De novo sequencing of complex genomes is one of the main challenges for researchers seeking high-quality reference sequences. Many de novo assemblies are based on short reads, producing fragmented ...genome sequences. Third-generation sequencing, with read lengths >10 kb, will improve the assembly of complex genomes, but these techniques require high-molecular-weight genomic DNA (gDNA), and gDNA extraction protocols used for obtaining smaller fragments for short-read sequencing are not suitable for this purpose. Methods of preparing gDNA for bacterial artificial chromosome (BAC) libraries could be adapted, but these approaches are time-consuming, and commercial kits for these methods are expensive. Here, we present a protocol for rapid, inexpensive extraction of high-molecular-weight gDNA from bacteria, plants, and animals. Our technique was validated using sunflower leaf samples, producing a mean read length of 12.6 kb and a maximum read length of 80 kb.
Summary
Despite the importance of plant–plant interactions on crop yield and plant community dynamics, our understanding of the genetic and molecular bases underlying natural variation of plant–plant ...interactions is largely limited in comparison with other types of biotic interactions. By listing 63 quantitative trait loci (QTL) mapping and global gene expression studies based on plants directly challenged by other plants, we explored whether the genetic architecture and the function of the candidate genes underlying natural plant–plant interactions depend on the type of interactions between two plants (competition versus commensalism versus reciprocal helping versus asymmetry). The 16 transcriptomic studies are unevenly distributed between competitive interactions (n = 12) and asymmetric interactions (n = 4, all focusing on response to parasitic plants). By contrast, 17 and 30 QTL studies were identified for competitive interactions and asymmetric interactions (either weed suppressive ability or response to parasitic plants), respectively. Surprisingly, no studies have been carried out on the identification of genetic and molecular bases underlying natural variation in positive interactions. The candidate genes underlying natural plant–plant interactions can be classified into seven categories of plant function that have been identified in artificial environments simulating plant–plant interactions either frequently (photosynthesis, hormones), only recently (cell wall modification and degradation, defense pathways against pathogens) or rarely (ABC transporters, histone modification and meristem identity/life history traits). Finally, we introduce several avenues that need to be explored in the future to obtain a thorough understanding of the genetic and molecular bases underlying plant–plant interactions within the context of realistic community complexity.
Significance Statement
While plant–plant interactions are recognized as a major factor responsible for crop yield and plant community dynamics, their underlying genetic and molecular mechanisms still deserve a deeper investigation. By considering studies where plants have been directly challenged by other plants, we identified plant functions that have been rarely identified in artificial environments simulating plant–plant interactions. The next challenge will be to understand the genetic and molecular mechanisms underlying positive interactions.
The herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) is applied directly to aquatic and conventional farming systems to control weeds, and is among the most widely distributed pollutants in the ...environment. Non-target organisms are exposed to 2,4-D via several ways, which could produce toxic effects depending on the dose, frequency of exposure, and the host factors that influence susceptibility and sensitivity. An increasing number of experimental evidences have shown concerns about its presence/detection in the environment, because several investigations have pointed out its potential lethal effects on non-target organisms. In this review, we critically evaluated the environmental fate and behavior of 2,4-D along with its eco-toxicological effects on aquatic, plants and human life to provide concise assessment in the light of recently published reports. The findings demonstrate that 2,4-D is present in a low concentration in surface water of regions where its usage is high. The highest concentrations of 2,4-D were detected in soil, air and surface water surrounded by crop fields, which suggest that mitigation strategies must be implanted locally to prevent the entry of 2,4-D into the environment. A general public may have frequent exposure to 2,4-D due to its wide applications at home lawns and public parks, etc. Various in vivo and in vitro investigations suggest that several species (or their organs) at different trophic levels are extremely sensitive to the 2,4-D exposure, which may explain variation in outcomes of reported investigations. However, implications for the prenatal exposure to 2,4-D remain unknown because 2,4-D-induced toxicity thresholds in organism have only been derived from juveniles or adults. In near future, introduction of 2,4-D resistant crops will increase its use in agriculture, which may cause relatively high and potentially unsafe residue levels in the environment. The recent findings indicate the urgent need to further explore fate, accumulation and its continuous low level exposure impacts on the environment to generate reliable database which is key in drafting new regulation and policies to protect the population from further exposure.
•Discusses the prevalence of 2,4-D in the environment•Describes current state of knowledge regarding 2,4-D use and its detrimental impacts•Highlights possible exposure routes to humans and potential for adverse impacts on farmers' health•Highlights risks associated with advance formulations of 2,4-D and herbicide resistant crops
Domestication of crop plants had effects on human lifestyle and agriculture. However, little is known about the underlying molecular mechanisms accompanying the changes in fruit appearance as a ...consequence of selection by early farmers. We report the fine mapping and cloning of a tomato (Solanum lycopersicum) fruit mass gene encoding the ortholog of KLUH, SIKLUH, a P450 enzyme of the CYP78A subfamily. The increase in fruit mass is predominantly the result of enlarged pericarp and septum tissues caused by increased cell number in the large fruited lines. SIKLUH also modulates plant architecture by regulating number and length of the side shoots, and ripening time, and these effects are particularly strong in plants that transgenically down-regulate SIKLUH expression carrying fruits of a dramatically reduced mass. Association mapping followed by segregation analyses revealed that a single nucleotide polymorphism in the promoter of the gene is highly associated with fruit mass. This single polymorphism may potentially underlie a regulatory mutation resulting in increased SIKLUH expression concomitant with increased fruit mass. Our findings suggest that the allele giving rise to large fruit arose in the early domesticates of tomato and becoming progressively more abundant upon further selections. We also detected association of fruit weight with CaKLUH in chile pepper (Capsicum annuum) suggesting that selection of the orthologous gene may have occurred independently in a separate domestication event. Altogether, our findings shed light on the molecular basis of fruit mass, a key domestication trait in tomato and other fruit and vegetable crops.
Key message
Or
Deb2
confers post-attachment resistance to
Orobanche cumana
and is located in a 1.38 Mbp genomic interval containing a cluster of receptor-like kinase and receptor-like protein genes ...with nine high-confidence candidates.
Sunflower broomrape is a holoparasitic angiosperm that parasitizes on sunflower roots, severely constraining crop yield. Breeding for resistance is the most effective method of control.
Or
Deb2
is a dominant resistance gene introgressed into cultivated sunflower from a wild-related species that confers resistance to highly virulent broomrape races. The objectives of this study were as follows: (i) locate
Or
Deb2
into the sunflower genome and determine putative candidate genes and (ii) characterize its underlying resistance mechanism. A segregating population from a cross between the sunflower resistant line DEB2, carrying
Or
Deb2
, and a susceptible line was phenotyped for broomrape resistance in four experiments, including different environments and two broomrape races (F
GV
and G
TK
). This population was also densely genotyped with microsatellite and SNP markers, which allowed locating
Or
Deb2
within a 0.9 cM interval in the upper half of Chromosome 4. This interval corresponded to a 1.38 Mbp genomic region of the sunflower reference genome that contained a cluster of genes encoding LRR (leucine-rich repeat) receptor-like proteins lacking a cytoplasmic kinase domain and receptor-like kinases with one or two kinase domains and lacking an extracellular LRR region, which were valuable candidates for
Or
Deb2
. Rhizotron and histological studies showed that
Or
Deb2
determines a post-attachment resistance response that blocks
O. cumana
development mainly at the cortex before the establishment of host-parasite vascular connections. This study will contribute to understand the interaction between crops and parasitic weeds, to establish durable breeding strategies based on genetic resistance and provide useful tools for marker-assisted selection and
Or
Deb2
map-based cloning.
Species often include multiple ecotypes that are adapted to different environments1. However, it is unclear how ecotypes arise and how their distinctive combinations of adaptive alleles are ...maintained despite hybridization with non-adapted populations2-4. Here, by resequencing 1,506 wild sunflowers from 3 species (Helianthus annuus, Helianthus petiolaris and Helianthus argophyllus), we identify 37 large (1-100 Mbp in size), non-recombining haplotype blocks that are associated with numerous ecologically relevant traits, as well as soil and climate characteristics. Limited recombination in these haplotype blocks keeps adaptive alleles together, and these regions differentiate sunflower ecotypes. For example, haplotype blocks control a 77-day difference in flowering between ecotypes ofthe silverleaf sunflower H. argophyllus (probably through deletion of a homologue of FLOWERING LOCUS T (FT)), and are associated with seed size, flowering time and soil fertility in dune-adapted sunflowers. These haplotypes are highly divergent, frequently associated with structural variants and often appear to represent introgressions from other-possibly now-extinct-congeners. These results highlight a pervasive role of structural variation in ecotypic adaptation.
Parasitic plants have a unique heterotrophic lifestyle based on the extraction of water and nutrients from host plants. Some parasitic plant species, particularly those of the family Orobanchaceae, ...attack crops and cause substantial yield losses. The breeding of resistant crop varieties is an inexpensive way to control parasitic weeds, but often does not provide a long-lasting solution because the parasites rapidly evolve to overcome resistance. Understanding mechanisms underlying naturally occurring parasitic plant resistance is of great interest and could help to develop methods to control parasitic plants. In this review, we describe the virulence mechanisms of parasitic plants and resistance mechanisms in their hosts, focusing on obligate root parasites of the genera
and
. We noticed that the resistance (R) genes in the host genome often encode proteins with nucleotide-binding and leucine-rich repeat domains (
proteins), hence we proposed a mechanism by which host plants use
proteins to activate downstream resistance gene expression. We speculated how parasitic plants and their hosts co-evolved and discussed what drives the evolution of virulence effectors in parasitic plants by considering concepts from similar studies of plant-microbe interaction. Most previous studies have focused on the host rather than the parasite, so we also provided an updated summary of genomic resources for parasitic plants and parasitic genes for further research to test our hypotheses. Finally, we discussed new approaches such as CRISPR/Cas9-mediated genome editing and RNAi silencing that can provide deeper insight into the intriguing life cycle of parasitic plants and could potentially contribute to the development of novel strategies for controlling parasitic weeds, thereby enhancing crop productivity and food security globally.
Resistance to downy mildew (
) in sunflower (
L.) is conferred by major resistance genes, denoted
Twenty-two
genes have been identified and genetically mapped so far. However, over the past 50 years, ...wide-scale presence of only a few of them in sunflower crops led to the appearance of new, more virulent pathotypes (races) so it is important for sunflower varieties to carry as wide a range of resistance genes as possible. We analyzed phenotypically 12 novel resistant sources discovered in breeding pools derived from two wild
species and in eight wild
ecotypes. All were effective against at least 16 downy mildew pathotypes. We mapped their resistance genes on the sunflower reference genome of 3,600 Mb, in intervals that varied from 75 Kb to 32 Mb using an AXIOM
genotyping array of 49,449 SNP. Ten probably new genes were identified according to resistance spectrum, map position, hypersensitive response to the transient expression of a
RXLR effector, or the ecotype/species from which they originated. The resistance source HAS6 was found to carry the first downy mildew resistance gene mapped on chromosome 11, whereas the other resistances were positioned on chromosomes 1, 2, 4, and 13 carrying already published
genes that we also mapped physically on the same reference genome. The new genes were designated
according to the current nomenclature. However, since sunflower downy mildew resistance genes have not yet been sequenced, rules for designation are discussed. This is the first large scale physical mapping of both 10 new and 10 already reported downy mildew resistance genes in sunflower.
Orobanche cumana (sunflower broomrape) is an obligatory and non-photosynthetic root parasitic plant that specifically infects the sunflower. It is located in Europe and in Asia, where it can cause ...yield losses of over 80%. More aggressive races have evolved, mainly around the Black Sea, and broomrape can rapidly spread to new areas. Breeding for resistance seems to be the most efficient and sustainable approach to control broomrape infestation. In our study, we used a population of 101 recombinant inbred lines (RILs), derived from a cross between the two lines HA89 and LR1 (a line derived from an interspecific cross with Helianthus debilis). Rhizotrons, pots and field experiments were used to characterize all RILs for their resistance to O. cumana race F parasitism at three post vascular connection life stages: (i) early attachment of the parasite to the sunflower roots, (ii) young tubercle and (iii) shoot emergence. In addition, RIL resistance to race G at young tubercle development stage was evaluated in pots. The entire population was genotyped, and QTLs were mapped. Different QTLs were identified for each race (F from Spain and G from Turkey) and for the three stages of broomrape development. The results indicate that there are several quantitative resistance mechanisms controlling the infection by O. cumana that can be used in sunflower breeding.
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