Whole-genome assembly and resequencing reveal genomic imprint and key genes of rapid domestication in narrow-leafed lupin

Shifting from a livestock-based protein diet to a plant-based protein diet has been proposed as an essential requirement to maintain global food sustainability, which requires the increased production of protein-rich crops for direct human consumption. Meanwhile, the lack of sufficient genetic diversity in crop varieties is an increasing concern for sustainable food supplies. Countering this concern requires a clear understanding of the domestication process and dynamics. Narrow-leafed lupin (Lupinus angustifolius L.) has experienced rapid domestication and has become a new legume crop over the past century, with the potential to provide protein-rich seeds. Here, using long-read whole-genome sequencing, we assembled the third-generation reference genome for the narrow-leafed lupin cultivar Tanjil, comprising 20 chromosomes with a total genome size of 615.8 Mb and contig N50 = 5.65 Mb. We characterized the original mutation and putative biological pathway resulting in low seed alkaloid level that initiated the recent domestication of narrow-leafed lupin. We identified a 1133-bp insertion in the cis-regulatory region of a putative gene that may be associated with reduced pod shattering (lentus). A comparative analysis of genomic diversity in cultivars and wild types identified an apparent domestication bottleneck, as precisely predicted by the original model of the bottleneck effect on genetic variability in populations. Our results identify the key domestication genetic loci and provide direct genomic evidence for a domestication bottleneck, and open up the possibility of knowledge-driven de novo domestication of wild plants as an avenue to broaden crop plant diversity to enhance food security and sustainable low-carbon emission agriculture.     

Screening of broad spectrum natural pesticides against conserved target arginine kinase in cotton pests by molecular modeling

Cotton is an economically important crop and its production is challenged by the diversity of pests and related insecticide resistance. Identification of the conserved target across the cotton pest will help to design broad spectrum insecticide. In this study, we have identified conserved sequences by Expressed Sequence Tag profiling from three cotton pests namely Aphis gossypii, Helicoverpa armigera, and Spodoptera exigua. One target protein arginine kinase having a key role in insect physiology and energy metabolism was studied further using homology modeling, virtual screening, molecular docking, and molecular dynamics simulation to identify potential biopesticide compounds from the Zinc natural database. We have identified four compounds having excellent inhibitor potential against the identified broad spectrum target which are highly specific to invertebrates.     

Functional Role of miRNAs: Key Players in Soybean Improvement

Soybean (Glycine max (L.) Merr) is an agro-economic crop growing across the world to cater nutrition for both human and animal feed due to the high oil and protein content in its edible seeds. The genes and QTLs associated with important agronomic traits in this crop have already been identified and validated for soybean cyst nematode (SCN), Phytophthora root and stem rot, Pythium root rot and aphid resistance, seed quality, nutrient values, and also employed for genetic improvement in soybean. In the last decade, micro RNAs (miRNAs) have been considered the effector molecules, so the detection and characterization of novel miRNAs in soybean have been taken up by several workers. The advancement in the strategy of sequencing and tools of bioinformatics during last decade has contributed to the discovery of many soybean miRNAs, thus miRNA can be used as a tool in molecular breeding studies, and this has opened new vistas for miRNA mediated genetic improvement of soybean to augment crop productivity as well as nutritional quality. This review addresses the current state of understanding of miRNAmediated stress responses, nutrient acquisition, plant development and crop production processes in soybean.     

Isolation and characterisation of a NBS-LRR resistance gene analogue from pearl millet

Plant resistance (R) proteins belonging to nucleotide-binding site–leucine-rich repeat (NBS–LRR) family are mainly involved in recognition of effectors secreted by pathogens. Pearl millet [Pennisetum glaucum (L.) R.Br] is one of the most drought tolerant cereals, staple food crop of the semi-arid tropics but is highly susceptible to the downy mildew disease caused by oomycetous Sclerospora graminicola (Sacc) schroet. Earlier studies have identified several resistance gene analogues (RGAs) in pearl millet which may be involved in resistance against downy mildew. Of these, a clone RGPM213 was shown to have more than 60% identity with R-proteins coding for NBS–LRR-like protein kinase. The exact nature and function of the R-protein encoded by this gene was not known. In the present study, the cDNA of RGPM213 encompassing NBS–LRR region was inserted into an expression vector pRSET-A and transformed into BL21 E.coli cells. The expressed recombinant fusion protein with a His tag was purified using nickel affinity purification and it had a molecular weight of 35 kDa on SDS-PAGE. Immunoaffinity purification using antibodies raised against this recombinant R-protein identified two proteins of molecular weights 55 kDa and 66 kDa from pearl millet seedling extracts. Peptide mass fingerprinting of these proteins followed by homology search in database revealed similarity of the 55 kDa protein with a protein kinase from Brassica oleracia containing serine/ threonine kinase domain.     

A novel tomato spotted wilt virus isolate encoding a noncanonical NSm C118F substitution associated with Sw-5 tomato gene resistance breaking

The nonstructural protein NSm of tomato spotted wilt virus (TSWV) has been identified as the avirulence determinant of the tomato single dominant Sw-5 resistance gene. Although Sw-5 effectiveness has been shown for most TSWV isolates, the emergence of resistance-breaking (RB) isolates has been observed. It is strongly associated with two point mutations (C118Y or T120N) in the NSm viral protein. TSWV-like symptoms were observed in tomato crop cultivars (+Sw-5) in the Baja California peninsula, Mexico, and molecular methods confirmed the presence of TSWV. Sequence analysis of the NSm 118–120 motif and three-dimensional protein modelling exhibited a noncanonical C118F substitution in seven isolates, suggesting that this substitution could emulate the C118Y-related RB phenotype. Furthermore, phylogenetic and molecular analysis of the full-length genome (TSWV-MX) revealed its reassortment-related evolution and confirmed that putative RB-related features are restricted to the NSm protein. Biological and mutational NSm 118 residue assays in tomato (+Sw-5) confirmed the RB nature of TSWV-MX isolate, and the F118 residue plays a critical role in the RB phenotype. The discovery of a novel TSWV-RB Mexican isolate with the presence of C118F substitution highlights a not previously described viral adaptation in the genus Orthotospovirus, and hence, the necessity of further crop monitoring to alert the establishment of novel RB isolates in cultivated tomatoes.     

Identification of differentially accumulating pistil proteins associated with self‐incompatibility of non‐heading Chinese cabbage

Non‐heading Chinese cabbage (Brassica campestris L. ssp. chinensis Makino), an important vegetable crop in China, exhibits a typical sporophytic self‐incompatibility (SI) system. To better understand the mechanism of SI response and identify potential candidate proteins involved in the SI system of this vegetable crop, the proteomic approach was taken to identify differential accumulating pistil proteins. Pistils were collected at 0 h and 2 h after self‐pollination at anthesis in self‐incompatible and compatible lines of non‐heading Chinese cabbage, and total proteins were extracted and separated by two‐dimensional gel electrophoresis (2‐DE). A total of 25 protein spots that displayed differential abundance were identified by matrix‐assisted laser desorption/ionisation‐time of flight mass spectrometry (MALDI–TOF/TOF MS) and peptide mass fingerprinting (PMF). Among them, 22 protein spots were confidently established. The mRNA levels of the corresponding genes were detected by quantitative RT‐PCR. The 22 identified protein spots are involved in energy metabolism (four), protein biosynthesis (three), photosynthesis (six), stress response and defence (five), and protein degradation (four). Among these potential candidate proteins, UDP‐sugar pyrophosphorylase could be involved in sucrose degradation to influence pollen germination and growth. Glutathione S–transferases could be involved in pollen maturation, and affect pollen fertility. Senescence‐associated cysteine protease, which is related to programmed cell death, could be mainly related to self pollen recognition of non‐heading Chinese cabbage. The study will contribute to further investigations of molecular mechanism of sporophytic SI in Brassicaceae.     

Olfactory responses of Rhopalosiphum padi to three maize,potato, and wheat cultivars and the selection of prospective crop border plants

Understanding host plant volatile – aphid interactions can facilitate the selection of crop border plants as a strategy to reduce plant virus incidence in crops. Crop border plant species with attractive odours could be used to attract aphids into the border crop and away from the main crop. As different cultivars of the same crop can vary in their olfactory attractiveness to aphids, selecting an attractive cultivar as a border crop is important to increase aphid landing rates. This study evaluated olfactory responses of the bird cherry‐oat aphid, Rhopalosiphum padi (L.) (Hemiptera: Aphididae), to three cultivars each of maize [Zea mays L. (Poaceae)], potato [Solanum tuberosum L. (Solanaceae)], and wheat [Triticum aestivum L. (Poaceae)] with the aim of selecting an attractive crop border plant to reduce the incidence of the non‐persistent Potato virus Y [PVY (Potyviridae)] in seed potatoes. Volatiles emitted by the crop cultivars were collected and identified using coupled gas chromatography/mass spectrometry. Quantitative and qualitative differences were found among cultivars. Behavioural responses of alate R. padi to odours of the cultivars and synthetic compounds identified from the plants were determined with a four‐arm olfactometer. Rhopalosiphum padi was attracted to odours emitted from maize cultivar 6Q‐121, but did not respond to odours from the remaining eight crop cultivars. Volatile compounds from maize and wheat cultivars that elicited a behavioural response from R. padi and contributed to differences in plant volatile profiles included (Z)‐3‐hexenyl acetate (attractant) and α‐farnesene, (E)‐2‐hexenal, indole, and (3E,7E)‐4,8,12‐trimethyltrideca‐1,3,7,11‐tetraene (TMTT) (repellents). We conclude that maize cv. 6Q‐121 is potentially suitable as a crop border plant based on the behavioural response of R. padi to the olfactory cues emitted by this cultivar. The findings provide insight into selecting crop cultivars capable of attracting R. padi to crop border plants.     

Molecular interactions between rosy apple aphids, Dysaphis plantaginea, and resistant and susceptible cultivars of its primary host Malus domestica

The use of crop varieties resistant or tolerant to insect pests or other stress factors is one approach in non‐chemical crop‐protection. Knowledge of the biochemical and molecular background of insect–plant interactions is a prerequisite for optimizing breeding for resistance. However, the resistance genes involved in plant–aphid interactions have so far only been identified and characterized in very few plant species. Our work aims to elucidate the molecular and biochemical mechanisms involved in resistance of apple trees, Malus domestica L. (Rosaceae), against its primary aphid pest, the rosy apple aphid, Dysaphis plantaginea (Passerini) (Homoptera: Aphididae), which is considered a serious economic pest of apple. Gene expression in both resistant and susceptible apple cultivars after infestation with rosy apple aphids was investigated by employing the cDNA‐AFLP method (cDNA–Amplified Fragment Length Polymorphism). From approximately 12 500 cDNA fragments detected on polyacrylamide gels, 21 bands were apparently up‐ or down‐regulated only in the resistant cultivar ‘Florina’ after aphid infestation compared to the susceptible cultivar ‘Topaz’ and/or mechanically wounded or non‐infested leaves. These fragments were cloned, sequenced, and the pattern of gene expression for six fragments was subsequently verified by virtual Northern blots. Sequence comparisons of these fragments to GenBank accessions revealed homologies to already known genes, most of them isolated from Arabidopsis thaliana L. Among them, a putative RNase‐L‐inhibitor‐like protein, a pectinacetylesterase, an inositol‐phosphatase‐like protein, a precursor of the large chain of the ribulose‐1,5‐biphosphate‐carboxylase, and defence‐related genes such as a vacuolar H(+)‐ATPase subunit‐like protein and an ADP‐ribosylating enzyme were identified. The results are discussed in relation to a putative role of these genes in conferring aphid resistance in apple trees.     

A CIPK protein kinase targets sucrose transporter MdSUT2.2 at Ser254 for phosphorylation to enhance salt tolerance

Soil salinity is one of the major abiotic stressors that negatively affect crop growth and yield. Salt stress can regulate antioxidants and the accumulation of osmoprotectants. In the study, a sucrose transporter MdSUT2.2 was identified in apple. Overexpression of MdSUT2.2 gene increased salt tolerance in the transgenic apple, compared with the WT control “Gala.” In addition, it was found that protein MdSUT2.2 was phosphorylated at Ser²⁵⁴ site in response to salt. A DUAL membrane yeast hybridization system through an apple cDNA library demonstrated that a protein kinase MdCIPK13 interacted with MdSUT2.2. A series of transgenic analysis in apple calli showed that MdCIPK13 was required for the salt‐induced phosphorylation of MdSUT2.2 protein and enhanced its stability and transport activity. Finally, it was found that MdCIPK13 improved salt resistance in an MdSUT2.2‐dependent manner. These findings had enriched our understanding of the molecular mechanisms underlying abiotic stress.     

Development of an ELISA for estimation of the copper nutritional status of wheat and cotton

Studies were carried out with hydroponically grown wheat and cotton to develop the Cu-requiring protein phenolase as a biomarker of Cu nutrient status. Isozymes of phenolase whose levels were reduced by Cu deficiency were identified by Western blots. A competitive enzyme-linked, immunosorbent assay (ELISA) was developed that could detect as little as 25 ng of phenolase. The ELISA revealed that Cu-sufficient cotton leaves had about 4-fold more phenolase antigen than did Cu-sufficient wheat leaves. In both species, the level of phenolase was reduced by 2- to 5-fold in leaves of Cu-deficient plants. Because the immunoassay for phenolase protein is rapid, inexpensive, and can be carried out with small amounts of leaf material, it has potential as a tool for assessment of the Cu status of crop plants.Abbreviations ELISA enzyme-linked immunosorbent assay - HRP horseradish peroxidase - TBS Trisbuffered saline (20 mM Tris-HCl, pH 9.5, 150 mM NaCl) - TBST Tris-buffered saline containing 0.05% Tween-20     

Proteomic characterization of seeds from yellow lupin (Lupinus luteus L.)

Yellow lupin (Lupinus luteus L.) is a legume crop containing a large amount of protein in its seeds. In this study, we constructed a seed‐protein catalog to provide a foundation for further study of the seeds. A total of 736 proteins were identified in 341 2DE spots by nano‐LC‐MS/MS. Eight storage proteins were found as multiple spots in the 2DE gels. The 736 proteins correspond to 152 unique proteins as shown by UniRef50 clustering. Sixty‐seven of the 152 proteins were associated with KEGG‐defined pathways. Of the remaining proteins, 57 were classified according to a GO term. The functions of the remaining 28 proteins have yet to be determined. This is the first yellow lupin seed–protein catalog, and it contains considerably more data than previously reported for white lupin (L. albus L.).     

Genome‐wide association of drought‐related and biomass traits with HapMap SNPs in Medicago truncatula

Improving drought tolerance of crop plants is a major goal of plant breeders. In this study, we characterized biomass and drought‐related traits of 220 Medicago truncatula HapMap accessions. Characterized traits included shoot biomass, maximum leaf size, specific leaf weight, stomatal density, trichome density and shoot carbon‐13 isotope discrimination (δ¹³C) of well‐watered M. truncatula plants, and leaf performance in vitro under dehydration stress. Genome‐wide association analyses were carried out using the general linear model (GLM), the standard mixed linear model (MLM) and compressed MLM (CMLM) in TASSEL, which revealed significant overestimation of P‐values by CMLM. For each trait, candidate genes and chromosome regions containing SNP markers were found that are in significant association with the trait. For plant biomass, a 0.5 Mbp region on chromosome 2 harbouring a plasma membrane intrinsic protein, PIP2, was discovered that could potentially be targeted to increase dry matter yield. A protein disulfide isomerase‐like protein was found to be tightly associated with both shoot biomass and leaf size. A glutamate‐cysteine ligase and an aldehyde dehydrogenase family protein with Arabidopsis homologs strongly expressed in the guard cells were two of the top genes identified by stomata density genome‐wide association studies analysis.     

Improved germination efficiency of Salicornia ramosissima seeds inoculated with Bacillus aryabhattai SP1016‐20

Saline agriculture and the crop cultivation of halophytes represent an alternative for the reclamation of salinized soils and for the management of irrigation water. Halotolerant plant growth promoting bacteria with biocontrol effect, as an alternative to commercial fungicides, may contribute to improve crop productivity while mitigating saline stress effects. The objective of this work was to isolate autochthonous rhizobacteria with biocontrol features, to be used as germination enhancers and plant‐growth promoting agents in the crop cultivation of Salicornia ramosissima. A set of isolates obtained from the rhizosphere of S. ramosissima was characterised in terms of Gram, motility, salt tolerance and biocontrol traits (hydrogen cyanide production, antifungal activity and production of extracellular lipases and proteases). One Gram‐positive motile isolate that tested positive for all biocontrol traits was identified by 16S rRNA gene sequencing as Bacillus aryabhattai. The inoculation of S. ramosissima seeds with B. aryabhattai SP1016‐20 reduced the negative effect of salinity on the germination efficiency. At the highest tested salinity (30 g/L NaCl) the final germination efficiency of inoculated seeds doubled in relation to non‐inoculated controls. Although the mechanisms involved in the biocontrol effect were not defined in the current work, the results highlight the potential of Bacillus aryabhattai SP1016‐20 as a plant‐growth promoting agent for the crop cultivation of Salicornia and contribute to the strengthening of the scientific basis of biosaline agriculture and plant growth promoting rhizobacteria‐assisted crop cultivation of halophytes in saline soils and estuarine sediments.     

<i>Aspergillus tubingensis</i> Causes Leaf Spot of Cotton (<i>Gossypium hirsutum</i> L.) in Pakistan

Cotton (Gossypium hirsutum L.) is a key fiber crop of great commercial importance. Numerous phytopathogens decimate crop production by causing various diseases. During July-August 2018, leaf spot symptoms were recurrently observed on cotton leaves in Rahim Yar Khan, Pakistan and adjacent areas. Infected leaf samples were collected and plated on potato dextrose agar (PDA) media. Causal agent of cotton leaf spot was isolated, characterized and identified as Aspergillus tubingensis based on morphological and microscopic observations. Conclusive identification of pathogen was done on the comparative molecular analysis of CaM and β-tubulin gene sequences. BLAST analysis of both sequenced genes showed 99% similarity with A. tubingensis. Koch’s postulates were followed to confirm the pathogenicity of the isolated fungus. Healthy plants were inoculated with fungus and similar disease symptoms were observed. Fungus was re-isolated and identified to be identical to the inoculated fungus. To our knowledge, this is the first report describing the involvement of A. tubingensis in causing leaf spot disease of cotton in Pakistan and around the world.     

Measuring host plant selection and retention of Halyomorpha halys by a trap crop

Trap cropping may exploit a pest's dispersal and host selection behavior in order to protect a desired crop. Here, we used a combination of visual sampling, immunomarking, and harmonic radar to assess host plant selection and retention time of the highly mobile and invasive Halyomorpha halys (Stål) (Hemiptera: Pentatomidae), as it moves within and between a polyculture trap crop of sorghum and sunflower, and a bell pepper cash crop. Visual sampling demonstrated no significant differences in H. halys densities across crops, whereas dislodging stink bugs to collect for protein analysis revealed ca. 4× more bugs in the trap crop plants than in the peppers. In total 145 H. halys were collected and of these 6% were doubly marked with proteins, demonstrating that minimal movement occurred between the two planting systems. Tracking tagged H. halys with harmonic radar revealed that the trap crop retained adult H. halys within the plots 1.5× longer and reduced their movement by nearly half compared with bugs released in the pepper cash crop. The data suggest the trap crop of sunflower plus sorghum has the potential to attract and arrest the invasive H. halys, demonstrating that trap cropping may operate as an effective management tool.     

QTL analysis of field resistance to Xanthomonas axonopodis pv. manihotis in cassava

We evaluated cassava bacterial blight (CBB) infection in an pair-cross population of 150 individuals derived from an intra-specific cross between two non-inbred cassava (Manihot esculenta Crantz) lines. The replicated trials were carried out in the field under high disease pressure over two consecutive crop cycles. Evaluations were conducted at 4 and 7 months after planting for the two cycles. Simple regression analysis and the nonparametric Kruskal-Wallis rank-sum test revealed that eight quantitative trait loci (QTLs) were involved in resistance. We detected changes in QTLs from crop cycle to crop cycle. The pathogen population (Xanthomonas axonopodis pv. manihotis) was also monitored over the period, using a restriction fragment length polymorphism probe and pathogenic tests. Changes in QTL detection over the 2 years could be correlated with changes in pathogen population structure. One QTL, located in linkage group D, was conserved over the two crop cycles, and in field to greenhouse evaluations. This study thus identified molecular markers useful for marker assisted-selection, a technique that can accelerate the long, multiple-season process of breeding for CBB resistance. Received: 1 January 2000 / Accepted: 25 June 2000