The Fastest and Most Reliable Identification of True Hybrids in the Genus Pisum L.

After crosses, the identification of true hybrids is not only the most important step in the initiation of a breeding program but also plays a crucial role in the improvement of hybrid varieties. However, current morphological or molecular-based hybrid identification methods are time-consuming and costly approaches that require knowledge and skill, as well as specific lab equipment. In the current study, xenia, direct or immediate effect of pollen on seeds was used to identify true hybrids in the genus Pisum L. for the first time without growing F1 plants. The current study was therefore aimed to (i) elucidate the xenia effect on seeds in intra- and interspecific crosses between P. sativum L. subsp. sativum var. sativum or var. arvense L. Poir. and its wild relatives, including P. sativum subsp. elatius (M. Bieb.) Aschers & Graebn. and P. fulvum Sibth. & Sm., and (ii) illuminate the beneficialness of the xenia effect in a practical improvement of the genus Pisum L. The pea cultivars, including P. sativum subsp. sativum var. sativum and P. sativum subsp. sativum var. arvense, were therefore crossed with P. sativum subsp. elatius and P. fulvum, and the occurrence of the xenia effect was studied on the seeds of fertilized female plants immediately after the crosses. It was concluded that using the xenia effect for the early detection of true hybrid immediately after crossing was not only the fastest, most reliable, and least expensive option as early selection criteria, but that xenia also provided information about dominant seed and pod traits after double fertilization.

Genome-wide discovery of di-nucleotide SSR markers based on whole genome re-sequencing data of Cicer arietinum L. and Cicer reticulatum Ladiz.

Simple sequence repeats (SSRs) are valuable genetic markers due to their co-dominant inheritance, multi-allelic and reproducible nature. They have been largely used for exploiting genetic architecture of plant germplasms, phylogenetic analysis, and mapping studies. Among the SSRs, di-nucleotide repeats are the most frequent of the simple repeats distributed throughout the plant genomes. In present study, we aimed to discover and develop di-nucleotide SSR markers by using the whole genome re-sequencing (WGRS) data from Cicer arietinum L. and C. reticulatum Ladiz. A total of 35,329 InDels were obtained in C. arietinum, whereas 44,331 InDels in C. reticulatum. 3387 InDels with 2 bp length were detected in C. arietinum, there were 4704 in C. reticulatum. Among 8091 InDels, 58 di-nucleotide regions that were polymorphic between two species were selected and used for validation. We tested primers for evaluation of genetic diversity in 30 chickpea genotypes including C. arietinum, C. reticulatum, C. echinospermum P.H. Davis, C. anatolicum Alef., C. canariense A. Santos & G.P. Lewis, C. microphyllum Benth., C. multijugum Maesen, C. oxyodon Boiss. & Hohen. and C. songaricum Steph ex DC. A total of 244 alleles were obtained for 58 SSR markers giving an average of 2.36 alleles per locus. The observed heterozygosity was 0.08 while the expected heterozygosity was 0.345. Polymorphism information content was found to be 0.73 across all loci. Phylogenetic tree and principal coordinate analysis clearly divided the accessions into four groups. The SSR markers were also evaluated in 30 genotypes of a RIL population obtained from an interspecific cross between C. arietinum and C. reticulatum. Chi-square (χ2) test revealed an expected 1:1 segregation ratio in the population. These results demonstrated the success of SSR identification and marker development for chickpea with the use of WGRS data. The newly developed 58 SSR markers are expected to be useful for chickpea breeders.

A kabuli chickpea ideotype.

The concept of 'crop ideotype' is coined as a desirable plant model expected to better perform for seed yield, oils and other useful characteristics when developed as a cultivar, and it consists of two major approaches, namely, (i) 'defect elimination', that is, integration of disease resistance to a susceptible genotype from a resistant genotype and (ii) 'selection for yield' by improving yield after crosses between desirable parents. For consideration of these approaches, here we introduced an ideotype in kabuli chickpea (Cicer arietinum L.) which is high-yielding, extra-large-seeded, and double- or multi-podded, has high plant height and imparipinnate-leafed traits, and is heat tolerant and resistant to ascochyta blight [Ascochyta rabiei (Pass.) Labr.], which causes considerable yield losses, via marker-assisted selection. F3 and F4 lines were evaluated for agro-morphological traits divided into six classes, namely, (i) imparipinnate-leafed and single-podded progeny, (ii) imparipinnate-leafed and double-podded progeny, (iii) imparipinnate-leafed and multi-podded progeny, (iv) unifoliolate-leafed and single-podded progeny, (v) unifoliolate-leafed and double-podded progeny, (vi) unifoliolate-leafed and multi-podded progeny. F3:4 lines having 100-seed weight ≥ 45 g and double- or multi-podded traits were additionally assessed for resistance to ascochyta blight using molecular markers including SCY17590 and CaETR-1. Superior lines having higher values than their best parents were determined for all studied traits indicating that economic and important traits including yield and seed size in chickpea could be improved by crossing suitable parents. Imparipinnate-leafed and multi-podded plants had not only the highest number of pods and seeds per plant but also the highest yield. On the other hand, imparipinnate-leafed and single podded progeny had the largest seed size, followed by imparipinnate-leafed and double-podded progeny. Multi-podded plants produced 23% more seed yield than that of single-podded plants, while multi-podded plants attained 7.6% more seed yield than that of double-podded plants. SCY17590 and CaETR-1 markers located on LG4 related to QTLAR2 and QTLAR1 were found in 14 lines among 152 F3:4 lines. Six superior lines were selected for being double- or multi-podded, imparipinnate-leafed, suitable for combine harvest, heat-tolerant, and resistant to ascochyta blight, and having both of two resistance markers and extra-large seeds as high as 50-60 g per 100-seed weight. Resistance alleles from two different backgrounds for resistance to ascochyta blight were integrated with double- or multi-podded kabuli chickpea lines having high yield, extra-large seeds, high plant height, imparipinnate-leaves and high heat tolerance, playing a crucial role for future demands of population and food security. These approaches seem to be applicable in ideotype breeding for other important crop plants.

De novo super-early progeny in interspecific crosses Pisum sativum L. × P. fulvum Sibth. et Sm.

Earliness in crop plants has a crucial role in avoiding the stress of drought and heat, which are the most important challenging stressors in crop production and are predicted to increase in the near future due to global warming. Furthermore, it provides a guarantee of vegetable production in the short growing season of agricultural lands in the northern hemisphere and at high altitudes. The growing human population needs super early plant cultivars for these agricultural lands to meet future global demands. This study examined de novo super-early progeny, referred to as much earlier than that of the earlier parent, which flowered in 13-17 days and pod setting in 18-29 days after germination, discovered in F2 and studied up to F5 derived from interspecific crosses between garden pea (P. sativum L.) and the most distant relative of pea (P. fulvum Sibth. et Sm.). De novo super-early progeny were found to be earlier by about one month than P. sativum and two months than P. fulvum under short day conditions in the F5 population. In respect of days to flowering and pod setting, de novo super-early progeny had a relatively high level of narrow sense heritability (h2 = 82% and 80%, respectively), indicating that the selections for earliness in segregating populations was effective for improvement of extreme early maturing varieties. De novo super-early progeny could be grown under heat stress conditions due to the escape ability. Vegetable types were not only high yielding but also free of any known undesirable traits from the wild species, such as pod dehiscence and non-uniform maturity. It could be considered complementary to "speed breeding", possibly obtaining more than six generations per year in a suitable climate chamber. Not only de novo super-early progeny but also transgressive segregation for agro-morphological traits can be created via interspecific crosses between P. sativum and P. fulvum, a precious unopened treasure in the second gene pool. Useful progeny obtained from crossing wild species with cultivated species reveal the importance of wild species.

Cicer turcicum: A New Cicer Species and Its Potential to Improve Chickpea.

Genetic resources of the genus Cicer L. are not only limited when compared to other important food legumes and major cereal crops but also, they include several endemic species with endangered status based on the criteria of the International Union for Conservation of Nature. The chief threats to endemic and endangered Cicer species are over-grazing and habitat change in their natural environments driven by climate changes. During a collection mission in east and south-east Anatolia (Turkey), a new Cicer species was discovered, proposed here as C. turcicum Toker, Berger & Gokturk. Here, we describe the morphological characteristics, images, and ecology of the species, and present preliminary evidence of its potential utility for chickpea improvement. C. turcicum is an annual species, endemic to southeast Anatolia and to date has only been located in a single population distant from any other known annual Cicer species. It belongs to section Cicer M. Pop. of the subgenus Pseudononis M. Pop. of the genus Cicer L. (Fabaceae) and on the basis of internal transcribed spacer (ITS) sequence similarity appears to be a sister species of C. reticulatum Ladiz. and C. echinospermum P.H. Davis, both of which are inter-fertile with domestic chickpea (C. arietinum L.). With the addition of C. turcicum, the genus Cicer now comprises 10 annual and 36 perennial species. As a preliminary evaluation of its potential for chickpea improvement two accessions of C. turcicum were field screened for reproductive heat tolerance and seeds were tested for bruchid resistance alongside a representative group of wild and domestic annual Cicer species. C. turcicum expressed the highest heat tolerance and similar bruchid resistance as C. judaicum Boiss. and C. pinnatifidum Juab. & Spach, neither of which are in the primary genepool of domestic chickpea. Given that C. arietinum and C. reticulatum returned the lowest and the second lowest tolerance and resistance scores, C. turcicum may hold much potential for chickpea improvement if its close relatedness supports interspecific hybridization with the cultigen. Crossing experiments are currently underway to explore this question.

Dopamine-imprinted monolithic column for capillary electrochromatography.

A dopamine-imprinted monolithic column was prepared and used in capillary electrochromatography as stationary phase for the first time. Dopamine was selectively separated from aqueous solution containing the competitor molecule norepinephrine, which is similar in size and shape to the template molecule. Morphology of the dopamine-imprinted column was observed by scanning electron microscopy. The influence of the organic solvent content of mobile phase, applied pressure and pH of the mobile phase on the recognition of dopamine by the imprinted monolithic column has been evaluated, and the imprinting effect in the dopamine-imprinted monolithic polymer was verified. Developed dopamine-imprinted monolithic column resulted in excellent separation of dopamine from structurally related competitor molecule, norepinephrine. Separation was achieved in a short period of 10 min, with the electrophoretic mobility of 5.81 × 10-5  m2 V-1 s-1 at pH 5.0 and 500 mbar pressure.

The adaptor molecule RIAM integrates signaling events critical for integrin-mediated control of immune function and cancer progression.

Lymphocyte activation requires adhesion to antigen-presenting cells. This is a critical event linking innate and adaptive immunity. Lymphocyte adhesion is accomplished through LFA-1, which must be activated by a process referred to as inside-out integrin signaling. Among the few signaling molecules that have been implicated in inside-out integrin activation in hematopoietic cells are the small guanosine triphosphatase (GTPase) Rap1 and its downstream effector Rap1-interacting molecule (RIAM), a multidomain protein that defined the Mig10-RIAM-lamellipodin (MRL) class of adaptor molecules. Through its various domains, RIAM is a critical node of signal integration for activation of T cells, recruits monomeric and polymerized actin to drive actin remodeling and cytoskeletal reorganization, and promotes inside-out integrin signaling in T cells. As a regulator of inside-out integrin activation, RIAM affects multiple functions of innate and adaptive immunity. The effects of RIAM on cytoskeletal reorganization and integrin activation have implications in cell migration and trafficking of cancer cells. We provide an overview of the structure and interactions of RIAM, and we discuss the implications of RIAM functions in innate and adaptive immunity and cancer.

The MultiBac Baculovirus/Insect Cell Expression Vector System for Producing Complex Protein Biologics.

Multiprotein complexes regulate most if not all cellular functions. Elucidating the structure and function of these complex cellular machines is essential for understanding biology. Moreover, multiprotein complexes by themselves constitute powerful reagents as biologics for the prevention and treatment of human diseases. Recombinant production by the baculovirus/insect cell expression system is particularly useful for expressing proteins of eukaryotic origin and their complexes. MultiBac, an advanced baculovirus/insect cell system, has been widely adopted in the last decade to produce multiprotein complexes with many subunits that were hitherto inaccessible, for academic and industrial research and development. The MultiBac system, its development and numerous applications are presented. Future opportunities for utilizing MultiBac to catalyze discovery are outlined.

PD-1 alters T-cell metabolic reprogramming by inhibiting glycolysis and promoting lipolysis and fatty acid oxidation.

During activation, T cells undergo metabolic reprogramming, which imprints distinct functional fates. We determined that on PD-1 ligation, activated T cells are unable to engage in glycolysis or amino acid metabolism but have an increased rate of fatty acid ß-oxidation (FAO). PD-1 promotes FAO of endogenous lipids by increasing expression of CPT1A, and inducing lipolysis as indicated by elevation of the lipase ATGL, the lipolysis marker glycerol and release of fatty acids. Conversely, CTLA-4 inhibits glycolysis without augmenting FAO, suggesting that CTLA-4 sustains the metabolic profile of non-activated cells. Because T cells utilize glycolysis during differentiation to effectors, our findings reveal a metabolic mechanism responsible for PD-1-mediated blockade of T-effector cell differentiation. The enhancement of FAO provides a mechanistic explanation for the longevity of T cells receiving PD-1 signals in patients with chronic infections and cancer, and for their capacity to be reinvigorated by PD-1 blockade.

PD-1 increases PTEN phosphatase activity while decreasing PTEN protein stability by inhibiting casein kinase 2.

Programmed death 1 (PD-1) is a potent inhibitor of T cell responses. PD-1 abrogates activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, but the mechanism remains unclear. We determined that during T cell receptor (TCR)/CD3- and CD28-mediated stimulation, PTEN is phosphorylated by casein kinase 2 (CK2) in the Ser380-Thr382-Thr383 cluster within the C-terminal regulatory domain, which stabilizes PTEN, resulting in increased protein abundance but suppressed PTEN phosphatase activity. PD-1 inhibited the stabilizing phosphorylation of the Ser380-Thr382-Thr383 cluster within the C-terminal domain of PTEN, thereby resulting in ubiquitin-dependent degradation and diminished abundance of PTEN protein but increased PTEN phosphatase activity. These effects on PTEN were secondary to PD-1-mediated inhibition of CK2 and were recapitulated by pharmacologic inhibition of CK2 during TCR/CD3- and CD28-mediated stimulation without PD-1. Furthermore, PD-1-mediated diminished abundance of PTEN was reversed by inhibition of ubiquitin-dependent proteasomal degradation. Our results identify CK2 as a new target of PD-1 and reveal an unexpected mechanism by which PD-1 decreases PTEN protein expression while increasing PTEN activity, thereby inhibiting the PI3K/Akt signaling axis.

PD-1 inhibits T cell proliferation by upregulating p27 and p15 and suppressing Cdc25A.

The programmed cell death-1 (PD)-1 receptor (CD279) is a potent T cell inhibitor with a critical role in peripheral tolerance, but it can also compromise anti-viral and antitumor T cell responses. The effects of PD-1 on the cell cycle leading to inhibition of T cell expansion are poorly understood. Recently, we examined the effects of PD-1 on the molecular control of the cell cycle machinery and on TCR-activated signaling pathways that regulate these downstream outcomes. Our studies showed that PD-1 blocks cell cycle progression in the G 1 phase. PD-1 did not alter the expression of G 1 phase cyclins or cyclin-dependent kinases (Cdks) but, instead, suppressed the transcription of SKP2, the substrate recognition component of the SCF (Skp2) ubiquitin ligase that leads p27 (kip1) to degradation and resulted in accumulation of p27 (kip1) . Subsequently, T cells receiving PD-1 signals displayed impaired Cdk2 activation and failed to phosphorylate two critical Cdk2 substrates, the retinoblastoma gene product (Rb) and the TGFß-specific transcription factor Smad3, leading to suppression of E2F target genes but enhanced Smad3 transactivation. These events resulted in upregulation of the Cdk4/6 inhibitor p15 (INK4B) and repression of the Cdk-activating phosphatase Cdc25A. The suppressive effect of PD-1 on Skp2 expression was mediated by inhibition of both PI3K/Akt and Ras/MEK/Erk pathways and was only partially reversed by IL-2, which restored activation of MEK/Erk but not Akt. Thus, PD-1 targets Ras and PI3K/Akt signaling to inhibit transcription of Skp2 and to activate Smad3 as an integral component of a pathway that regulates blockade of cell cycle progression in T lymphocytes. Here, we discuss the detailed sequence of these signaling events and their implications in mediating cell-intrinsic and -extrinsic mechanisms that inhibit proliferation of T effector cells in response to PD-1-mediated signaling.