Predictive ability of genomic selection models in a multi-population perennial ryegrass training set using genotyping-by-sequencing

Key message

Genomic prediction models for multi-year dry matter yield, via genotyping-by-sequencing in a composite training set, demonstrate potential for genetic gain improvement through within-half sibling family selection.

Abstract

Perennial ryegrass (Lolium perenne L.) is a key source of nutrition for ruminant livestock in temperate environments worldwide. Higher seasonal and annual yield of herbage dry matter (DMY) is a principal breeding objective but the historical realised rate of genetic gain for DMY is modest. Genomic selection was investigated as a tool to enhance the rate of genetic gain. Genotyping-by-sequencing (GBS) was undertaken in a multi-population (MP) training set of five populations, phenotyped as half-sibling (HS) families in five environments over 2 years for mean herbage accumulation (HA), a measure of DMY potential. GBS using the ApeKI enzyme yielded 1.02 million single-nucleotide polymorphism (SNP) markers from a training set of n = 517. MP-based genomic prediction models for HA were effective in all five populations, cross-validation-predictive ability (PA) ranging from 0.07 to 0.43, by trait and target population, and 0.40–0.52 for days-to-heading. Best linear unbiased predictor (BLUP)-based prediction methods, including GBLUP with either a standard or a recently developed (KGD) relatedness estimation, were marginally superior or equal to ridge regression and random forest computational approaches. PA was principally an outcome of SNP modelling genetic relationships between training and validation sets, which may limit application for long-term genomic selection, due to PA decay. However, simulation using data from the training experiment indicated a twofold increase in genetic gain for HA, when applying a prediction model with moderate PA in a single selection cycle, by combining among-HS family selection, based on phenotype, with within-HS family selection using genomic prediction.

     

Development of pipette tip gap closure migration assay (s-ARU method) for studying semi-adherent cell lines

This work presents a pipette tip gap closure migration assay prototype tool (semi-adherent relative upsurge—s-ARU—method) to study cell migration or wound healing in semi-adherent cell lines, such as lymph node carcinoma of the prostate (LNCaP). Basically, it consists of a 6-well cover plate modification, where pipette tips with the filter are shortened and fixed vertically to the inner surface of the cover plate, with their heights adjusted to touch the bottom of the well center. This provides a barrier for the inoculated cells to grow on, creating a cell-free gap. Such a uniform gap formed can be used to study migration assay for both adherent as well as semi-adherent cells. After performing time studies, effective measurement of gap area can be carried out conveniently through image analysis software. Here, the prototype was tested for LNCaP cells, treated with testosterone and flutamide as well as with bacteriophages T4 and M13. A scratch assay using PC3 adherent cells was also performed for comparison. It was observed that s-ARU method is suitable for studying LNCaP cells migration assay, as observed from our results with testosterone, flutamide, and bacteriophages (T4 and M13). Our method is a low-cost handmade prototype, which can be an alternative to the other migration assay protocol(s) for both adherent and semi-adherent cell cultures in oncological research along with other biological research applications.     

A novel templates of piperazinyl-1,2-dihydroquinoline-3-carboxylates: Synthesis,anti-microbial evaluation and molecular docking studies

A series of piperazinyl-1,2-dihydroquinoline carboxylates were synthesized by the reaction of ethyl 4-chloro-1-methyl-2-oxo-1,2-dihydroquinoline-3-carboxylates with various piperazines and their structures were confirmed by ¹H NMR, ¹³C NMR, IR and mass spectral analysis. All the synthesized compounds were screened for their in vitro antimicrobial activities. Further, the in silico molecular docking studies of the active compounds was performed to explore the binding interactions between piperazinyl-1,2-dihydroquinoline carboxylate derivatives and the active site of the Staphylococcus aureus (CrtM) dehydrosqualene synthase (PDB ID: 2ZCQ). The docking studies revealed that the synthesized derivatives showed high binding energies and strong H-bond interactions with the dehydrosqualene synthase validating the observed antimicrobial activity data. Based on antimicrobial activity and docking studies, the compounds 9b and 10c were identified as promising antimicrobial lead molecules. This study might provide insights to identify new drug candidates that target the S. aureus virulence factor, dehydrosqualene synthase.     

Thiazole- and selenazole-comprising high-affinity inhibitors possess bright microsecond-scale photoluminescence in complex with protein kinase CK2

A previously disclosed protein kinase (PK) CK2-selective inhibitor 4-(2-amino-1,3-thiazol-5-yl)benzoic acid (ATB) and its selenium-containing counterpart (ASB) revealed remarkable room temperature phosphorescence when bound to the ATP pocket of the protein kinase CK2. Conjugation of these fragments with a mimic of CK2 substrate peptide resulted in bisubstrate inhibitors with increased affinity towards the kinase. Attachment of the fluorescent acceptor dye 5-TAMRA to the conjugates led to significant enhancement of intensity of long-lifetime (microsecond-scale) photoluminescence of both sulfur- and selenium-containing compounds. The developed photoluminescent probes make possible selective determination of the concentration of CK2 in cell lysates and characterization of CK2 inhibitors by means of time-gated measurement of photoluminescence.     

Chill-responsive dehydrins in blueberry: Are they associated with cold hardiness or dormancy transitions?

To survive winters, woody perennials of temperate zones must enter into endodormancy. Resumption of spring growth requires sufficient exposure to low temperature (chill units, CUs) in winter (chilling requirement), which also plays a role in the development of cold hardiness (cold acclimation). Physiological studies on dormancy breaking have focused on identifying markers, such as appearance or disappearance of proteins in response to varying degrees of chill unit accumulation. However, whether these changes are associated with dormancy transitions or cold acclimation is not clear. In the present study, greenhouse-grown blueberry (Vaccinium section Cyanococcus) plants were used to address this question. Three blueberry cultivars, Bluecrop, Tifblue, and Gulfcoast having chilling requirement of approximately 1 200, 900 and 600 CUs, respectively, were first exposed to 4°C for long enough to provide chill units equivalent to one-half of their respective chilling requirement. This treatment was expected to result in cold acclimation. A fraction of plants was then subjected to a 15/12°C (light/dark) regime for 2 weeks, a treatment expected to be “dormancy-neutral” but cause deacclimation. Before and after each treatment, cold hardiness and dormancy status of floral buds were determined; proteins were extracted from the buds collected on the same sampling date, and separated by one-dimensional SDS-PAGE. Dehydrin-like proteins were identified by immunoblotting, using anti-dehydrin antiserum. Results indicate that the chilling treatment resulted in cold acclimation as indicated by increased bud hardiness in all three cultivars. Data also indicate a distinct accumulation of three dehydrin-like proteins of 65, 60, and 14 kDa during cold acclimation. The cold hardiness and levels of dehydrin proteins decreased during the exposure to 15/12°C for 2 weeks. Results also confirmed that this treatment had no negative effect on chill unit accumulation. Densitometric scans of protein gels indicated a close association between the abundance of dehydrins and degree of cold hardiness in these cultivars. In addition, levels of the dehydrin proteins and cold hardiness remained about the same between 100% and >100% satisfaction of chilling requirement. These results suggest that changes in dehydrin expression are more closely associated with cold hardiness than with dormancy transitions.     

Triplex formation at physiological pH by 5-Me-dC-N4-(spermine) [X] oligodeoxynucleotides: non protonation of N3 in X of X*G:C triad and effect of base mismatch/ionic strength on triplex stabilities.

Oligodeoxynucleotide (ODN) directed triplex formation has therapeutic importance and depends on Hoogsteen hydrogen bonds between a duplex DNA and a third DNA strand. T*A:T triplets are formed at neutral pH and C+*G:C are favoured at acidic pH. It is demonstrated that spermine conjugation at N4 of 5-Me-dC in ODNs 1-5 (sp-ODNs) imparts zwitterionic character, thus reducing the net negative charge of ODNs 1-5. sp-ODNs form triplexes with complementary 24mer duplex 8:9 show foremost stability at neutral pH 7.3 and decrease in stability towards lower pH, unlike the normal ODNs where optimal stability is found at an acidic pH 5.5. At pH 7.3, control ODNs 6 and 7 carrying dC or 5-Me-dC, respectively, do not show any triple helix formation. The stability order of triplex containing 5-Me-dC-N4-(spermine) with normal and mismatched duplex was found to be X*G:C approximately X*A:T > X*C:G > X*T:A. The hysteresis curve of sp-ODN triplex 3*8:9 indicated a better association with complementary duplex 8:9 as compared to unmodified ODN 6 in triplex 6*8:9. pH-dependent UV difference spectra suggest that N3 protonation is not a requirement for triplex formation by sp-ODN and interstrand interaction of conjugated spermine more than compensates for loss in stability due to absence of a single Hoogsteen hydrogen bond. These results may have importance in designing oligonucleotides for antigene applications.     

Novel thrombin inhibitors that are based on a macrocyclic tripeptide motif

A series of macrocyclic -keto amides containing the D-Phe-Pro-Arg (fPR) motif were synthesized and evaluated in vitro as inhibitors of human -thrombin and bovine trypsin. Structure-function studies, relating ring size and modifications at the P3 and P1' positions to enzyme inhibition, are described. An X-ray crystallo-graphic study was performed on a ternary complex formed from 3i, thrombin, and hirugen.     

Triplex formation at physiological pH: comparative studies on DNA triplexes containing 5-Me-dC tethered at N4 with spermine and tetraethyleneoxyamine.

Oligodeoxynucleotides with spermine conjugation at C4 of 5-Me-dC ( sp -ODN) exhibit triple helix formation with complementary Watson-Crick duplexes, and were optimally stable at physiological pH 7.3 and low salt concentration. This was attributed to a favored reassociation of the polycationic third strand with the anionic DNA duplex. To gain further insights into the factors that contribute to the enhancement of triplex stability and for engineering improved triplex systems, the spermine appendage at C4 of 5-Me-dC was replaced with 1,11-diamino-3,6,9-trioxaundecane to create teg -ODNs. From the triple helix forming abilities of these modified ODNs studied by hysteresis behaviour and the effect of salts on triplex stability, it is demonstrated here that teg- ODNs stabilise triplexes through hydrophobic desolvation while sp -ODNs stabilise triplexes by charge effects. The results imply that factors in addition to base stacking effects and interstrand hydrogen bonds are significantly involved in modulation of triplex stability by base modified oligonucleotides.