Discovery of polymorphisms in starch-related genes in rice germplasm by amplification of pooled DNA and deeply parallel sequencing

High-throughput sequencing of pooled DNA was applied to polymorphism discovery in candidate genes involved in starch synthesis. This approach employed semi- to long-range PCR (LR-PCR) followed by next-generation sequencing technology. A total of 17 rice starch synthesis genes encoding seven classes of enzymes, including ADP-glucose pyrophosphorylase (AGPase), granule starch synthase (GBSS), soluble starch synthase (SS), starch branching enzyme (BE), starch debranching enzyme (DBE) and starch phosphorylase (SPHOL) and phosphate translocator (GPT1) from 233 genotypes were PCR amplified using semi- to long-range PCR. The amplification products were equimolarly pooled and sequenced using massively parallel sequencing technology (MPS). By detecting single nucleotide polymorphism (SNP)/Indels in both coding and noncoding areas of the genes, we identified genetic differences and characterized the SNP/Indel variation and distribution patterns among individual starch candidate genes. Approximately, 60.9 million reads were generated, of which 54.8 million (90%) mapped to the reference sequences. The average coverage rate ranged from 12,708 to 38,300 times for SSIIa and SSIIIb, respectively. SNPs and single/multiple-base Indels were analysed in a total assembled length of 116,403 bp. In total, 501 SNPs and 113 Indels were detected across the 17 starch-related loci. The ratio of synonymous to nonsynonymous SNPs (Ka/Ks) test indicated GBSSI and isoamylase 1 (ISA1) as the least diversified (most purified) and conservative genes as the studied populations have been through cycles of selection. This report demonstrates a useful strategy for screening germplasm by MPS to discover variants in a specific target group of genes.     

A high-throughput assay for rapid and simultaneous analysis of perfect markers for important quality and agronomic traits in rice using multiplexed MALDI-TOF mass spectrometry

The application of single nucleotide polymorphisms (SNPs) in plant breeding involves the analysis of a large number of samples, and therefore requires rapid, inexpensive and highly automated multiplex methods to genotype the sequence variants. We have optimized a high-throughput multiplexed SNP assay for eight polymorphisms which explain two agronomic and three grain quality traits in rice. Gene fragments coding for the agronomic traits plant height (semi-dwarf, sd-1 ) and blast disease resistance ( Pi-ta ) and the quality traits amylose content ( waxy ), gelatinization temperature ( alk ) and fragrance ( fgr ) were amplified in a multiplex polymerase chain reaction. A single base extension reaction carried out at the polymorphism responsible for each of these phenotypes within these genes generated extension products which were quantified by a matrix-assisted laser desorption ionization-time of flight system. The assay detects both SNPs and indels and is co-dominant, simultaneously detecting both homozygous and heterozygous samples in a multiplex system. This assay analyses eight functional polymorphisms in one 5 µL reaction, demonstrating the high-throughput and cost-effective capability of this system. At this conservative level of multiplexing, 3072 assays can be performed in a single 384-well microtitre plate, allowing the rapid production of valuable information for selection in rice breeding.     

Characterization of Some Chromosomal Aberrations in Regenerated Rice Plants (<Emphasis Type="Italic">Oryza sativa</Emphasis>)

Somaclonal variation was studied in two Iranian land races of O. sativa spp. japonica var Hassani and O. sativa spp. indica var Sadridomsiah and 2000 plants of each cultivar were cytogenetically examined in two steps. In the first step, chromosome counts of root tips was used to detect ploidy levels and aneuploids of regenerated plants. In the second step, chromosomal aberrations were characterized by pachytene analysis of PMC’s. Ploidy levels were seen between n and 4n (haploids to tetraploids) in both cultivars with diploid resource (2n = 2x = 24). The total rate of variation for Hassani (japonica) was 13.7% including 10.8% for changes in chromosome number (ploidy levels and aneuploids) and 2.9% for chromosomal aberrations such as deficiency. A total rate of variation for Sadridomsiah (indica) was 15.6% including 12.4% for change in numbers and 3.2% for aberrations in construction. Most of important cytological mutations were observed in various chromosomes among regenerated plants of cultivars. Neither nullisomics nor inversions were distinguished in any samples.     

Effects of Corticosterone and Dietary Energy on Immune Function of Broiler Chickens

An experiment was conducted to investigate the effects of dietary energy level on the performance and immune function of stressed broiler chickens (Gallus gallus domesticus). A total of 96 three-day-old male broiler chickens (Ross × Ross) were divided into two groups. One group received a high energy (HE) diet and the other group received a low energy (LE) diet for 7 days. At 5 days of age, the chickens from each group were further divided into two sub-groups and received one of the following two treatments for 3 days: (1) subcutaneous injection of corticosterone, twice per day (CORT group; 2 mg of CORT/kg BW in corn oil) and (2) subcutaneous injection of corn oil, twice per day (Control/Sham treatment group). At 10 days of age, samples of blood, duodenum, jejunum, and ileum were obtained. Compared with the other three groups, the LE group treated with CORT had the lowest average daily gain (ADG) and the poorest feed conversion ratio (FCR, P < 0.05). Furthermore, CORT treatment decreased the relative weight (RW) of the bursa independent of the dietary energy level, but it decreased the RW of the thymus only in the chickens fed the LE diet. By contrast, CORT administration decreased the RW of the spleen only in the chickens fed the HE diet (P < 0.05). The plasma total protein, albumin, tumor necrosis factor alpha, interleukin 2 and immunoglobulin G (IgG) levels were affected by the CORT treatment (P < 0.05); however, these factors were not significantly affected by the dietary energy level. Toll-like receptor-5 mRNA level was down-regulated by CORT injection in the duodenum and ileum (P < 0.05) and showed a trend of down-regulation in the jejunum (P=0.0846). The present study showed that CORT treatment induced immunosuppressive effects on the innate immune system of broiler chickens, which were ameliorated by consumption of higher dietary energy.     

Attenuating Effect of Zinc and Vitamin E on the Intestinal Oxidative Stress Induced by Silver Nanoparticles in Broiler Chickens

The aim of this study was to assess the relationship between magnesium status and oxidative stress in obese and nonobese women. This cross-sectional study included 83 women, aged between 20 and 50 years, who were divided into two groups: the obese group (n = 31) and the control group (n = 52). The control group was age-matched with the obese group. Magnesium intake was monitored using 3-day food records and NutWin software version 1.5. The plasma and erythrocyte magnesium concentrations were determined by flame atomic absorption spectrophotometry. Plasma levels of thiobarbituric acid reactive substances (TBARS) were determined as biomarkers for lipid peroxidation and therefore of oxidative stress. The mean values of the magnesium content in the diet were found to be lower than those recommended, though there was no significant difference between groups (p > 0.05). The mean concentrations of plasma and erythrocyte magnesium were within the normal range, with no significant difference between groups (p > 0.05). The mean concentration of plasma TBARS was higher in obese woman, and the difference between the groups was statistically different (p < 0.05). There was a positive correlation between erythrocyte magnesium and plasma TBARS in the obese group (p = 0.021). Obese patients ingest low dietary magnesium content, which does not seem to affect the plasma and erythrocyte concentrations of the mineral. The study showed a negative correlation between erythrocyte magnesium concentrations and plasma TBARS, suggesting the influence of magnesium status on the parameters of oxidative stress in obese women.