Functional microsatellites in cassava (Manihot esculenta Crantz): genomic mapping and genetic characterisation in cultivars from Misiones

The cassava breeding program in Uganda has released many improved cultivars resistant/tolerant to cassava brown streak disease (CBSD) and cassava mosaic disease (CMD). However, many farmers have continued to use cultivars that are susceptible to these major viral diseases but with diverse attributes. There is a need to understand farmers’ cassava cultivar attribute preferences and CBSD, CMD prevalence on the preferred cultivars. A total of 150 cassava farmer fields (74 in Bukedea district and 76 in Kumi district) located in eastern Uganda were evaluated for farmers’ cultivars and attribute preferences, as well as prevalence of CBSD and CMD on the farmer selected cultivars. The eastern region was of interest in the study, due to it-being the major cassava producing area in the country. In addition, 30 cassava plants of different genotypes were chosen randomly along transects of each field and assessed for CBSD/CMD incidence and severity on preferred cultivars. Results showed that more than 64% of the farmers in eastern Uganda preferred NASE 03 due to its sweet taste and high yields, followed by NASE 14 (21%) because of its high yields. There was a significant (P≤0.001) correlation between cassava cultivars preferred and CBSD severity (r = -0.56), CBSD incidence (r = -0.53), CMD severity (r = -0.51) and CMD incidence (r = -0.39). In corroboration, the most preferred cultivar, NASE 03 was found most susceptible to CBSD and CMD in both Bukedea and Kumi districts with CBSD incidence of 62.2% and 52.7% and CMD incidence of 56.9% and 34.3% respectively. The results showed that CBSD and CMD are prevalent on farmer preferred cassava cultivars, and that farmer cultivar preference depends not only on disease resistance but also other attributes.

Bacterial blight of cassava is one of the most important diseases of cassava worldwide due to its growing concern, widespread and destructive nature. Even though the use of resistant cultivars is the most effective management strategy for the disease, such cultivars are not well identified. Therefore, the objective of this study was to screen 21 cassava cultivars collected from major growing regions of Kenya, for reaction against bacterial blight caused by Xanthomonas axonopodis pv. manihotis (Xam). The cultivars were inoculated with Xam by leaf clipping and stem puncturing inoculation methods, for in vitro and potted plants, respectively. The cassava cultivars varied in their reaction to the pathogen, including incubation period, wilt incidence and area under the disease progress curve (AUDPC) values. Four groups of cultivars with differential reactions to Xam isolate were identified. Four cultivars (TME419, 30572, 98/0505 and Kibaha) were resistant, 4 cultivars (Albert, Ebwanatereka, Karibuni and 92/0326) moderately resistance, 11 cultivars (Serere, Muzege, TME7, 98/0581, Tajirika, Namikonga, Kibandameno, Mzalauka, AR40-6, Shibe and 01/1371) susceptible and the other 2 cultivars (Kiroba and Numbari) were highly susceptible. The resistant cultivars should be multiplied and made available as clean planting materials to cassava producing farmers and integrated as one of the options in disease management measures. These genotypes could also form vital germplasm of cassava bacterial blight disease resistance breeding programs. The cassava cultivars that showed a resistant reaction to the bacterial blight pathogen should be further evaluated against a large number of Xam isolates.

High-throughput genomics of SARS-CoV-2 is essential to characterize virus evolution and to identify adaptations that affect pathogenicity or transmission. While single-nucleotide variations (SNVs) are commonly considered as driving virus adaption, RNA recombination events that delete or insert nucleic acid sequences are also critical. Whole genome targeting sequencing of SARS-CoV-2 is typically achieved using pairs of primers to generate cDNA amplicons suitable for next-generation sequencing (NGS). However, paired-primer approaches impose constraints on where primers can be designed, how many amplicons are synthesized and requires multiple PCR reactions with non-overlapping primer pools. This imparts sensitivity to underlying SNVs and fails to resolve RNA recombination junctions that are not flanked by primer pairs. To address these limitations, we have designed an approach called ‘Tiled-ClickSeq’, which uses hundreds of tiled-primers spaced evenly along the virus genome in a single reverse-transcription reaction. The other end of the cDNA amplicon is generated by azido-nucleotides that stochastically terminate cDNA synthesis, removing the need for a paired-primer. A sequencing adaptor containing a Unique Molecular Identifier (UMI) is appended to the cDNA fragment using click-chemistry and a PCR reaction generates a final NGS library. Tiled-ClickSeq provides complete genome coverage, including the 5’UTR, at high depth and specificity to the virus on both Illumina and Nanopore NGS platforms. Here, we analyze multiple SARS-CoV-2 isolates and clinical samples to simultaneously characterize minority variants, sub-genomic mRNAs (sgmRNAs), structural variants (SVs) and D-RNAs. Tiled-ClickSeq therefore provides a convenient and robust platform for SARS-CoV-2 genomics that captures the full range of RNA species in a single, simple assay.

SummaryField experiments were made in the rainforest zone of Western Nigeria over three cropping seasons on the effects of potassium fertilizer of one local cassava cultivar 53101 and two improved cultivars 60506 and 60447 which wereF1hybrids of the former. The results showed that K-fertilizer increased the starch yields only slightly. Cultivar 60506 produced significantly higher starch yields (P< 0–01) than the local strain, 53101, while cultivar 60447 gave lower starch recovery than the local cultivar.A definite influence of K application on the cyanide (HCN) content of the tuber roots could not be established. The peeled tubers of the improved cultivars contained much less cyanide than those of the local 53101 strain (P< 0–01). Potassium had the highest (0·72–1·28% dry weight basis) while phosphorus had the lowest concentration (0·06–0·12%) in the peeled tuber. Owing to its higher yielding capability the unfertilized 60506 removed larger amounts of potash (average 110·9 kg k20/ha) from the soil than unfertilized 53101 (average 82·2 kg k20/ha).

BackgroundCassava is a major staple food crop for the people in tropical and sub-tropical areas, a non-traditional vegetable root crop in Egypt. It has important industrial raw materials for the production of starch, alcohol, pharmaceuticals, and livestock feed. The current study is evaluating two cassava cultivars: American and Brazilian types under three bio-stimulants: glutamine, lysine, and active dry yeast, as foliar applications. Vegetative growth characters, tuber yield, and its quality as well as chemical contents of cassava tubers were evaluated.ResultsThe results showed that American cassava cultivar recorded the highest vegetative growth characters (plant height, leaves number, main stems, lateral branches, leaf area, and chlorophyll content), tuber yield (number and weight), and tuber quality (length, diameter, dry matter, and tubers-shoots ratio) as well as chemical composition (starch, total carbohydrates, N, P, and K). The foliar application of glutamine at the rate of 200 mg/L increased vegetative growth characters and tuber yield as well as tuber quality and improved chemical contents of cassava tubers. On the contrary, the lowest values of cassava growth and its productivity as well as chemical composition were associated to Brazilian cultivar when foliar sprayed by yeast at the concentration of 2 g/L.ConclusionFrom this study, it could be concluded that American type of cassava is the most suitable under Egyptian conditions, which ranked the first in all vegetative growth characters, tuber yield, tuber quality, and chemical composition, with foliar sprayed by glutamine at the rate of 200 mg/L.

A new human genome reference sequence--GRCh37--was recently generated and made available by the Genome Reference Consortium. Since the prior disposable human reference sequence--hg18--was previously used for the mitochondrial DNA primer BLAST validation, a revision of those previously published primer pairs is required. Thus, the aim of this Short Communication is to perform an in silico BLAST test of the published disposable nine primer pairs using the new human reference sequence and to report the pertinent modifications. The new analysis showed that one of the tested primer pairs requires a revision. Therefore, a new validated primer pair, which specifically amplifies the mitochondrial region located between positions 6520 and 9184, is presented.

Selection of cultivars for minimization of waste and of water consumption in cassava starch production

Cassava is predominantly an upland crop that is also cultivated in inland valley swamps (IVS). Identifying physiological traits that can withstand excess moisture stress can aid in the selection and use of stable cassava cultivars in IVS. Three cassava cultivars were evaluated for growth and yield in the upland and IVS ecologies in the 1993–94 crop seasons using a randomized complete block design. In the upland, 80/40 outyielded 87/29 and ‘coco’, while in the IVS, 87/29 had the highest yield. Highest yielding cultivars in each ecology also had the highest tuberous root bulking rate (TBR), dry matter (DM) production, crop growth rate (CGR), relative growth rate, net assimilation rate (NAR), leaf area index, leaf production rate and leaf life. Positive correlations between these parameters and tuberous root yield were noted. High yield of 87/29 in IVS was partly due to its low tuberous root rotting. High root rotting in 80/40 was partly as a result of the greater depth of tuberous roots in the soil (0.3–0.6 m) as compared to the other cultivars (0.15–0.3 m) where roots were in contact with stressful water levels earlier than others. The yield and yield components, growth and leaf characters were all drastically reduced when the cultivars were grown in IVS as compared to upland. For example, root yields of ‘coco’, 87/29 and 80/40 were reduced by 53 %, 60 % and 92 %, respectively, in IVS. Selection of cassava cultivars with longer leaf life and leaf area maintenance leading to high CGR, TBR, NAR and harvest index (in IVS and upland), coupled with a shallow tuberous root formation zone (in IVS) can give higher root and leaf yields in the two ecologies.

An Oligonucleotide Microarray for High-Throughput Sequencing of the Mitochondrial Genome

<p indent="0mm">Sugarcane is one of the most important sugar crops in the world. However, it is difficult to develop SSR on a large scale since the genome of cultivar has not been sequenced, which limits the genetic improvement of sugarcane. In this study, a template of monoploid sugarcane genome was assembled using a set of 4660 BAC library sequences (with a cumulative length of 382 Mb, predicting 25,316 genes) from cultivar ‘R570’. SSR loci were identified by using MISA (Microsatellite identification tool) software. The distribution characteristics of the monoploid genome ‘R570’ was comprehensively analyzed by comparing with the SSR loci of four Gramineae plants (<italic>Sorghum bicolor</italic>,<italic> Zea mays</italic>,<italic> Oyrza sativa</italic>, and <italic>Brachypodium distachyon</italic>). Fifty pairs of primers with TG and AG repeat motifs were designed to verify the amplification efficiency and polymorphism by PCR amplification in four <italic>Saccharum</italic> clones (R570, ROC1, LA purple, and SES208) and twenty four core parents of sugarcane. A total of 27,241 SSR loci were identified, with an average of 6.29 SSR loci per BAC clone and an average density of 71.33 SSR Mb^–1 which was much lower than that of sorghum (350.00 SSR Mb^–1). The mono-nucleotide (11,079) and tri-nucleotide repeat motifs (6447) accounted for 64.33% of the total SSR loci. The number and proportion of tri-nucleotide repeat motifs were the largest in the four Gramineae plants. In addition, A/T (accounting for 84.8%) motif had the highest proportion and C/G (accounting for 15.2%) motif the lowest proportion in the mono-nucleotide repeat motifs and TGT/ACA (accounting for 16.04%) motif had the highest proportion in the trinucleotide repeat motifs. In general, the genomes in Gramineae plants are rich in A/T repeat motifs. In the polymorphism validation of 50 pairs of primers (41 pairs of TG motif and 9 pairs of AG motif), 45 pairs of primers (90%) were found to be able to amplify successfully, of which 35 (70%) were polymorphic in 4 sugarcane clones. Furthermore, 20 pairs of polymorphic SSR primers were used to detect 24 core parents of sugarcane, a total of 95 alleles were amplified with an average of 4.75 alleles per primer, verifying the application feasibility of these primers for the genetic diversity analysis in sugarcane. The development of SSR markers from the monoploid genome of cultivars ‘R570’ not only enriches the number of SSR markers available in sugarcane genetic analysis, but also facilitates the genetic diversity analysis of sugarcane population and the genetic mechanism dissection of important agronomic traits, which provides a foundation for the in-depth research of molecular breeding in sugarcane.

Protein phosphorylation on tyrosine has been originally characterized in animal systems and has been shown to be involved in several fundamental processes including signal transduction, growth control, and malignancy. It has been later demonstrated to occur also in a number of bacteria, and recent data suggest that it may participate in the control of bacterial pathogenicity. In this work, we provide evidence that the gram-positive human pathogen Staphylococcus aureus harbors a protein-tyrosine kinase activity. This activity is borne by a protein, termed Cap5B2, whose phosphorylating capacity is expressed only in the presence of a stimulatory protein, either Cap5A1 or Cap5A2, that enhances its affinity for the phosphoryl donor ATP. In fact, the last 27/29 amino acids of the C-terminal domain of either polypeptide are sufficient for stimulating Cap5B2 activity. The stimulation of Cap5B2 by Cap5A1 involves essentially three amino acid residues in a helix of Cap5A1 (Asp202, Glu203, and Asp205) and three residues in a helix (helix 7) of Cap5B2 (Glu190, Lys192, and Lys193), thus suggesting helix-helix interaction between these two proteins. This type of helix-helix interaction resembles the interaction required for the activation of MinD ATPase by MinE protein in the process of septum-site determination, MinD sharing sequence similarity with Cap5B2. Such activation mechanism is described here in a gram-positive bacterial tyrosine kinase, and differs from the activation mechanism previously proposed for gram-negative bacteria. Therefore, it appears that S. aureus, and possibly other gram-positive bacteria, utilizes a specific molecular mechanism for triggering protein-tyrosine kinase activity.

SummaryThe results of field experiments on the influence of nitrogen fertilization on the yield components, starch and prussic acid content of two of the cassava cultivars recommended for cultivation in Nigeria are presented. The two cultivars 53101 (local) and 60506 (improved) were harvested at 9, 12 and 15 months. In general, the improved cultivars appeared to respond better to N fertilization, but high rates of N (150 kg N/ha) tended to reduce the yield performance of the two cultivars. The local cultivar produced maximum yields of 31·5, 37·5 and 55·7 fresh tuber t/ha with 60 kg N/ha at 9, 12 and 15 months, respectively, while the improved cultivar gave maximum yields of 31·5 and 48·4 t/ha with 120 kg N/ha at 9 and 12 months, respectively, and 64·1 t/ha with 90 kg N/ha at 15 months. By allowing the crop to grow up to 15 months, the starch yield was more than doubled and about three times the yield obtained at 9 months. The 53101 strain seemed to produce maximum starch yields with 60 kg N/ha while the 60506 type tended to give maximum starch yields with 90 kg N/ha. Influence of N treatments was significant up to 12 months of age but not at 15 months.The HCN content of both cultivars diminished considerably with the age of the plant, dropping at 15 months to about half the level at 9 months. While the HCN concentration of the local cultivar tended to increase with higher N-fertilization, that of the improved cultivar diminished, particularly at 12 months after planting. At 15 months however, the HCN content of both cultivars rose with increasing N. fertilization.

Cassava cultivation faces increasing challenges from diseases, particularly cassava mosaic virus. Efficient propagation systems are essential for producing disease-free plants and sustaining production. However, information on the agronomic, physiological, growth traits, and yield of cassava plantlets produced through rapid propagation remains limited. This study aimed to investigate the photosynthetic and agronomic traits, as well as the growth and yield, of cassava plantlets derived from an aeroponic system under both pot and field conditions, using a 3 × 4 factorial arrangement in a randomized complete block design (RCBD). Three cassava varieties—Kasetsart 50 (KU50), Rayong 9 (RY9), and Huay Bong 60 (HB60)—were assigned as Factor A, while four plantlet sources—three derived from aeroponic systems (leaf bud cuttings, mini-cuttings, and normal cuttings) and one from conventional cuttings—were assigned as Factor B. The results indicated that HB60 and RY9 exhibited superior growth, stem diameter, photosynthesis rate, stomatal conductance, transpiration, and higher yields. Notably, RY9 also showed greater plant height and stem diameter, contributing to a higher multiplication rate. Plantlet sources did not significantly affect photosynthetic traits under either pot or field conditions but did increase canopy height, starch yield in the field, and starch content in the pot. Interestingly, the performance of leaf bud cuttings, mini-cuttings, and normal cuttings was comparable to conventional planting methods in terms of photosynthetic traits, yield traits, and harvest index. These findings suggest similarities among cassava cultivars in their responses to different plantlet sources and highlight the potential value of plantlet sources as a consideration for plant propagation programs.

Isotype-specific agglutination-PCR (ISAP): A sensitive and multiplex method for measuring allergen-specific IgE

Tuber yield, starch content, starch yield and chip color are complex traits that are important for industrial uses and food processing of potato. Chip color depends on the quantity of reducing sugars glucose and fructose in the tubers, which are generated by starch degradation. Reducing sugars accumulate when tubers are stored at low temperatures. Early and efficient selection of cultivars with superior yield, starch yield and chip color is hampered by the fact that reliable phenotypic selection requires multiple year and location trials. Application of DNA-based markers early in the breeding cycle, which are diagnostic for superior alleles of genes that control natural variation of tuber quality, will reduce the number of clones to be evaluated in field trials. Association mapping using genes functional in carbohydrate metabolism as markers has discovered alleles of invertases and starch phosphorylases that are associated with tuber quality traits. Here, we report on new DNA variants at loci encoding ADP-glucose pyrophosphorylase and the invertase Pain-1, which are associated with positive or negative effect with chip color, tuber starch content and starch yield. Marker-assisted selection (MAS) and marker validation were performed in tetraploid breeding populations, using various combinations of 11 allele-specific markers associated with tuber quality traits. To facilitate MAS, user-friendly PCR assays were developed for specific candidate gene alleles. In a multi-parental population of advanced breeding clones, genotypes were selected for having different combinations of five positive and the corresponding negative marker alleles. Genotypes combining five positive marker alleles performed on average better than genotypes with four negative alleles and one positive allele. When tested individually, seven of eight markers showed an effect on at least one quality trait. The direction of effect was as expected. Combinations of two to three marker alleles were identified that significantly improved average chip quality after cold storage and tuber starch content. In F1 progeny of a single-cross combination, MAS with six markers did not give the expected result. Reasons and implications for MAS in potato are discussed.Electronic supplementary materialThe online version of this article (doi:10.1007/s00122-012-2035-z) contains supplementary material, which is available to authorized users.