RNAseq-based Expression Analysis Research Articles

Genome-wide association study for phosphate responsive root hair length and density in bread wheat

The increase in root hair length and density is considered an effective way to deal with external phosphorous (P) limitation. The identification of useful loci for root hair traits is crucial for wheat breeding. Here, We employed a simple, cost-effective, and medium-throughput screening method using cigar rolls to assess P-responsive root hair length (RHL) and density (RHD) in bread wheat at the seedling stage. A set of 113 historical bread wheat cultivars was screened for root hair length and density (RHL/D) under low (0.005 mM KH2PO4) and high P-levels (0.25 mM KH2PO4). Significant variations (p < 0.001) were found among genotypes for length and density under both P treatments. Genotyping was performed using 50 K SNP array data to conduct the genome-wide association study (GWAS) using a multi-locus model (mrMLM) and 67 SNPs linked to RHL/D under phosphorous treatment were identified. Of these, 33 SNPs were associated with RHL and 34 were linked with RHD. The maximum number of SNPs (43) linked to target traits were observed under high-P levels following 24 were identified under low-P conditions. We also identified 6 SNPs associated with target traits causing missense mutations and predicted their candidate genes of which 5 were protein coding. RNA-seq based expression analysis indicated that all genes were expressed in roots of 24 wheat varieties ranging from 0.38 to 101.7 tpm. The least expression (0.41–10.28 tpm) was observed in TraesCS4D02G066200 which consisted of non-translating CDS and was highly expressed in leaf tissues with 14.55–53.67 tpm. Of these genes, TraesCS1A02G313600 solely expressed in roots with no expression (0 tpm) in leaf tissues. This first GWAS study on root hairs in bread wheat illustrates the genetic basis of RHL and RHD and identifies loci that could be an invaluable resource for further functional investigation.

Identification of Anthocyanins-Related Glutathione S-Transferase (GST) Genes in the Genome of Cultivated Strawberry (Fragaria × ananassa).

Anthocyanins are responsible for the red color of strawberry, they are a subclass of flavonoids synthesized in cytosol and transferred to vacuole to form the visible color. Previous studies in model and ornamental plants indicated members of the glutathione S-transferase (GST) gene family were involved in vacuolar accumulation of anthocyanins. In the present study, a total of 130 FaGST genes were identified in the genome of cultivated strawberry (Fragaria × ananassa), which were unevenly distributed across the 28 chromosomes from the four subgenomes. Evolutionary analysis revealed the expansion of FaGST family was under stable selection and mainly drove by WGD/segmental duplication event. Classification and phylogenetic analysis indicated that all the FaGST genes were clarified into seven subclasses, among which FaGST1, FaGST37, and FaGST97 belonging to Phi class were closely related to FvRAP, an anthocyanin-related GST of wildwood strawberry, and this clade was clustered with other known anthocyanin-related GSTs. RNAseq-based expression analysis at different developmental stages of strawberry revealed that the expression of FaGST1, FaGST37, FaGST39, FaGST73, and FaGST97 was gradually increased during the fruit ripening, consistent with the anthocyanins accumulation. These expression patterns of those five FaGST genes were also significantly correlated with those of other anthocyanin biosynthetic genes such as FaCHI, FaCHS, and FaANS, as well as anthocyanin regulatory gene FaMYB10. These results indicated FaGST1, FaGST37, FaGST39, FaGST73, and FaGST97 may function in vacuolar anthocyanin accumulation in cultivated strawberry.

Accurate characterization of the IFITM locus using MiSeq and PacBio sequencing shows genetic variation in Galliformes

BackgroundInterferon inducible transmembrane (IFITM) proteins are effectors of the immune system widely characterized for their role in restricting infection by diverse enveloped and non-enveloped viruses. The chicken IFITM (chIFITM) genes are clustered on chromosome 5 and to date four genes have been annotated, namely chIFITM1, chIFITM3, chIFITM5 and chIFITM10. However, due to poor assembly of this locus in the Gallus Gallus v4 genome, accurate characterization has so far proven problematic. Recently, a new chicken reference genome assembly Gallus Gallus v5 was generated using Sanger, 454, Illumina and PacBio sequencing technologies identifying considerable differences in the chIFITM locus over the previous genome releases.MethodsWe re-sequenced the locus using both Illumina MiSeq and PacBio RS II sequencing technologies and we mapped RNA-seq data from the European Nucleotide Archive (ENA) to this finalized chIFITM locus. Using SureSelect probes capture probes designed to the finalized chIFITM locus, we sequenced the locus of a different chicken breed, namely a White Leghorn, and a turkey.ResultsWe confirmed the Gallus Gallus v5 consensus except for two insertions of 5 and 1 base pair within the chIFITM3 and B4GALNT4 genes, respectively, and a single base pair deletion within the B4GALNT4 gene. The pull down revealed a single amino acid substitution of A63V in the CIL domain of IFITM2 compared to Red Jungle fowl and 13, 13 and 11 differences between IFITM1, 2 and 3 of chickens and turkeys, respectively. RNA-seq shows chIFITM2 and chIFITM3 expression in numerous tissue types of different chicken breeds and avian cell lines, while the expression of the putative chIFITM1 is limited to the testis, caecum and ileum tissues.ConclusionsLocus resequencing using these capture probes and RNA-seq based expression analysis will allow the further characterization of genetic diversity within Galliformes.