Comparison of whole-genome amplifications for microsatellite genotyping of \u3ci\u3eRotylenchulus reniformis\u3c/i\u3e

Currently, a large number of microsatellites are available for Rotylenchulus reniformis (reniform nematode); however, two barriers exist for genotyping samples from different geographical areas. The limited amount of nucleic acids obtained from single nematodes which would require their multiplication to obtain enough DNA for testing; and the strictly regulated transport of live samples and multiplication in greenhouse for being a plant pathogen. Whole-genome amplification (WGA) of samples consisting of one and five dead gravid females with their associated egg masses was successfully performed on disrupted tissue using three commercial kits. DNA yield after WGA ranged from 0.5 to 8 μg and was used to test 96 microsatellite markers we previously developed for the reniform nematode. The results were compared to those of fingerprinting the original population (MSRR03). Out of 96 markers tested, 71 had amplicons in MSRR03. Using WGA of single gravid females with their associated egg masses, 86-93% of the alleles found on MSRR03 were detected, and 87-88% of the alleles found on MSRR03 when using WGA of samples composed of five gravid females with their associated egg masses as template. Our results indicate that reniform nematode samples as small as a single gravid female with her associated egg mass can be used in WGA and direct testing with microsatellites, giving consistent results when compared to the original population

Comparison of whole-genome amplifications for microsatellite genotyping of \u3ci\u3eRotylenchulus reniformis\u3c/i\u3e

Currently, a large number of microsatellites are available for Rotylenchulus reniformis (reniform nematode); however, two barriers exist for genotyping samples from different geographical areas. The limited amount of nucleic acids obtained from single nematodes which would require their multiplication to obtain enough DNA for testing; and the strictly regulated transport of live samples and multiplication in greenhouse for being a plant pathogen. Whole-genome amplification (WGA) of samples consisting of one and five dead gravid females with their associated egg masses was successfully performed on disrupted tissue using three commercial kits. DNA yield after WGA ranged from 0.5 to 8 μg and was used to test 96 microsatellite markers we previously developed for the reniform nematode. The results were compared to those of fingerprinting the original population (MSRR03). Out of 96 markers tested, 71 had amplicons in MSRR03. Using WGA of single gravid females with their associated egg masses, 86-93% of the alleles found on MSRR03 were detected, and 87-88% of the alleles found on MSRR03 when using WGA of samples composed of five gravid females with their associated egg masses as template. Our results indicate that reniform nematode samples as small as a single gravid female with her associated egg mass can be used in WGA and direct testing with microsatellites, giving consistent results when compared to the original population

A Genetic Screen for Dominant Enhancers of the Cell-Cycle Regulator α-Endosulfine Identifies Matrimony as a Strong Functional Interactor in Drosophila

The coordination of cell-cycle events with developmental processes is essential for the reproductive success of organisms. In Drosophila melanogaster, meiosis is tightly coupled to oocyte development, and early embryos undergo specialized S-M mitoses that are supported by maternal products. We previously showed that the small phosphoprotein α-endosulfine (Endos) is required for normal oocyte meiotic maturation and early embryonic mitoses in Drosophila. In this study, we performed a genetic screen for dominant enhancers of endos00003 and identified several genomic regions that, when deleted, lead to impaired fertility of endos00003/+ heterozygous females. We uncovered matrimony (mtrm), which encodes a Polo kinase inhibitor, as a strong dominant enhancer of endos. mtrm126 +/+ endos00003 females are sterile because of defects in early embryonic mitoses, and this phenotype is reverted by removal of one copy of polo. These results provide compelling genetic evidence that excessive Polo activity underlies the strong functional interaction between endos00003 and mtrm126. Moreover, we show that endos is required for the increased expression of Mtrm in mature oocytes, which is presumably loaded into early embryos. These data are consistent with the model that maternal endos antagonizes Polo function in the early embryo to ensure normal mitoses through its effects on Mtrm expression during late oogenesis. Finally, we also identified genomic deletions that lead to loss of viability of endos00003/+ heterozygotes, consistent with recently published studies showing that endos is required zygotically to regulate the cell cycle during development

Comparison of whole-genome amplifications for microsatellite genotyping of Rotylenchulus reniformis

Currently, a large number of microsatellites are available for Rotylenchulus reniformis (reniform nematode); however, two barriers exist for genotyping samples from different geographical areas. The limited amount of nucleic acids obtained from single nematodes which would require their multiplication to obtain enough DNA for testing; and the strictly regulated transport of live samples and multiplication in greenhouse for being a plant pathogen. Whole-genome amplification (WGA) of samples consisting of one and five dead gravid females with their associated egg masses was successfully performed on disrupted tissue using three commercial kits. DNA yield after WGA ranged from 0.5 to 8 \ub5g and was used to test 96 microsatellite markers we previously developed for the reniform nematode. The results were compared to those of fingerprinting the original population (MSRR03). Out of 96 markers tested, 71 had amplicons in MSRR03. Using WGA of single gravid females with their associated egg masses, 86-93% of the alleles found on MSRR03 were detected, and 87-88% of the alleles found on MSRR03 when using WGA of samples composed of five gravid females with their associated egg masses as template. Our results indicate that reniform nematode samples as small as a single gravid female with her associated egg mass can be used in WGA and direct testing with microsatellites, giving consistent results when compared to the original population

Identification of PNG kinase substrates uncovers interactions with the translational repressor TRAL in the oocyte-to-embryo transition

The Drosophila Pan Gu (PNG) kinase complex regulates hundreds of maternal mRNAs that become translationally repressed or activated as the oocyte transitions to an embryo. In a previous paper (Hara et al., 2017), we demonstrated PNG activity is under tight developmental control and restricted to this transition. Here, examination of PNG specificity showed it to be a Thrkinase yet lacking a clear phosphorylation site consensus sequence. An unbiased biochemical screen for PNG substrates identified the conserved translational repressor Trailer Hitch (TRAL). Phosphomimetic mutation of the PNG phospho-sites in TRAL reduced its ability to inhibit translation in vitro. In vivo, mutation of tral dominantly suppressed png mutants and restored Cyclin B protein levels. The repressor Pumilio (PUM) has the same relationship with PNG, and we also show that PUM is a PNG substrate. Furthermore, PNG can phosphorylate BICC and ME31B, repressors that bind TRAL in cytoplasmic RNPs. Therefore, PNG likely promotes translation at the oocyte-to-embryo transition by phosphorylating and inactivating translational repressors.National Institutes of Health (U.S.) (Grant GM39341)National Institutes of Health (U.S.) (Grant GM118090

Cotton ( Gossypium hirsutum

A field study was conducted over a two-year period (2006-2007) at the Delta Research and Extension Center, Stoneville, MS, USA to screen selected entries in the 2006 Mississippi Cotton Variety Trials for tolerance to the reniform nematode (Rotylenchulus reniformis). Trials were conducted in nonirrigated fields with primarily sandy loam soils. Though some variability was noted between test locations and years, six of 13 cotton (Gossypium hirsutum) cultivars tested were considered tolerant to the reniform nematode: “Cropland Genetics 3520 B2RF,” “DynaGrow 2520 B2RF,” “Stoneville 5242 BR,” “Stoneville 5599 BR,” “Deltapine 488 BG/RR,” and “Fibermax 960 B2R.” Of these, the first three exhibited yields similar to the productive cultivar “Deltapine 445 BG/RR” in all environments. Though they will not suppress the reniform nematode population, these cultivars can help reduce economic losses attributed to this pathogen in the Midsouth region of the USA