Chromatographic printed array strip (C-PAS) method for cultivar-specific identification of sweetpotato cultivars 'Beniharuka' and 'Fukumurasaki'.

Sweetpotato (Ipomoea batatas) cultivars grown in Japan are highly valued for their excellent sweetness, high quality, and good texture. The export volume of sweetpotato from Japan has been rising rapidly, with a 10-fold increase on a weight basis over the last 10 years. However, since sweetpotato is propagated vegetatively from storage roots, it is easy to cultivate and propagate this crop, prompting concerns that Japanese sweetpotato cultivars/lines are being exported overseas, cultivated without permission, or reimported. Therefore, a rapid and accurate cultivar identification methodology is needed. In this study, we comprehensively analyzed the insertion sites of Cl8 retrotransposon to develop a cultivar identification technique for the Japanese cultivars 'Beniharuka' and 'Fukumurasaki'. These two cultivars were successfully distinguished from other cultivars using a minimum of two marker sets. Using the chromatographic printed array strip (C-PAS) method for DNA signal detection, 'Beniharuka' and 'Fukumurasaki' can be precisely identified using a single strip of chromatographic paper based on multiplex DNA signals derived from the amplicons of the Cl8 insertion sites. Since this method can detect DNA signals in only ~15 minutes, we expect that our method will facilitate rapid, reliable, and convenient cultivar discrimination for on-site inspection of sweetpotato.

Polyploid GWAS reveals the basis of molecular marker development for complex breeding traits including starch content in the storage roots of sweet potato.

Given the importance of prioritizing genome-based breeding of sweet potato to enable the promotion of food and nutritional security for future human societies, here, we aimed to dissect the genetic basis of storage root starch content (SC) when associated with a complex set of breeding traits including dry matter (DM) rate, storage root fresh weight (SRFW), and anthocyanin (AN) content in a mapping population containing purple-fleshed sweet potato. A polyploid genome-wide association study (GWAS) was extensively exploited using 90,222 single-nucleotide polymorphisms (SNPs) obtained from a bi-parental 204 F1 population between 'Konaishin' (having high SC but no AN) and 'Akemurasaki' (having high AN content but moderate SC). Through the comparison of polyploid GWAS on the whole set of the 204 F1, 93 high-AN-containing F1, and 111 low-AN-containing F1 populations, a total of two (consists of six SNPs), two (14 SNPs), four (eight SNPs), and nine (214 SNPs) significantly associated signals were identified for the variations of SC, DM, SRFW, and the relative AN content, respectively. Of them, a novel signal associated with SC, which was most consistent in 2019 and 2020 in both the 204 F1 and 111 low-AN-containing F1 populations, was identified in homologous group 15. The five SNP markers associated with homologous group 15 could affect SC improvement with a degree of positive effect (~4.33) and screen high-starch-containing lines with higher efficiency (~68%). In a database search of 62 genes involved in starch metabolism, five genes including enzyme genes granule-bound starch synthase I (IbGBSSI), α-amylase 1D, α-amylase 1E, and α-amylase 3, and one transporter gene ATP/ADP-transporter were located on homologous group 15. In an extensive qRT-PCR of these genes using the storage roots harvested at 2, 3, and 4 months after field transplantation in 2022, IbGBSSI, which encodes the starch synthase isozyme that catalyzes the biosynthesis of amylose molecule, was most consistently elevated during starch accumulation in sweet potato. These results would enhance our understanding of the underlying genetic basis of a complex set of breeding traits in the starchy roots of sweet potato, and the molecular information, particularly for SC, would be a potential platform for molecular marker development for this trait.

Polyploid QTL-seq towards rapid development of tightly linked DNA markers for potato and sweetpotato breeding through whole-genome resequencing.

Potato (Solanum tuberosum L.) and sweetpotato (Ipomoea batatas L.), which are nutritionally and commercially important tuberous crops, possess a perplexing heredity because of their autopolyploid genomes. To reduce cross-breeding efforts for selecting superior cultivars from progenies with innumerable combinations of traits, DNA markers tightly linked to agronomical traits are required. To develop DNA markers, we developed a method for quantitative trait loci (QTL) mapping using whole-genome next-generation sequencing (NGS) in autopolyploid crops. To apply the NGS-based bulked segregant method, QTL-seq was modified. (1) Single parent-specific simplex (unique for one homologous chromosome) single-nucleotide polymorphisms (SNPs), which present a simple segregation ratio in the progenies, were exploited by filtering SNPs by SNP index (allele frequency). (2) Clusters of SNPs, which were inherited unevenly between bulked progenies with opposite phenotypes, especially those with an SNP index of 0 for the bulk that did not display the phenotypes of interest, were explored. These modifications allowed for separate tracking of alleles located on each of the multiple homologous chromosomes. By applying this method, clusters of SNPs linked to the potato cyst nematode resistance H1 gene and storage root anthocyanin (AN) content were identified in tetraploid potato and hexaploid sweetpotato, respectively, and completely linked DNA markers were developed at the site of the presented SNPs. Thus, polyploid QTL-seq is a versatile method that is free from specialized manipulation for sequencing and construction of elaborate linkage maps and facilitates rapid development of tightly linked DNA markers in autopolyploid crops, such as potato and sweetpotato.

QTL analysis and GWAS of agronomic traits in sweetpotato (Ipomoea batatas L.) using genome wide SNPs.

While sweetpotato (Ipomoea batatas L.) improvement has generally been done by field-based selection, molecular genetic studies on traits of interest, i.e., molecular markers are needed for enhancing the breeding program of this world's 7th most important crop, as such markers facilitate marker-assisted selection. Here, we performed a combined approach of QTLs analyses and GWAS of storage root ß-carotene content (BC), dry-matter (DM) and starch content (SC) using the genetic linkage maps constructed with 5,952 and 5,640 SNPs obtained from F1 progenies between cultivars 'J-Red' and 'Choshu'. BC was negatively correlated with DM (r = -0.45) and SC (r = -0.51), while DM was positively correlated with SC (r = 0.94). In both parental maps, a total of five, two and five QTL regions on linkage groups 7 and 8 were associated with BC, DM and SC, respectively. In GWAS of BC, one strong signal (P = 1.04 × 10-9) was observed on linkage group 8, which co-located with one of the above QTL regions. The SNPs markers found here, particularly for ß-carotene, would be useful base resources for future marker-assisted selection program with this trait.

Molecular Detection and Antibiotyping of Multidrug-Resistant Salmonella Isolated from Houseflies in a Fish Market.

Houseflies (Musca domestica) are well-known mechanical vectors for spreading multidrug-resistant bacteria. Fish sold in open markets are exposed to houseflies. The present study investigated the prevalence and antibiotypes of multidrug-resistant (MDR) Salmonella spp. in houseflies captured from a fish market. Direct interviews with fish vendors and consumers were also performed to draw their perceptions about the role of flies in spreading antibiotic-resistant bacteria. A total of 60 houseflies were captured from a local fish market in Bangladesh. The presence of Salmonella spp. was confirmed using PCR method. Antibiogram was determined by the disk diffusion method, followed by the detection of tetA, tetB, and qnrA resistance genes by PCR. From the interview, it was found that most of the consumers and vendors were not aware of antibiotic resistance, but reported that flies can carry pathogens. Salmonella spp. were identified from the surface of 34 (56.7%) houseflies, of which 31 (91.2%) were found to be MDR. This study revealed 25 antibiotypes among the isolated Salmonella spp. All tested isolates were found to be resistant to tetracycline. tetA and tetB were detected in 100% and 47.1% of the isolates, respectively. Among the 10 isolates phenotypically found resistant to ciprofloxacin, six (60%) were found to be positive for qnrA gene. As far as we know, this is the first study from Bangladesh to report and describe the molecular detection of multidrug-resistant Salmonella spp. in houseflies in a fish market facility. The occurrence of a high level of MDR Salmonella in houseflies in the fish market is of great public health concerns.

Genome-Edited Triple-Recessive Mutation Alters Seed Dormancy in Wheat.

Common wheat has three sets of sub-genomes, making mutations difficult to observe, especially for traits controlled by recessive genes. Here, we produced hexaploid wheat lines with loss of function of homeoalleles of Qsd1, which controls seed dormancy in barley, by Agrobacterium-mediated CRISPR/Cas9. Of the eight transformed wheat events produced, three independent events carrying multiple mutations in wheat Qsd1 homeoalleles were obtained. Notably, one line had mutations in every homeoallele. We crossed this plant with wild-type cultivar Fielder to generate a transgene-free triple-recessive mutant, as revealed by Mendelian segregation. The mutant showed a significantly longer seed dormancy period than wild-type, which may result in reduced pre-harvest sprouting of grains on spikes. PCR, southern blotting, and whole-genome shotgun sequencing revealed that this segregant lacked transgenes in its genomic sequence. This technique serves as a model for trait improvement in wheat, particularly for genetically recessive traits, based on locus information from diploid barley.

Role of courtyard counselling meeting in improving household food safety knowledge and practices in Munshiganj district of Bangladesh.

Unsafe food is linked to the deaths of an estimated two million people annually. Food containing harmful agents is responsible for more than 200 diseases ranging from diarrhoea to cancers. A one-sample pilot intervention study was conducted to evaluate the role of courtyard counselling meetings as the means of intervention for improving food safety knowledge and practices among household food handlers in a district of Bangladesh. The study was conducted in three phases: a baseline survey, the intervention and an end-line survey between April and November 2015 where 194 food handlers took part. Data were collected through observations and face-to-face interviews. The mean age of the respondents was 38.8 (±12.4) years, all of whom were females. Hand washing before eating, and washing utensils with soap were significantly improved at the end-line in comparison to the baseline (57% vs. 40% and 83% vs. 69%, respectively). Hand washing with soap was increased by 4%. The mean score of food handling practices was significantly increased after the intervention (20.5 vs. 22.1; P<0.001). However, hand washing after use of toilet was unchanged after the intervention (75% vs.76%). Knowledge about safe food and the necessity of thorough cooking were significantly increased after the intervention (88% from 64% and 34% from 21%, respectively). Mean scores of knowledge and practice on food safety were significantly increased by 1.9 and 1.6, respectively after the one month intervention. Thus this food safety education in rural communities should be scaled up and, indeed, strengthened using the courtyard counselling meetings in Bangladesh.

Development of pre-breeding technology for root system study and selection of Kihara Afghan wheat landraces (KAWLR) to enhance wheat breeding in the rain-fed region.

To enhance a root trait-based selection program for rain-fed wheat breeding in Afghanistan, we simulated an efficient pre-breeding drought system. Plants were grown in 1 m pipes as control or 2 m pipes to simulate drought conditions soaking ground water up by capillary action supplemented by two different life supporting irrigations from top of the pipes (T1 and T2 droughts). T1 was used for studying genetic diversity in 360 Kihara Afghan wheat landraces (KAWLR). Both drought treatments were used to evaluate root traits in 30 selected genotypes. KAWLR showed large root length variations under T1, categorized as long root (>200 cm; LR), medium root (100-150 cm; MR) and short root (20-100 cm; SR) systems. LR genotypes were more drought resistant in terms of greater plant survivability under T1 and T2 compared with other groups and were capable of adjusting their root biomass partitioning at deepest part of the soil profile. Majority of the LR genotypes originated from predominantly rain-fed provinces, and most of their agronomic traits were strongly correlated with root biomass deep in the soil in response to drought. Three LR genotypes, including the longest root genotype LR-871 (KU7604), are recommended for rain-fed wheat breeding in Afghanistan.

Expression of trehalose-6-phosphate phosphatase in maize ears improves yield in well-watered and drought conditions.

Maize, the highest-yielding cereal crop worldwide, is particularly susceptible to drought during its 2- to 3-week flowering period. Many genetic engineering strategies for drought tolerance impinge on plant development, reduce maximum yield potential or do not translate from laboratory conditions to the field. We overexpressed a gene encoding a rice trehalose-6-phosphate phosphatase (TPP) in developing maize ears using a floral promoter. This reduced the concentration of trehalose-6-phosphate (T6P), a sugar signal that regulates growth and development, and increased the concentration of sucrose in ear spikelets. Overexpression of TPP increased both kernel set and harvest index. Field data at several sites and over multiple seasons showed that the engineered trait improved yields from 9% to 49% under non-drought or mild drought conditions, and from 31% to 123% under more severe drought conditions, relative to yields from nontransgenic controls.

Cryo-EM structure of the small subunit of the mammalian mitochondrial ribosome.

The mammalian mitochondrial ribosomes (mitoribosomes) are responsible for synthesizing 13 membrane proteins that form essential components of the complexes involved in oxidative phosphorylation or ATP generation for the eukaryotic cell. The mammalian 55S mitoribosome contains significantly smaller rRNAs and a large mass of mitochondrial ribosomal proteins (MRPs), including large mito-specific amino acid extensions and insertions in MRPs that are homologous to bacterial ribosomal proteins and an additional 35 mito-specific MRPs. Here we present the cryo-EM structure analysis of the small (28S) subunit (SSU) of the 55S mitoribosome. We find that the mito-specific extensions in homologous MRPs generally are involved in inter-MRP contacts and in contacts with mito-specific MRPs, suggesting a stepwise evolution of the current architecture of the mitoribosome. Although most of the mito-specific MRPs and extensions of homologous MRPs are situated on the peripheral regions, they also contribute significantly to the formation of linings of the mRNA and tRNA paths, suggesting a tailor-made structural organization of the mito-SSU for the recruitment of mito-specific mRNAs, most of which do not possess a 5' leader sequence. In addition, docking of previously published coordinates of the large (39S) subunit (LSU) into the cryo-EM map of the 55S mitoribosome reveals that mito-specific MRPs of both the SSU and LSU are involved directly in the formation of six of the 15 intersubunit bridges.

Quantitative Proteomics of the Root of Transgenic Wheat Expressing TaBWPR-1.2 Genes in Response to Waterlogging.

Once candidate genes are available, the application of genetic transformation plays a major part to study their function in plants for adaptation to respective environmental stresses, including waterlogging (WL). The introduction of stress-inducible genes into wheat remains difficult because of low transformation and plant regeneration efficiencies and expression variability and instability. Earlier, we found two cDNAs encoding WL stress-responsive wheat pathogenesis-related proteins 1.2 (TaBWPR-1.2), TaBWPR-1.2#2 and TaBWPR-1.2#13. Using microprojectile bombardment, both cDNAs were introduced into "Bobwhite". Despite low transformation efficiency, four independent T2 homozygous lines for each gene were isolated, where transgenes were ubiquitously and variously expressed. The highest transgene expression was obtained in Ubi:TaBWPR-1.2#2 L#11a and Ubi:TaBWPR-1.2#13 L#4a. Using quantitative proteomics, the root proteins of L#11a were analyzed to explore possible physiological pathways regulated by TaBWPR-1.2 under normal and waterlogged conditions. In L#11a, the abundance of proteasome subunit alpha type-3 decreased under normal conditions, whereas that of ferredoxin precursor and elongation factor-2 increased under waterlogged conditions in comparison with normal plants. Proteomic results suggest that L#11a is one of the engineered wheat plants where TaBWPR-1.2#2 is most probably involved in proteolysis, protein synthesis and alteration in the energy pathway in root tissues via the above proteins in order to gain metabolic adjustment to WL.

Interaction of α-synuclein with vesicles that mimic mitochondrial membranes.

α-Synuclein, an intrinsically-disordered protein associated with Parkinson's disease, interacts with mitochondria, but the details of this interaction are unknown. We probed the interaction of α-synuclein and its A30P variant with lipid vesicles by using fluorescence anisotropy and (19)F nuclear magnetic resonance. Both proteins interact strongly with large unilamellar vesicles whose composition is similar to that of the inner mitochondrial membrane, which contains cardiolipin. However, the proteins have no affinity for vesicles mimicking the outer mitochondrial membrane, which lacks cardiolipin. The (19)F data show that the interaction involves α-synuclein's N-terminal region. These data indicate that the middle of the N-terminal region, which contains the KAKEGVVAAAE repeats, is involved in binding, probably via electrostatic interactions between the lysines and cardiolipin. We also found that the strength of α-synuclein binding depends on the nature of the cardiolipin acyl side chains. Eliminating one double bond increases affinity, while complete saturation dramatically decreases affinity. Increasing the temperature increases the binding of wild-type, but not the A30P variant. The data are interpreted in terms of the properties of the protein, cardiolipin demixing within the vesicles upon binding of α-synuclein, and packing density. The results advance our understanding of α-synuclein's interaction with mitochondrial membranes.

Ribosome shifting or splitting: it is all up to the EF-G.

Mitochondria possess two elongation factor Gs: one with translocation activity (EF-G1(mt)) and the other with no confirmed activity (EF-G2(mt)). Tsuboi et al. (2009) now show that the function of EF-G2(mt) is not in elongation but, rather, in ribosome recycling.

Role of Cdk5-mediated phosphorylation of Prx2 in MPTP toxicity and Parkinson's disease.

We reported previously that calpain-mediated Cdk5 activation is critical for mitochondrial toxin-induced dopaminergic death. Here, we report a target that mediates this loss. Prx2, an antioxidant enzyme, binds Cdk5/p35. Prx2 is phosphorylated at T89 in neurons treated with MPP+ and/or MPTP in animals in a calpain/Cdk5/p35-dependent manner. This phosphorylation reduces Prx2 peroxidase activity. Consistent with this, p35-/- neurons show reduced oxidative stress upon MPP+ treatment. Expression of Prx2 and Prx2T89A, but not the phosphorylation mimic Prx2T89E, protects cultured and adult neurons following mitochondrial insult. Finally, downregulation of Prx2 increases oxidative stress and sensitivity to MPP+. We propose a mechanistic model by which mitochondrial toxin leads to calpain-mediated Cdk5 activation, reduced Prx2 activity, and decreased capacity to eliminate ROS. Importantly, increased Prx2 phosphorylation also occurs in nigral neurons from postmortem tissue from Parkinson's disease patients when compared to control, suggesting the relevance of this pathway in the human condition.

Low nutrient intakes among infants in rural Bangladesh are attributable to low intake and micronutrient density of complementary foods.

We assessed the adequacy of nutrient intakes of 135 rural Bangladeshi breast-fed infants 6-12 mo of age and examined nutritional trade-offs due to possible displacement of breast milk by complementary foods. Observers completed 12-h daytime measurements of breast milk and complementary food intakes; data for the previous 12 h were obtained from maternal recall, yielding estimates of total 24-h intakes. On average, infants were mildly wasted (mean +/- SD weight-for-length Z-score = -0.92 +/- 0.88) and moderately stunted (length-for-age Z-score = -1.49 +/- 0.96). Total energy intakes at 6-8 and 9-12 mo were 88 and 86% of absolute energy requirements (kJ/d), 106 and 105% of requirements per kg body weight, and 97 and 94% of requirements per kg median weight-for-length, respectively. Breast milk contributed 78% of energy intake at 6-8 mo and 75% at 9-12 mo. Mean meal frequency and energy density of complementary foods were generally consistent with recommendations, but only small amounts of food were offered. Nevertheless, only 72% of the food energy offered was consumed. Total energy intake was positively correlated with meal frequency, quantity consumed per meal, and energy intake from breast milk, but not with energy density of complementary foods. Energy intake from complementary foods was inversely related to energy intake from breast milk. The diets fell short of recommended intakes for numerous vitamins and minerals. We conclude that although greater intakes of complementary foods were associated with higher total energy intake, micronutrient intake remained low due to the low micronutrient density of the complementary foods consumed and the partial displacement of breast milk.

Behavior-change trials to assess the feasibility of improving complementary feeding practices and micronutrient intake of infants in rural Bangladesh.

This study used simple rapid-assessment techniques to test the feasibility of increasing the consumption of complementary foods by infants by asking mothers to increase meal quantity or frequency or by altering the viscosity/energy density of the food. The feasibility of using micronutrient supplements either added directly to food or administered as liquid drops was also examined. The study was conducted in rural Bangladesh and involved four separate short-term behavioral change trials. Depending on the trial, fieldworkers recruited 30 to 45 infants 6 to 12 months of age. Following recommendations to increase the amount of food provided to infants, the mean intakes from single meals increased from 40 +/- 23 g on day 1 to 64 +/- 30 g on day 7 (p < 0.05). In a second trial, the mean meal frequency increased from 2.2 +/- 1.3 on day 1 to 4.1 +/- 1.3 on day 7 (p < 0.05). Provision of high-energy-density diets, prepared by decreasing viscosity with alpha-amylase or by hand-mashing rice and dhal into a paste before feeding, increased single-meal energy consumption from 54 +/- 35 kcal to 79 +/- 52 kcal or 75 +/- 37 kcal (p < 0.05), respectively. Both types of micronutrient supplements were well accepted and used according to recommendations. In conclusion, it was possible to change short-term child-feeding behaviors to promote increased food intake, mealfrequency, energy density, and micronutrient consumption. Because each of these interventions lasted for only about 1 week, however, the long-term sustainability of these changes is not known. Moreover, the effect of increased feeding of complementary foods on intakes of breastmilk and total daily consumption of energy and nutrients requires further study.