Identification of limb-specific Lmx1b auto-regulatory modules with Nail-patella syndrome pathogenicity

LMX1B haploinsufficiency causes Nail-patella syndrome (NPS; MIM 161200), characterized by nail dysplasia, absent/hypoplastic patellae, chronic kidney disease, and glaucoma. Accordingly in mice, Lmx1b has been shown to play crucial roles in the development of the limb, kidney and eye. Although one functional allele of Lmx1b appears adequate for development, Lmx1b null mice display ventral-ventral distal limbs with abnormal kidney, eye and cerebellar development, more disruptive, but fully concordant with NPS. In Lmx1b functional knockouts (KOs), Lmx1b transcription in the limb is decreased nearly 6-fold, indicating autoregulation. Herein, we report on two conserved Lmx1b-associated cis-regulatory modules (LARM1 and LARM2) that are bound by Lmx1b, amplify Lmx1b expression with unique spatial modularity in the limb, and are necessary for Lmx1b-mediated limb dorsalization. These enhancers, being conserved across vertebrates (including coelacanth, but not other fish species), and required for normal locomotion, provide a unique opportunity to study the role of dorsalization in the fin to limb transition. We also report on two NPS patient families with normal LMX1B coding sequence, but with loss-of-function variations in the LARM1/2 region, stressing the role of regulatory modules in disease pathogenesis.

The Response of Wheat with Different Allele Statuses of the Gpc-B1 Gene under Zinc Deficiency

The aim of this study was to investigate the effect of zinc (Zn) deficiency on the growth and grain yield of wheat with different allele statuses of the Gpc-B1 gene. For this research, common wild emmer wheat (Triticum turgidum ssp. dicoccoides (Koern. ex Asch. &Graebn.) Schweinf.), bread wheat (Triticum aestivum L. cv. Festivalnaya), and two intogressive lines were used. T. dicoccoides and introgressive line 15-7-1 carry a functional allele of the Gpc-B1 gene, while the T. aestivum cv. Festivalnaya and introgressive line 15-7-2 carry the non-functional Gpc-B1 allele. Zn deficiency did not affect the shoot height or fresh weight of any of the studied plants. The only exception was T. dicoccoides, where a small decrease in shoot height was registered. Additionally, under Zn deficiency T. dicoccoides had an increase in flag leaf area, spike length, and dry weight, as well as in grain number and grain yield per spike. The other variants did not experience changes in the above-described parameters under Zn deficiency. Under Zn deficiency, the Zn concentration in the grains was higher in the plants with a functional allele of the Gpc-B1 gene compared to the plants with a non-functional allele. These results show that wheat with a functional allele of the Gpc-B1 gene growing under Zn deficiency is capable of grain production with a sufficient Zn concentration without a decrease in yield.

Validation of a Novel Fgf10Cre–ERT2 Knock-in Mouse Line Targeting FGF10Pos Cells Postnatally

Fgf10 is a key gene during development, homeostasis and repair after injury. We previously reported a knock-in Fgf10Cre–ERT2 line (with the Cre-ERT2 cassette inserted in frame with the start codon of exon 1), called thereafter Fgf10Ki–v1, to target FGF10Pos cells. While this line allowed fairly efficient and specific labeling of FGF10Pos cells during the embryonic stage, it failed to target these cells after birth, particularly in the postnatal lung, which has been the focus of our research. We report here the generation and validation of a new knock-in Fgf10Cre–ERT2 line (called thereafter Fgf10Ki–v2) with the insertion of the expression cassette in frame with the stop codon of exon 3. Fgf10Ki−v2/+ heterozygous mice exhibited comparable Fgf10 expression levels to wild type animals. However, a mismatch between Fgf10 and Cre expression levels was observed in Fgf10Ki–v2/+ lungs. In addition, lung and limb agenesis were observed in homozygous embryos suggesting a loss of Fgf10 functional allele in Fgf10Ki–v2 mice. Bioinformatic analysis shows that the 3′UTR, where the Cre-ERT2 cassette is inserted, contains numerous putative transcription factor binding sites. By crossing this line with tdTomato reporter line, we demonstrated that tdTomato expression faithfully recapitulated Fgf10 expression during development. Importantly, Fgf10Ki–v2 mouse is capable of significantly targeting FGF10Pos cells in the adult lung. Therefore, despite the aforementioned limitations, this new Fgf10Ki–v2 line opens the way for future mechanistic experiments involving the postnatal lung.

Characterization of a novel Fgf10CreERT2 knock-in mouse line targeting postnatal lung Fgf10 lineages

Fgf10 is a key gene during development, homeostasis and repair after injury. We previously reported a Fgf10CreERT2 line (with the CreERT2 cassette inserted in frame with the start codon of exon 1), called thereafter Fgf10Ki-v1, to target Fgf10Pos cells. While this line allowed fairly efficient and specific labeling of Fgf10Pos cells during the embryonic stage, it failed to target these cells after birth, particularly in the postnatal lung, which has been the focus on our research. We report here the generation and validation of a new Fgf10CreERT2 (called thereafter Fgf10Ki-v2) with the insertion of the expression cassette in frame with the stop codon of exon 3. This new Fgf10Ki-v2 line exhibited comparable Fgf10 expression level to their wild type counterpart. However, a disconnection between the Fgf10 and the Cre expression was observed in Fgf10Ki-v2/+ lungs. In addition, lung and limb agenesis were observed in homozygous embryos suggesting a loss of Fgf10 functional allele in Fgf10Ki-v2 mice. Bio-informatics analysis shows that the 3’UTR, where the CreERT2 cassette is inserted, contains numerous putative transcription factor binding sites. By crossing this line with tdTomato reporter line, we demonstrated that tdTomato expression faithfully recapitulated Fgf10 expression during development. Significantly, Fgf10Ki-v2 mouse is capable of significantly targeting Fgf10Pos cells in the adult lung. Therefore, despite the aforementioned limitations, this new Fgf10Ki-v2 line opens the way for future mechanistic experiments involving the postnatal lung.

The genetics and physiology of seed dormancy, a crucial trait in common bean domestication

Background Physical seed dormancy is an important trait in legume domestication. Although seed dormancy is beneficial in wild ecosystems, it is generally considered to be an undesirable trait in crops due to reduction in yield and / or quality. The physiological mechanism and underlying genetic factor(s) of seed dormancy is largely unknown in several legume species. Here we employed an integrative approach to understand the mechanisms controlling physical seed dormancy in common bean (Phaseolus vulgaris L.). Results Using an innovative CT scan imaging system, we were able to track water movements inside the seed coat. We found that water uptake initiates from the bean seed lens. Using a scanning electron microscopy (SEM) we further identified several micro-cracks on the lens surface of non-dormant bean genotypes. Bulked segregant analysis (BSA) was conducted on a bi-parental RIL (recombinant inbred line) population, segregating for seed dormancy. This analysis revealed that the seed water uptake is associated with a single major QTL on Pv03. The QTL region was fine-mapped to a 118 Kb interval possessing 11 genes. Coding sequence analysis of candidate genes revealed a 5-bp insertion in an ortholog of pectin acetylesterase 8 that causes a frame shift, loss-of-function mutation in non-dormant genotype. Gene expression analysis of the candidate genes in the seed coat of contrasting genotypes indicated 21-fold lower expression of pectin acetylesterase 8 in non-dormant genotype. An analysis of mutational polymorphism was conducted among wild and domesticated beans. Although all the wild beans possessed the functional allele of pectin acetylesterase 8, the majority (77%) of domesticated beans had the non-functional allele suggesting that this variant was under strong selection pressure through domestication. Conclusions In this study, we identified the physiological mechanism of physical seed dormancy and have identified a candidate allele causing variation in this trait. Our findings suggest that a 5-bp insertion in an ortholog of pectin acetylesterase 8 is likely a major causative mutation underlying the loss of seed dormancy during domestication. Although the results of current study provide strong evidences for the role of pectin acetylesterase 8 in seed dormancy, further confirmations seem necessary by employing transgenic approaches.

Characteristics of promising strawberry varieties and elite forms by chemical composition and genes of the aromatic complex of fruits

The results of the analysis of promising strawberry genotypes by the chemical composition and genes, involved in the determination of fruit aroma, are presented. By the complex of chemical traits, the strawberry varieties and elite forms of breeding of the I.V. Michurin Federal Scientific Center are highlighted: Flora (sugars – 9.2%, ascorbic acid – 65.0 mg/100 g, anthocyanins – 74.2 mg/100 g), elite seedling 56-5 (Gigantella Maxim × Privlekatelnaya) (sugars – 8.1%, ascorbic acid – 83.5 mg/100 g, anthocyanins – 64.3 mg/100 g). These strawberry forms are also characterized by a homozygous state of the functional allele of the FaOMT gene of the aromatic complex of fruits, which indicates high mesifurane content in fruits. The research results can be used in further work on the creation of strawberry varieties with a high level of taste and aromatic qualities, and also the antioxidant value of the fruits.

Compromised chitin synthesis in lager yeast affects its Congo red resistance and release of mannoproteins from the cells

ABSTRACT A mutant lager strain resistant to the cell wall-perturbing agent Congo red (CR) was isolated and the genetic alterations underlying CR resistance were investigated by whole genome sequencing. The parental lager strain was found to contain three distinct Saccharomyces cerevisiae (Sc)-type CHS6 (CHitin Synthase-related 6) alleles, two of which have one or two nonsense mutations in the open reading frame, leaving only one functional allele, whereas the functional allele was missing in the isolated CR-resistant strain. On the other hand, the Saccharomyces eubayanus-type CHS6 alleles shared by both the parental and mutant strains appeared to contribute poorly to chitin synthase-activating function. Therefore, the CR resistance of the mutant strain was attributable to the overall compromised activity of CHS6 gene products. The CR-resistant mutant cells exhibited less chitin production on the cell surface and smaller amounts of mannoprotein release into the medium. All these traits, in addition to the CR resistance, were complemented by the functional ScCHS6 gene. It is of great interest whether the frequent nonsense mutations found in ScCHS6 open reading frame in lager yeast strains are a consequence of the domestication process of lager yeast.