To accurately quantify gene expression using quantitative PCR amplification, it is vital that one or more ideal internal control genes are used to normalize the samples to be compared. Ideally, the expression level of those internal control genes should vary as little as possible between tissues, developmental stages and environmental conditions. In this study, 32 candidate genes for internal control were obtained from the analysis of nine independent experiments which included 333 Affymetrix GeneChip Wheat Genome arrays. Expression levels of the selected genes were then evaluated by quantitative real-time PCR with cDNA samples from different tissues, stages of development and environmental conditions. Finally, fifteen novel internal control genes were selected and their respective expression profiles were compared using NormFinder, geNorm, Pearson correlation coefficients and the twofold-change method. The novel internal control genes from this study were compared with thirteen traditional ones for their expression stability. It was observed that seven of the novel internal control genes were better than the traditional ones in expression stability under all the tested cDNA samples. Among the traditional internal control genes, the elongation factor 1-alpha exhibited strong expression stability, whereas the 18S rRNA, Alpha-tubulin, Actin and GAPDH genes had very poor expression stability in the range of wheat samples tested. Therefore, the use of the novel internal control genes for normalization should improve the accuracy and validity of gene expression analysis.
Common oat (Avena sativa) is an important cereal crop serving as a valuable source of forage and human food. Although reference genomes of many important crops have been generated, such work in oat has lagged behind, primarily owing to its large, repeat-rich polyploid genome. Here, using Oxford Nanopore ultralong sequencing and Hi-C technologies, we have generated a reference-quality genome assembly of hulless common oat, comprising 21 pseudomolecules with a total length of 10.76 Gb and contig N50 of 75.27 Mb. We also produced genome assemblies for diploid and tetraploid Avena ancestors, which enabled the identification of oat subgenomes and provided insights into oat chromosomal evolution. The origin of hexaploid oat is inferred from whole-genome sequencing, chloroplast genomes and transcriptome assemblies of different Avena species. These findings and the high-quality reference genomes presented here will facilitate the full use of crop genetic resources to accelerate oat improvement.
A high-density genetic map constructed with a wheat 55 K SNP array was highly consistent with the physical map of this species and it facilitated the identification of a novel major QTL for productive tiller number. Productive tiller number (PTN) plays a key role in wheat grain yield. In this study, a recombinant inbred line population with 199 lines derived from a cross between '20828' and 'Chuannong16' was used to construct a high-density genetic map using wheat 55 K single nucleotide polymorphism (SNP) array. The constructed genetic map contains 12,109 SNP markers spanning 3021.04 cM across the 21 wheat chromosomes. The orders of the genetic and physical positions of these markers are generally in agreement, and they also match well with those based on the 660 K SNP array from which the one used in this study was derived. The ratios of SNPs located in each of the wheat deletion bins were similar among the wheat 9 K, 55 K, 90 K, 660 K and 820 K SNP arrays. Based on the constructed maps, a novel major quantitative trait locus QPtn.sau-4B for PTN was detected across multi-environments in a 0.55 cM interval on 4B and it explained 17.23-45.46% of the phenotypic variance. Twenty common genes in the physical interval between the flanking markers were identified on chromosome 4B of 'Chinese Spring' and wild emmer. These results indicate that wheat 55 K SNP array could be an ideal tool in primary mapping of target genes and the identification of QPtn.sau-4B laid a foundation for the following fine mapping and cloning work.
Pre-harvest sprouting (PHS) is mainly caused by the breaking of seed dormancy in high rainfall regions, which leads to huge economic losses in wheat. In this study, we evaluated 717 Chinese wheat landraces for PHS resistance and carried out genome-wide association studies (GWAS) using to 9,740 DArT-seq and 178,803 SNP markers. Landraces were grown across six environments in China and germination testing of harvest-ripe grain was used to calculate the germination rate (GR) for each accession at each site. GR was highly correlated across all environments. A large number of landraces (194) displayed high levels of PHS resistance (i.e., mean GR < 0.20), which included nine white-grained accessions. Overall, white-grained accessions displayed a significantly higher mean GR (42.7–79.6%) compared to red-grained accessions (19.1–56.0%) across the six environments. Landraces from mesic growing zones in southern China showed higher levels of PHS resistance than those sourced from xeric areas in northern and north-western China. Three main quantitative trait loci (QTL) were detected by GWAS: one on 5D that appeared to be novel and two co-located with the grain color transcription factor Tamyb10 on 3A and 3D. An additional 32 grain color related QTL (GCR-QTL) were detected when the set of red-grained landraces were analyzed separately. GCR-QTL occurred at high frequencies in the red-grained accessions and a strong correlation was observed between the number of GCR-QTL and GR (R2 = 0.62). These additional factors could be critical for maintaining high levels of PHS resistance and represent targets for introgression into white-grained wheat cultivars. Further, investigation of the origin of haplotypes associated with the three main QTL revealed that favorable haplotypes for PHS resistance were more common in accessions from higher rainfall zones in China. Thus, a combination of natural and artificial selection likely resulted in landraces incorporating PHS resistance in China.
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