Pyramiding of multiple genes generates rapeseed introgression lines with clubroot and herbicide resistance, high oleic acid content, and early maturity

Clubroot and herbicide resistance,high oleic acid(OA)content,and early maturity are targets of rapeseed(Brassica napus L.)breeding.The objective of this study was to develop new male-fertility restorer lines by pyramiding favorable genes to improve these traits simultaneously.Seven elite alleles for the four traits were introduced into the restorer line 621R by speed breeding with marker-assisted and phenotypic selection.Six introgression lines(ILs)were developed with four-to seven-gene combinations and crossed with two elite parents to develop hybrids.All ILs and their corresponding hybrids displayed high resistance to both clubroot pathotype 4 and sulfonylurea herbicides.Three ILs and their hybrids showed large increases in OA contents and four showed earlier maturity.These new ILs may be useful in rapeseed hybrid breeding for the target traits.

Precise base editing of non-allelic acetolactate synthase genes confers sulfonylurea herbicide resistance in maize

Single-nucleotide polymorphisms contribute to phenotypic diversity in maize. Creation and functional annotation of point mutations has been limited by the low efficiency of conventional methods based on random mutation. An efficient tool for generating targeted single-base mutations is desirable for both functional genomics and precise genetic improvement. The objective of this study was to test the efficiency of targeted C-to-T base editing of two non-allelic acetolactate synthase(ALS) in generating sulfonylurea herbicide-resistant mutants. A CRISPR/Cas9 nickase-cytidine deaminase fused with uracil DNA glycosylase inhibitor(UGI) was employed to achieve targeted conversion of cytosine to thymine in ZmALS1 and ZmALS2. Both protoplasts and recovered mutant plants showed the activity of the cytosine base editor, with an in vivo efficiency of up to 13.8%. Transgene-free edited plants harboring a homozygous ZmALS1 mutation or a ZmALS1 and ZmALS2 double mutation were tested for their resistance at a dose of up to 15-fold the recommended limit of chlorsulfuron, a sulfonylurea herbicide widely used in agriculture. Targeted base editing of C-to-T per se and a phenotype verified in the generated mutants demonstrates the power of base editing in precise maize breeding.

Development of herbicide resistance genes and their application in rice

Rice is one of the most important food crops in the world.Weeds seriously affect the rice yield and grain quality.In recent years,there are tremendous progresses in the research and application of herbicideresistant genes in rice worldwide.This article reviews the working mechanisms of six herbicides(glyphosate,glufosinate,acetolactate synthase inhibitor herbicides,acetyl-Co A carboxylase inhibitor herbicides,hydroxyhenylpyruvate dioxygenase(HPPD)inhibitor herbicides and dinitroaniline herbicides),the resistance mutations of the corresponding herbicide-target genes,and the herbicide detoxification mechanisms by non-target genes.Examples are provided on herbicide-resistant rice materials obtained by transformation of exogenous resistance genes,by artificial mutagenesis and mutant screening,and by modifying the target genes through gene editing.This paper also introduces the current application of herbicide-resistant rice,points out problems that may be caused by utilization of herbicide resistant rice and solutions to the problems,and discusses the future prospects for the development of herbicideresistant rice.

Genomic insights into the origin, adaptive evolution, and herbicide resistance of Leptochloa chinensis, a devastating tetraploid weedy grass in rice fields

Chinese sprangletop (Leptochloa chinensis), belonging to the grass subfamily Chloridoideae, is one of the most notorious weeds in rice ecosystems. Here, we report a chromosome-scale reference genome assembly and a genomic variation map of the tetraploid L. chinensis. The L. chinensis genome is derived from two diploid progenitors that diverged ∼10.9 million years ago, and its two subgenomes display neither fractionation bias nor overall gene expression dominance. Comparative genomic analyses reveal substantial genome rearrangements in L. chinensis after its divergence from the common ancestor of Chloridoideae and, together with transcriptome profiling, demonstrate the important contribution of tetraploidization to the gene sources for the herbicide resistance of L. chinensis. Population genomic analyses of 89 accessions from China reveal that L. chinensis accessions collected from southern/southwestern provinces have substantially higher nucleotide diversity than those from the middle and lower reaches of the Yangtze River, suggesting that L. chinensis spread in China from the southern/southwestern provinces to the middle and lower reaches of the Yangtze River. During this spread, L. chinensis developed significantly increased herbicide resistance, accompanied by the selection of numerous genes involved in herbicide resistance. Taken together, our study generated valuable genomic resources for future fundamental research and agricultural management of L. chinensis, and provides significant new insights into the herbicide resistance as well as the origin and adaptive evolution of L. chinensis.

Control of Glyphosate-Resistant Giant Ragweed (Ambrosia trifida L.) with Isoxaflutole and Metribuzin Tankmix

Five field trials were conducted over a two-year period (2013, 2014) to determine the control of glyphosate-resistant (GR) giant ragweed with isoxaflutole (IFT) and metribuzin (MTZ) applied alone and in combination. Treatments were designed to assess the dose response of an IFT plus MTZ tank-mix as well as each chemical applied alone to classify the response using Flint’s adaptation of Colby’s equation. Two factor factorial experiments were performed in the growth room to ascertain the response of IFT versus glyphosate, IFT versus MTZ, and IFT plus MTZ versus glyphosate on single plants. Field experiments evaluated the control of GR giant ragweed with IFT plus MTZ in tank-mix in a 1:4 ratio. The rate of IFT plus MTZ for 80% control of GR giant ragweed at 4 and 8 weeks after application (WAA) was 518 (104 g a.i. ha-1 IFT + 414 g a.i. ha-1 MTZ) and 631 g a.i. ha-1 (126 g a.i. ha-1 IFT + 505 g a.i. ha-1 MTZ), respectively. A rate of 668 and 467 g a.i. ha-1 was required to reduce GR giant ragweed density and biomass by 80%, respectively. Field experiments evaluating the control of GR giant ragweed with tank-mixes of IFT plus MTZ, where glyphosate was a constant tank-mix partner, were mostly synergistic. However, the low tank-mix rate (52.5 + 210 g a.i. ha-1) had an additive response for GR giant ragweed biomass reduction. When tested in the greenhouse and growth room, glyphosate susceptible (GS) giant ragweed showed some antagonism with glyphosate and isoxaflutole tank-mixes at rates less than commercial field rates. GR giant ragweed showed an additive response across all treatments in the growth room. Greenhouse experiments evaluating IFT versus MTZ and IFT plus MTZ versus glyphosate revealed all tank-mix treatments to be synergistic at 2 WAA.

CRISPR/Cas9-based Editing of Acetyl-CoA Carboxylase (ACC1) Gene in Barley

Plastid localized acetyl-CoA carboxylase(ACCase;EC 6.4.1.2)is a target for aryloxyphenoxypropionates(APPs)and cyclohexanediones(CHDs),two groups of selective herbicides used in controlling grassy weeds.Wheat and barley are important cereal crops in the grass(Poaceae or Gramineae)family,and thus sensitive for those herbicides.Characterization of this form of ACCase(or ACC1)in wheat and barley is essential if these agents are used in the sustainable agriculture.In this study,it was confirmed that a single ACC1 gene presented on the second chromosome per homologous group in common wheat,wild emmer wheat,goat grass and barley.Using CRISPR/Cas9,the barley ACC1 gene was edited,specifically in the carboxyl transferase(CT)domain that was critical for herbicide responses in grass species.Two new alleles were generated,one with a 3-bp deletion leading to ACC1:p.Ile1878del and one with a 26-bp deletion causing ACC1:p.Ser2099_Lys2311del.Both were recovered as heterozygotes in the T0 generation.All the seven T0 plants harboring the 3-bp deletion grew normally,but the only T0 plant with 26-bp deletion died at the extension stage(Zadoks 32),probably because there was inadequate ACC1 activity when the plant was big.In the T1 generation,the 3-bp deletion(or Ile1878del)did not impact the edited plants in tiller numbers,tiller height,spike length and spikelet numbers,when compared to the wild-type allele in the non-edited segregants.This study demonstrated that CRISPR/Cas9 was practical to generate single amino acid deletions in the ACC1 protein and the Ile1878 deletion did not compromise plant growth.Unfortunately,the ACC1:p.Ile1878del protein did not confer resistance to the currently tested APP herbicides,including clethodim,haloxyfop,quizalofop-Pethyl and sethoxydim.

Marker gene excision in transgenic Brassica napus via Agrobacterium-mediated Cre/lox transient expression system

Oilseed rape(Brassica napus L.)is one of the most important oil crops in the world.However,study on marker-free transgene of B.napus for bio-safety purpose is limited in this allotetraploid crop.In order to advance marker gene excision research,a newly designed Cre/lox system combining crossing and auto-excision strategy was introduced into B.napus.The system consists of 2 sets of independent vectors including pC35 Spro::T7 RP carrying T7 RNA polymerase and pCT7 pro::Cre carrying T7 promoter respectively.After hybridization of 2 according types of transgenic B.napus,marker gene would be removed as T7 RNA polymerase facilitate T7 promoter to promote Cre gene expression.Totally 52 and 46 positive To transgenic lines of these 2 vectors were obtained after identification by PCR and test trip.T1 plants from 3 T0 positive pC35 Spro::T7 RP lines and 2 T0 positive pC35 Spro::T7 RP lines were identified as single copy according to segregation ratio and were chosen for crossing.However,expression of CP4 EPSPS(glyphosate resistance gene)and OXY(bromoxynil resistance gene)were not found in F1 progeny,which proved that the excision was not complete.The possible reasons for our limited success were investigated and detailed analyses were performed.Although this system is not applicable for generating transgenic B.napus free from selectable marker gene,it provided valuable experience and clue for further improvement of this technique.Many other advantages and further improvement will be progressed in future work.

Allelopathic Potential of Wheat on Sourgrass Resistant to Glyphosate

We aimed with this study to assay the allelopathic potential of wheat genotypes in inhibiting germination and initial growth of sourgrass (Digitaria insularis) resistant to the herbicide glyphosate. Two experiments were installed under greenhouse conditions: the first one aimed to assay the potential of wheat genotypes in inhibiting emergence and initial growth of two test plants, and the second aimed to evaluate the potential of those wheat genotypes which performed better at the first experiment, inhibiting the emergence and initial growth of sourgrass biotypes resistant to the herbicide glyphosate. Five doses of aqueous cool-extracted extract from 32 wheat genotypes were applied to tomato and cucumber (Experiment 1) aiming to identify the ones with higher inhibitory effect. The five most inhibitory wheat genotypes were selected for Experiment 2, where the same doses of extract were applied on sourgrass. For both experiments, shoot fresh and dry mass, as well as water content, were assessed. Sourgrass germination is affected by wheat extracts, but initial seedling growth seems not to be affected;wheat genotypes differ in terms of their allelopathic potential in inhibiting sourgrass germination;wheat presents little inhibitory effect on sourgrass.

Base-Editing-Mediated Artificial Evolution of OsALS1 In Planta to Develop Novel Herbicide-Tolerant Rice Germplasms

Recently developed CRISPR-mediated base editors,which enable the generation of num erous nucleotide changes in target genomic regions,have been widely adopted for gene correction and generation of crop germ plasms containing im portant gain-of-function genetic variations.How ever,to engineer target genes with unknown functional SNPs remains challenging.To address this issue,we present here abase-e diting-mediated gene evolution(BEMGE)m ethod,employing both Cas9n-based cytosine and adenine base editors as well as a single-guide RNA(sgRNA)library tiling the full-length coding region,for developing novel rice germ plasm swith mutations in any endogenous gene.To this end,OsALS1 was artificially evolved in rice cells using BEMGE through both Agrobacterium-mediated and particle-bom bardment-mediated transform ation.Four different types of amino acid substitutions in the evolved OsALS1,derived from two sites that have never been targeted by natural or human selection during rice dom estication,were identified,conferring varying levels of tolerance to the herbicide bispyribac-sodium.Furtherm ore,the P171F substitution identified in a strong OsALS1 allele was quickly introduced into the commercial rice cultivar Nangeng 46 through precise base editing w ith the corresponding base editor and sgRNA.Collectively,these data indicate great potential of BEMGE in creating important genetic variants of target genes for crop improvement.

Developing herbicide resistant Sri-lankan rice(Oryza sativa L.)varieties:An application of Self Organizing Map

Application of high concentrations of post-emergent broad spectrum systemic herbicide,glyphosate is prevalently used to control rice weeds in Asian countries including Sri Lanka.Off target movements of glyphosate adversely affect growth of the rice plant reducing the yield.Inducing herbicide resistance(HR)in cultivated rice is a novel approach to enhance selectivity and crop safety.Studies on induced HR in Sri Lankan rice varieties are limited and studies are required to include HR rice in a cropping program.Ethyl Methyl Sulfonate(EMS),a chemical mutagen is used for functional mutations.The present study is an attempt of raising HR rice lines through conventional breeding methods.A suitable glyphosate concentration was determined by a preliminary study using five rice varieties(Bg300,Bg352,At362,Bg379-2 and H4)and varying glyphosate concentrations(0.25 g/l,0.5 g/l,1 g/l,2 g/l and 3 g/l).Resistance percentage
50%was arbitrary considered as resistant varieties.Complete Randomized Design with three replicates was used in the experiment.Agromorphological characters were recorded for the survived plants.Twelve varieties(Bg359,At362,Bw364,Ld365,Bg366,Bg369,Bg379-2,Bg403,Bg454“Pachcha perumal”,“Kalu heenati”and“Kurulu thuda”)showed natural resistance to glyphosate and fourteen varieties have increased resistance after mutagenesis by EMS(S0-First generation).HR resistance percentage of S1(Second generation)plants was similar to S0 plants indicating HR was transferred to new generation.Conventional statistics was supplemented with Self-Organizing Maps(SOM)to visualize variation of agro-morphological characters under treatment.The result proved that EMS application is an effective method in breeding new rice germplasm with HR and SOM is an important tool for visualizing the multi-dimensional dataset in lower dimensions.

Optimized prime editing efficiently generates heritable mutations in maize

Low efficiency is the main obstacle to using prime editing in maize(Zea mays).Recently,prime-editing efficiency was greatly improved in mammalian cells and rice(Oryza sativa)plants by engineering primeediting guide RNAs(pegRNAs),optimizing the prime editor(PE)protein,and manipulating cellular determinants of prime editing.In this study,we tested PEs optimized via these three strategies in maize.We demonstrated that the ePE5max system,composed of PEmax,epegRNAs(pegRNA-evopreQ.1),nicking single guide RNAs(sgRNAs),and MLH1dn,efficiently generated heritable mutations that conferred resistance to herbicides that inhibit 5-enolpyruvylshikimate-3-phosphate synthase(EPSPS),acetolactate synthase(ALS),or acetyl CoA carboxylase(ACCase)activity.Collectively,we demonstrate that the ePE5max system has sufficient efficiency to generate heritable(homozygous or heterozygous)mutations in maize target genes and that the main obstacle to using PEs in maize has thus been removed.

Ground-based hyperspectral remote sensing for weed management in crop production

Agricultural remote sensing has been developed and applied in monitoring soil,crop growth,weed infestation,insects,diseases and water status in farm fields to provide data and information to guide agricultural management practices.Precision agriculture has been implemented through prescription mapping of crop fields at different scales with the data remotely sensed from space-borne,airborne and ground-based platforms.Ground-based remote sensing techniques offer portability,flexibility and controllability in applications for precision agriculture.In weed management,crop injury from off-target herbicide spray drift and herbicide resistance in weeds are two important issues.For precision weed management,ground-based hyperspectral remote sensing techniques were developed for detection of crop injury from dicamba and differentiation between glyphosate resistant and sensitive weeds.This research presents the techniques for ground-based hyperspectral remote sensing for these two applications.Results illustrate the advantages of ground-based hyperspectral remote sensing for precision weed management.