provide rich genetic sources of tolerance to biotic and abiotic stresses, and they can be useful resources for the improvement of wheat production in Iran and other countries.

Guihua Bai

Hard Winter Wheat Genetics Research Unit

United States Department of Agriculture - Agricultural Research Service

A Fast Silver Staining Protocol Enabling Simple and Efficient Detection of SSR Markers using a Non-denaturing Polyacrylamide Gel

Guihua Bai

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Hard Winter Wheat Genetics Research Unit

United States Department of Agriculture - Agricultural Research Service

A Fast Silver Staining Protocol Enabling Simple and Efficient Detection of SSR Markers using a Non-denaturing Polyacrylamide Gel

Hard Winter Wheat Genetics Research Unit