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 ...

Guangzhou University

To understand the mechanisms of aluminum (Al) tolerance in wheat (Triticum aestivum L.), suppression subtractive hybridization (SSH) libraries were constructed from Al-stressed roots of two near-isogenic lines (NILs). A total of 1,065 putative genes from the SSH libraries was printed in a cDNA array. Relative expression levels of those genes were compared between two NILs at seven time points of Al stress from 15 min to 7 days. Fifty-seven genes were differentially expressed for at least one time point of Al treatment. Among them, 28 genes including genes for aluminum-activated malate transporter-1, ent-kaurenoic acid oxidase-1, beta-glucosidase, lectin, histidine kinase, and phospoenolpyruvate carboxylase showed more abundant transcripts in Chisholm-T and therefore may facilitate Al tolerance. In addition, a set of genes related to senescence and starvation of nitrogen, iron, and sulfur, such as copper chaperone homolog, nitrogen regulatory gene-2, yellow stripe-1, and methylthioribose kinase, was highly expressed in Chisholm-S under Al stress. The results suggest that Al tolerance may be co-regulated by multiple genes with diverse functions, and those genes abundantly expressed in Chisholm-T may play important roles in enhancing Al tolerance. The down-regulated genes in Chisholm-S may repress root growth and restrict uptake of essential nutrient elements, and lead to root senescence.

Transcriptional analysis between two wheat near-isogenic lines contrasting in aluminum tolerance under aluminum stress.

Drought tolerance is a key trait for increasing and stabilizing barley productivity in dry areas worldwide. Identification of the genes responsible for drought tolerance in barley (Hordeum vulgare L.) will facilitate understanding of the molecular mechanisms of drought tolerance, and also facilitate the genetic improvement of barley through marker-assisted selection or gene transformation. To monitor the changes in gene expression at the transcriptional level in barley leaves during the reproductive stage under drought conditions, the 22K Affymetrix Barley 1 microarray was used to screen two drought-tolerant barley genotypes, Martin and Hordeum spontaneum 41-1 (HS41-1), and one drought-sensitive genotype Moroc9-75. Seventeen genes were expressed exclusively in the two drought-tolerant genotypes under drought stress, and their encoded proteins may play significant roles in enhancing drought tolerance through controlling stomatal closure via carbon metabolism (NADP malic enzyme, NADP-ME, and pyruvate dehydrogenase, PDH), synthesizing the osmoprotectant glycine-betaine (C-4 sterol methyl oxidase, CSMO), generating protectants against reactive-oxygen-species scavenging (aldehyde dehydrogenase,ALDH, ascorbate-dependent oxidoreductase, ADOR), and stabilizing membranes and proteins (heat-shock protein 17.8, HSP17.8, and dehydrin 3, DHN3). Moreover, 17 genes were abundantly expressed in Martin and HS41-1 compared with Moroc9-75 under both drought and control conditions. These genes were possibly constitutively expressed in drought-tolerant genotypes. Among them, seven known annotated genes might enhance drought tolerance through signalling [such as calcium-dependent protein kinase (CDPK) and membrane steroid binding protein (MSBP)], anti-senescence (G2 pea dark accumulated protein, GDA2), and detoxification (glutathione S-transferase, GST) pathways. In addition, 18 genes, including those encoding Delta(l)-pyrroline-5-carboxylate synthetase (P5CS), protein phosphatase 2C-like protein (PP2C), and several chaperones, were differentially expressed in all genotypes under drought; thus they were more likely to be general drought-responsive genes in barley. These results could provide new insights into further understanding of drought-tolerance mechanisms in barley.

Differentially expressed genes between drought-tolerant and drought-sensitive barley genotypes in response to drought stress during the reproductive stage.

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.

Delta-1-pyrroline-5-carboxylate synthase gene1 (P5CS1) is the key gene involved in the biosynthesis of proline and is significantly induced by drought stress. The exploration of genetic variation in HvP5CS1 may facilitate a better understanding of the mechanism of drought adaptation in barley. In the current study, 41 polymorphisms including 16 single nucleotide polymorphisms (SNPs) and 25 insertions/deletions (indels) were detected in HvP5CS1 among 287 barley (Hordeum vulgare L.) accessions collected worldwide, with 13 distinct haplotypes identified in the barley collection. Five polymorphisms in HvP5CS1 were significantly (P < 0.001) associated with drought tolerance related traits in barley. The phenotypic variation of a given trait explained by each associated polymorphism ranged from 4.43% to 9.81%. Two sequence variations that were significantly (P < 0.0001) associated with grain yield had marginally significant positive Tajima's D values in the sliding window, so they might have been selected for environmental adaptation. Meanwhile, two haplotypes HvP5CS1_H1 and HvP5CS1_H4, which contained desired alleles of the two variations mentioned above, were significantly (P < 0.001) associated with drought tolerance related traits, and explained 5.00~11.89% of the phenotypic variations. These variations associated with drought tolerance related traits can be used as potential markers for improving drought tolerance in barley.

Genetic variations of HvP5CS1 and their association with drought tolerance related traits in barley (Hordeum vulgare L.).

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.

Ronghua Li

College of Life Sciences

Guangzhou University

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

Ronghua Li

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Guangzhou University

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

College of Life Sciences