Novel genetic variants of GA-insensitive Rht-1 genes in hexaploid wheat and their potential agronomic value.

This study has found numerous novel genetic variants of GA-insensitive dwarfing genes with potential agricultural value for crop improvement. The cultivar, Spica is a tall genotype and possesses the wild-type genes of Rht-A1a, Rht-B1a and Rht-D1a. The cultivar Quarrion possesses a null mutant in the DELLA motif in each of the 3 genomes. This is a first report of a null mutant of Rht-A1. In addition, novel null mutants which differ from reported null alleles of Rht-B1b, Rht-B1e and Rht-D1b have been found in Quarrion, Carnamah and Whistler. The accession, Aus1408 has an allele of Rht-B1 with a mutation in the conserved 'TVHYNP' N-terminal signal binding domain with possible implications on its sensitivity to GA. Mutations in the conserved C-terminal GRAS domain of Rht-A1 alleles with possible effects on expression have been found in WW1842, Quarrion and Drysdale. Genetic variants with putative spliceosomal introns in the GRAS domain have been found in all accessions except Spica. Genome-specific cis-sequences about 124 bp upstream of the start codon of the Rht-1 gene have been identified for each of the three genomes

Novel genetic variants at 5' end of Rht-1 genes.

<p>Alignment of the amino acid sequences translated from DNA sequence amplicons from primer pair, Dell_RhtF2/Dell_RhtR2 (RP 2293−2565 of FN649763 or RP 260−532 in Table S2) of accessions Aus1408 (AU), Whistler (WH), WW1842 (WW), Carnamah (CA), Drysdale (DR), Spica (SP) and Quarrion (QU) with homologous regions of published Rht-A1a, Rht-B1and Rht-D1 sequences. Percentage denotes the relative frequency of the amplicon. * denotes a stop codon or an amber mutation. GenBank accession numbers for the sequences areas denoted. Amplicon sequences with Rht-A1a sequence are AU-37%, WH-45%, WW-30%, DR-25%, CA-30% and SP-42% (GenBank JX255420−JX255425 respectively) and Quarrion-35%. Sequences with Rht-B1a include WH-38%, DR-55% and SP-47% (JX255430−JX255432 respectively). Sequences with Rht-B1b include WW-27%, QU-33% and CA-11% (JX255436). Sequences with Rht-D1a include SP-5% (JX255448); CA-7%, WW-5% and Quarrion-5%. Sequences with Rht-D1b include WH-3% and DR-1%. Sequences in Rht Group 1 include CA-11%, WW-7% (JX255456), SP-6% (JX255457), and WH-4% (JX255458). Sequences in Rht Group 3 include AU-15% (JX255464) and Quarrion-3%.</p

Alignment of 5′ UTR sequences from the cultivar, Whistler (WH) with homologous regions of <i>Rht-1</i> alleles in the A, B and D genomes.

<p>The percentage denotes the relative proportions of the amplicon from the accession. The RP of the reference sequence, FN649763 is 2034−2136. The GenBank accession numbers for the sequences from Whistler are as denoted.</p

HRM analysis of <i>Rht-1</i> variants at the C-terminal GRAS domain in WW1842 X Whistler DH population.

<p>Normalized HRM curve for genotyping genetic variation in the <i>Rht-1</i> gene bounded by the primers Rht-F6R6_F1a, Rht-F6R6_F1b and Rht-F6R6_FR1 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069690#pone-0069690-t001" target="_blank">Table 1</a>). The results are shown for parents, WW1842 and Whistler and eight progeny lines of the DH population (180 lines). Genotype data enabled the mapping of the molecular variation on chromosome 4A.</p

Putative spliceosomal introns in GRAS domain of <i>Rht-1</i> genes.

<p>Alignment of (A) DNA and (B) amino acid (from translation) sequences of representative amplicons from the primer pair, Dell_RhtF4/Dell_RhtR4 (RP 2797−3171 of FN649763 or RP 764−1138 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069690#pone.0069690.s003" target="_blank">Table S2</a>). Bases in lower case are putative intron sequences. Amino acid sequences are translated in Frame 1 from (A). Amplicons with the typical <i>Rht-1</i> sequence include SP-100%; WH-97%, WW-95%, DR-88%, AU-68%, CA-50% and QU-12% (JX255470−JX255476 respectively). Amplicons that have the putative intron sequence like QU-56% (JX255483) include WH-2%, WW-1%, DR-6%, AU-12% and CA-45% ((JX255477−JX255482 respectively). Amplicons that have a second intron like AU-2% (JX255486) include WH-0.4% (JX255484) and WW-1% (JX255485). Please refer to nt sequences in GenBank accession numbers for SNPs as annotated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069690#pone.0069690.s003" target="_blank">Table S2</a>.</p

Sequences of the primers used in the amplification of <i>Rht</i>-1 gene fragments from hexaploid wheat.

*<p>nucleotide.</p

Rare predicted loss-of-function variants of type I IFN immunity genes are associated with life-threatening COVID-19

BackgroundWe previously reported that impaired type I IFN activity, due to inborn errors of TLR3- and TLR7-dependent type I interferon (IFN) immunity or to autoantibodies against type I IFN, account for 15-20% of cases of life-threatening COVID-19 in unvaccinated patients. Therefore, the determinants of life-threatening COVID-19 remain to be identified in similar to 80% of cases.MethodsWe report here a genome-wide rare variant burden association analysis in 3269 unvaccinated patients with life-threatening COVID-19, and 1373 unvaccinated SARS-CoV-2-infected individuals without pneumonia. Among the 928 patients tested for autoantibodies against type I IFN, a quarter (234) were positive and were excluded.ResultsNo gene reached genome-wide significance. Under a recessive model, the most significant gene with at-risk variants was TLR7, with an OR of 27.68 (95%CI 1.5-528.7, P=1.1x10(-4)) for biochemically loss-of-function (bLOF) variants. We replicated the enrichment in rare predicted LOF (pLOF) variants at 13 influenza susceptibility loci involved in TLR3-dependent type I IFN immunity (OR=3.70[95%CI 1.3-8.2], P=2.1x10(-4)). This enrichment was further strengthened by (1) adding the recently reported TYK2 and TLR7 COVID-19 loci, particularly under a recessive model (OR=19.65[95%CI 2.1-2635.4], P=3.4x10(-3)), and (2) considering as pLOF branchpoint variants with potentially strong impacts on splicing among the 15 loci (OR=4.40[9%CI 2.3-8.4], P=7.7x10(-8)). Finally, the patients with pLOF/bLOF variants at these 15 loci were significantly younger (mean age [SD]=43.3 [20.3] years) than the other patients (56.0 [17.3] years; P=1.68x10(-5)).ConclusionsRare variants of TLR3- and TLR7-dependent type I IFN immunity genes can underlie life-threatening COVID-19, particularly with recessive inheritance, in patients under 60 years old