R1 Populations Research Articles

Confirmation and characterization of the first case of acetolactate synthase (ALS)-inhibitor resistance in Japanese brome (Bromus japonicus) in the US.

Japanese brome (Bromus japonicus Thumb.) is one of the problematic annual weeds in winter wheat (Triticum aestivum L.) and is generally controlled by acetolactate synthase (ALS) inhibitors. Repeated use of the ALS inhibitor propoxycarbazone-Na resulted in the evolution of resistance to this herbicide in three B. japonicus populations, i.e., R1, R2, and R3 in Kansas (KS). However, the level of resistance and mechanism conferring resistance in these populations is unknown. The objectives of this research were to (i) evaluate the level of resistance to propoxycarbazone-Na in R1, R2, and R3 in comparison with a known susceptible population (S1), (ii) investigate the mechanism of resistance involved in conferring ALS-inhibitor resistance, and (iii) investigate the cross-resistance to other ALS inhibitors. Dose-response (0 to 16x; x = 44 g ai ha-1 of propoxycarbazone-Na) assay indicated 167, 125, and 667-fold resistance in R1, R2 and R3 populations, respectively, compared to S1 population. ALS gene sequencing confirmed the mutations resulting in amino acid substitutions, i.e., Pro-197-Thr (R3, R1)/Ser (R2, R1) bestowing resistance to these ALS inhibitors. Such amino acid substitutions also showed differential cross-resistance to sulfosulfuron, mesosulfuron-methyl, pyroxsulam, and imazamox among resistant populations. Pretreatment with malathion (a cytochrome P450 enzyme-inhibitor) followed by imazamox treatment suggested cross-resistance to this herbicide possibly via metabolism only in R3 population. Overall, these results confirm the first case of target-site based resistance to ALS inhibitors in B. japonicus in the US, highlighting the need for exploring herbicides with alternative modes of action to enhance weed control in winter wheat. © 2024 Society of Chemical Industry.

Non–target site mechanisms of cross-resistance evolution to florpyrauxifen-benzyl in 2,4-D–resistant Palmer amaranth (Amaranthus palmeri)

AbstractThe evolution of herbicide resistance in weeds can reduce the herbicide’s efficacy, depleting crop yield and quality. Our group previously confirmed 2,4-D resistance in three Palmer amaranth (Amaranthus palmeri S. Watson) populations (R1 to R3). In the current study, the first filial (F1) seeds of 2,4-D–resistant populations were subjected to screening tests for resistance to other auxin-mimicking herbicides, florpyrauxifen-benzyl (FPB; 30 g ai ha−1) and dicamba (560 g ae ha−1). Dicamba killed all resistant populations. FPB provided 100% control of only the R3 population. Sensitivities to FPB were reduced by 2 and 35 percentage points in R1 and R2 populations, respectively. Pretreatment with malathion increased FPB sensitivity by 15 percentage points in the R2 population. FPB resistance characterization and mechanism were evaluated using a purified line of the R2 population (F2). The FPB sensitivity was 29-fold lower in the F2 line than in the susceptible (S) standard. Absorption, translocation, and total metabolism of FPB were similar for S and R2 populations. However, less florpyrauxifen-acid (FPA) was detected in the R2 population (17.0% to 25.4%) than in the S population (22.8% to 33.2%), due to its rapid metabolism and/or reduced production with resistance evolution. Because the results of the non–target site resistance mechanism evaluation observed in this study were insufficient to account for the 29-fold reduced sensitivity of the R2 population to FPB, further genetic studies are needed to investigate the presence of target-site resistance in that population.

Trp-574-Leu mutation and metabolic resistance by cytochrome P450 gene conferred high resistance to ALS-inhibiting herbicides in Descurainia sophia

Descurainia sophia (flixweed) is a troublesome weed in winter wheat fields in North China. Resistant D. sophia populations with different acetolactate synthetase (ALS) mutations have been reported in recent years. In addition, metabolic resistance to ALS-inhibiting herbicides has also been identified. In this study, we collected and purified two resistant D. sophia populations (R1 and R2), which were collected from winter wheat fields where tribenuron-methyl provided no control of D. sophia at 30 g a.i. ha−1. Whole plant bioassay and ALS activity assay results showed the R1 and R2 populations had evolved high-level resistance to tribenuron-methyl and florasulam and cross-resistance to imazethapyr and pyrithiobac‑sodium. The two ALS genes were cloned from the leaves of R1 and R2 populations, ALS1 (2004 bp) and ALS2 (1998 bp). A mutation of Trp 574 to Leu in ALS1 was present in both R1 and R2. ALS1 and ALS2 were cloned from R1 and R2 populations respectively and transferred into Arabidopsis thaliana. Homozygous T3 transgenic seedlings with ALS1 of R1 or R2 were resistant to ALS-inhibiting herbicides and the resistant levels were the same. Transgenic seedlings with ALS2 from R1 or R2 were susceptible to ALS-inhibiting herbicides. Treatment with cytochrome P450 inhibitor malathion decreased the resistant levels to tribenuron-methyl in R1 and R2. RNA-Seq was used to identify target cytochrome P450 genes possibly involved in resistance to ALS-inhibiting herbicides. There were five up-regulated differentially expressed cytochrome P450 genes: CYP72A15, CYP83B1, CYP81D8, CYP72A13 and CYP71A12. Among of them, CYP72A15 had the highest expression level in R1 and R2 populations. The R1 and R2 populations of D. sophia have evolved resistance to ALS-inhibiting herbicides due to Trp 574 Leu mutation in ALS1 and possibly other mechanisms. The resistant function of CYP72A15 needs further research.

Pro197 and Trp574 substitutions in the acetolactate synthase of corn marigold (Glebionis segetum) and their impact on competitive ability against barley

AbstractThree suspected resistant (R1, R2, and R3) corn marigold populations collected from winter cereal fields located in central Greece were studied to confirm and elucidate the mechanisms of resistance to acetolactate synthase (ALS) inhibitors and their competitive ability against barley. Whole-plant dose–response assays proved that the three suspected R populations were highly cross-resistant to the ALS inhibitors tribenuron, pyroxsulam + florasulam, and imazamox, whereas their control with synthetic auxin plus ALS inhibitors co-formulated mixtures was increased in the order of tritosulfuron + dicamba < florasulam + clopyralid < tribenuron + mecoprop-P < florasulam + aminopyralid. The ALS gene sequence revealed a point mutation in 11 plants of the R1, R2, and R3 populations, which resulted in the substitution of Pro-197-Thr or Trp-574-Leu. By contrast, all three sequenced plants of the susceptible (S) population were found with the wild-type allele encoding Pro-197 and Trp-574. This is the first report of ALS-inhibitor resistance in corn marigold. The competition study between barley and four densities of the S, R2, or R3 populations indicated similar biomass rates for all three populations, suggesting lack of association between the competitive ability of the R populations and the target-site resistance mechanism, which was also confirmed by the similar biomass reduction rates of barley grown in competition with S or R populations.

Reduced Glyphosate Movement and Mutation of the EPSPS Gene (Pro106Ser) Endow Resistance in Conyza canadensis Harvested in Mexico

Glyphosate has been the most widely used herbicide for decades providing a unique tool, alone or in mixtures, to control weeds on citrus in Veracruz. Conyza canadensis has developed glyphosate resistance for the first time in Mexico. The level and mechanisms of resistance of four resistant populations Rs (R1, R2, R3, and R4) were studied and compared with that of a susceptible population (S). Resistance factor levels showed two moderately resistant populations (R2 and R3) and two highly resistant populations (R1 and R4). Glyphosate translocation through leaves to roots was ∼2.8 times higher in the S population than in the four R populations. A mutation (Pro106Ser) in the EPSPS2 gene was identified in the R1 and R4 populations. Mutation in the target site associated with reduced translocation is involved in increased glyphosate resistance in the R1 and R4 populations; whereas for the R2 and R3 populations, it was only mediated by reduced translocation. This is the first study of glyphosate resistance in C. canadensis from Mexico in which the resistance mechanisms involved are described in detail and control alternatives are proposed.

Constitutive overexpression of EPSPS by gene duplication is involved in glyphosate resistance in Salsola tragus.

Glyphosate-resistant Salsola tragus accessions have been identified in the USA and Argentina; however, the mechanisms of glyphosate resistance have not been elucidated. The goal of this study was to determine the mechanism/s of glyphosate resistance involved in two S.tragus populations (R1 and R2) from Argentina. Both glyphosate-resistant populations had a six-fold lower sensitivity to glyphosate than the S population (i.e. resistance index). No evidence of differential absorption, translocation or metabolism of glyphosate was found in the R1 and R2 populations compared to a susceptible population (S). No 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) mutations were detected, but S.tragus R1 and R2 plants had ≈14-fold higher EPSPS gene relative copy number compared to the S counterpart. In R1 and R2, EPSPS duplication entailed a greater constitutive EPSPS transcript abundance by approximately seven-fold and a basal EPSPS activity approximately three-fold higher than the S population. The current study reports EPSPS gene duplication for the first time as a mechanism of glyphosate resistance in S.tragus populations. The increase of glyphosate dose needed to kill R1 and R2 plants was linked to the EPSPS transcript abundance and level of EPSPS activity. This evidence supports the convergent evolution of the overexpression of the EPSPS gene in several Chenopodiaceae/Amaranthaceae species adapted to drought environments and the role of gene duplication as an adaptive advantage for plants to withstand stress. © 2022 Society of Chemical Industry.

An Asp376Glu substitution in ALS gene and enhanced metabolism confers high tribenuron-methyl resistance in Sinapis alba.

Acetolactate synthase (ALS) inhibiting herbicides (group 2) have been widely applied for the last 20 years to control Sinapis alba in cereal crops from southern Spain. In 2008, a tribenuron-methyl (TM) resistant (R) S. alba population was first reported in a cereal field in Malaga (southern Spain). In 2018, three suspected R S. alba populations (R1, R2 and R3) to TM were collected from three different fields in Granada (southern Spain, 100 km away from Malaga). The present work aims to confirm the putative resistance of these populations to TM and explore their resistance mechanisms. Dose-response assays showed that the R1, R2 and R3 populations ranging between 57.4, 44.4 and 57.1 times more resistance to TM than the susceptible population (S). A mutation in the ALS gene (Asp376Glu) was detected in the Rs S. alba populations. 14C-metabolism studies show that metabolites and TM were changing significantly faster in the R than in the S plants. Alternative chemical control trials showed that 2,4-D and MCPA (auxin mimics), glyphosate (enolpyruvyl shikimate phosphate synthase,EPSPS, inhibitor-group 9), metribuzin (PSII inhibitors/Serine 264 Binders, -group 5) and mesotrione (hydroxyphenyl pyruvate dioxygenase, HPPD, inhibitor-group 27) presented a high control of the four populations of S. alba tested, both S and R. Based on these results, it is the first case described where the Asp376Glu mutation and P450-mediated metabolism participates in resistance to TM in S. alba. Comparing these results with those found in the S. alba population in Malaga in 2008, where the resistance was TSR type (Pro197Ser), we can suggest that despite the geographical proximity (over 100 km), the resistance in these cases was due to different evolutionary events.

Investigation of resistance mechanisms to bentazone in multiple resistant Amaranthus retroflexus populations

Redroot amaranth (Amaranthus retroflexus L.) is a noxious weed that affects soybean production in China. Experiments were conducted to determine the molecular basis of resistance to bentazone. Whole-plant dose-response experiments showed that two populations (R1 and R2) exhibited resistance to bentazone with resistance indices of 9.01 and 6.85, respectively. Sequencing of the psbA gene revealed no amino acid substitution in the two populations. qRT–PCR analysis verified that psbA gene expression in R1 and R2 populations was increased significantly after treatment with bentazone, which was 3-fold and 5-fold higher than that in S1 and S2 populations, respectively. The P450 inhibitor malathion significantly reduced the level of resistance in the R1 and R2 populations when used prior to bentazone treatment. The R1 population exhibited multiple resistance to thifensulfuron-methyl and lactofen, caused by target site mutations (Asp-376-Glu in ALS, Arg-128-Gly in PPO2). In conclusion, increased gene expression of the psbA gene and enhanced herbicide metabolism seem to be the basis of resistance to bentazone in these A. retroflexus populations.

Characterization of glyphosate and quizalofop-p-ethyl multiple resistance in Eleusine indica

Glyphosate and Acetyl-coenzyme A Carboxylase (ACCase) inhibitors are popular herbicides that control goosegrass. However, some populations are difficult to control due to resistance resulting from the increasing selection pressure. The objectives of this research were to detect the multiple resistance levels, resistance mechanisms, and fitness costs of two goosegrass populations collected in China. The resistance indices of two resistant populations (denominated as R1 and R2) to glyphosate were 3.8 and 2.3, respectively; and it was 18.0 and 14.2 to quizalofop-p-ethyl, respectively. Shikimate accumulation in R1 and R2 populations was only 8% of that of the susceptible population after glyphosate treatment. A Pro-106-Ala mutation in EPSPS and an Asp-2078-Gly mutation in ACCase were present in both resistant populations. Both the expression level of EPSPS and ACCase in resistant populations were similar to that of susceptible populations. The leaf area of the individuals in wild-type populations was more than three times of the leaf area in the resistant populations. Similarly, resistant plants were 45–49% shorter, had 70–76% less fresh shoot weight, and 67–69% fewer seeds than wild-type plants. Goosegrass populations have evolved multiple resistance to glyphosate and the ACCase inhibitor quizalofop-p-ethyl in China. The Pro-106-Ala mutation in the EPSPS and the Asp-2078-Gly mutation in the ACCase were responsible for this resistance. In addition, a fitness cost exists in the resistant populations, and more work should conduct to clear which mutation is responsible for the fitness penalty.

Molecular basis of cross-resistance to acetohydroxy acid synthase-inhibiting herbicides in Sagittaria trifolia L.

Acetohydroxy acid synthase (AHAS)-inhibiting herbicides are one of the most commonly used herbicides for controlling the growth of Sagittaria trifolia L. in paddy fields in Northeastern China. In this study, we collected five suspected resistant populations of S. trifolia (R1-R5) from three different provinces of Northeastern China. The results of whole-plant bioassays revealed that those populations showed high level of resistance to bensulfuron-methyl with resistance index (GR50 R/S) ranging from 39.90 to 88.50. The results of AHAS-activity assays were consistent with the results of the whole-plant bioassays. The AHAS gene analysis showed that R2 and R3 populations contained Pro-197-Leu mutations that were highly resistant to penoxsulam; R1 and R4 populations contained Pro-197-Ser mutations that were highly resistant to bispyribac‑sodium; R5 population contained Trp-574-Leu mutation that showed high resistance to IMI, PT, PTB and SU herbicides. The AHAS with resistance mutations showed less sensitivity to feedback inhibition by BCAAs and R genotypes had increased free BCAAs

Glyphosate Resistance in Amaranthus viridis in Brazilian Citrus Orchards

Glyphosate is the main tool for weed management in Brazilian citrus orchards, where weeds, such as Conyza bonariensis and Digitaria insularis, have been found with resistance to this herbicide. Field prospections have allowed the identification of a possible new case of glyphosate resistance. In this work, the susceptibility levels to glyphosate on three Amaranthus viridis L. populations, with suspected resistance (R1, R2, and R-IAC), collected in citrus orchards from the São Paulo State, Brazil, as well as their accumulation rates of shikimic acid, were determined. The fresh weight of the susceptible population (S) was reduced by 50% (GR50) with ~30 g ea ha−1 glyphosate, while the GR50 values of the R populations were between 5.4 and 11.3 times higher than that for S population. The LD50 (herbicide dose to kill 50% of individuals of a weed population) values of the S population were ≤150 g ea ha−1 glyphosate, while the LD50 of the R populations ranged from 600 to 920 g ea ha−1. Based on the reduction of fresh weight and the survival rate, the R1 population showed the highest level of glyphosate resistance, which had GR50 and LD50 values of 248 and 918 g ea ha−1 glyphosate, respectively. The S population accumulated 240 µg shikimic acid at 1000 µM glyphosate, while the R1, R2, and R-IAC populations accumulated only 16, 43, and 33 µg shikimic acid, respectively (between 5.6 to 15 times less than the S population). Enzyme activity assays suggested that at least one target site-type mechanism was involved in resistance. This result revealed the first report of glyphosate resistance in A. viridis reported in the world.

Potato virus Y; the Andean connection.

Potato virus Y (PVY) causes disease in potatoes and other solanaceous crops. The appearance of its necrogenic strains in the 1980s made it the most economically important virus of potatoes. We report the isolation and genomic sequences of 32 Peruvian isolates of PVY which, together with 428 published PVY genomic sequences, gave an alignment of 460 sequences. Of these 190 (41%) were non-recombinant, and 162 of these provided a dated phylogeny, that corresponds well with the likely history of PVY, and show that PVY originated in South America which is where potatoes were first domesticated. The most basal divergences of the PVY population produced the N and C: O phylogroups; the origin of the N phylogroup is clearly Andean, but that of the O and C phylogroups is unknown, although they may have been first to establish in European crops. The current PVY population originated around 156 CE. PVY was probably first taken from South America to Europe in the 16th century in tubers. Most of the present PVY diversity emerged in the second half of the 19th century, after the Phytophthora infestans epidemics of the mid-19th century destroyed the European crop and stimulated potato breeding. Imported breeding lines were shared, and there was no quarantine. The early O population was joined later by N phylogroup isolates and their recombinants generated the R1 and R2 populations of damaging necrogenic strains. Our dating study has confirmed that human activity has dominated the phylodynamics of PVY for the last two millennia.

Nicosulfuron-resistant Amaranthus retroflexus L. in Northeast China

Amaranthus retroflexus L. is a troublesome broadleaf weed in autumn crop fields in China. Farmers complain that A. retroflexus cannot be controlled by nicosulfuron at the recommended field rate in maize fields in Heilongjiang Province. The aim of this study was to determine the molecular basis of nicosulfuron resistance in A. retroflexus. Whole-plant response assays revealed that two resistant populations (R1 and R2) exhibited resistance (14.50- and 44.24-fold) to nicosulfuron. In vitro acetolactate synthase (ALS) activity assays indicated that the nicosulfuron I50 values for R1 and R2 populations were 8.12 and 22.32 times higher than that for the susceptible population (S), respectively. Sequence analysis of ALS showed amino acid mutations Ser-653-Asn in R1 and Trp-574-Leu in R2. These two target-site mutations seem to play a primary role in A. retroflexus resistance to nicosulfuron, and this is the first time that the Ser-653-Asn mutation has been reported in this species.

Non-target-site and target-site resistance to AHAS inhibitors in American sloughgrass (Beckmannia syzigachne)

American sloughgrass (Beckmannia syzigachne (Steud.) Fernald) is one of the most competitive and malignant weeds in rice-wheat rotation fields in China. American sloughgrass populations in the Jiangsu Province of China became less sensitive to acetohydroxyacid synthase (AHAS) inhibitors after repeated application for many years in these areas. Two suspected resistant American sloughgrass populations (R1 and R2) collected in the field were detected the resistance to inhibitors of AHAS in whole-plant dose-response assays, compared to the susceptible (S) population. These assays indicated that R1 showed low resistance to mesosulfuron-methyl (3.32-fold), imazapic (2.84-fold) and pyroxsulam (1.55-fold), moderate resistance to flazasulfuron (4.67-fold) and pyribenzoxim (7.41-fold), and high resistance to flucarbazone (11.73-fold). However, using a combination of the cytochrome P450 inhibitor, malathion, with mesosulfuron-methyl resulted in a reduction in R1 resistance relative to mesosulfuron-methyl alone. Furthermore, R2 was highly resistant to flazasulfuron (34.90-fold), imazapic (11.30-fold), flucarbazone (49.20-fold), pyribenzoxim (12.94-fold), moderately resistant to mesosulfuron-methyl (9.77-fold) and pyroxsulam (6.26-fold), and malathion had no effect on R2 resistance to mesosulfuron-methyl. The full-length of AHAS genes was sequenced and the AHAS enzymes were assayed in vitro in order to clarify the mechanism of resistance to AHAS inhibitors in R1 and R2 populations. The results demonstrated that R2 had a Pro-197-Ser mutation in the AHAS gene, and the sensitivity of R2 to the five AHAS inhibitors was decreased, which may result in R2 resistance to AHAS inhibitors. There was no mutation in the AHAS gene of R1, and there were no significant differences in enzyme sensitivity between susceptible (S) and resistant (R1) populations. An enhanced metabolism may be the main mechanism of R1 resistance to AHAS inhibitors.

From tolerance to resistance: mechanisms governing the differential response to glyphosate in Chloris barbata.

Susceptibility and the mechanism (s) governing tolerance/resistance to glyphosate were characterized in two putative-glyphosate-resistant Chloris barbata populations (R1 and R2), collected in Persian lime orchards from Colima State, Mexico, comparing them with one non-treated population (referred to as S). Glyphosate doses required to reduce fresh weight or cause mortality by 50% were 4.2-6.4 times higher in resistant populations than in the S population. The S population accumulated 4.3 and 5.2 times more shikimate than the R2 and R1 populations, respectively. There were no differences in 14 C-glyphosate uptake between R and S populations, but the R plants translocated at least 12% less herbicide to the rest of plant and roots 96 h after treatment. Insignificant amounts of glyphosate were metabolized to aminomethyl phosphonate and glyoxylate in both R and S plants. The 5-enolpyruvylshikimate-3-phosphate synthase gene of the R populations contained the Pro106-Ser mutation, giving them a resistance 12 (R2) and 14.7 (R1) times greater at target-site level compared with the S population. The Pro106-Ser mutation governs the resistance to glyphosate of the R1 and R2 C barbata populations, but the impaired translocation could contribute to the resistance. These results confirm the first case of glyphosate resistance evolved in this species. © 2018 Society of Chemical Industry.

Target and Non-target Site Mechanisms Developed by Glyphosate-Resistant Hairy beggarticks (Bidens pilosa L.) Populations from Mexico.

In 2014 hairy beggarticks (Bidens pilosa L.) has been identified as being glyphosate-resistant in citrus orchards from Mexico. The target and non-target site mechanisms involved in the response to glyphosate of two resistant populations (R1 and R2) and one susceptible (S) were studied. Experiments of dose-response, shikimic acid accumulation, uptake-translocation, enzyme activity and 5-enolpyruvyl shikimate-3-phosphate synthase (EPSPS) gene sequencing were carried out in each population. The R1 and R2 populations were 20.4 and 2.8-fold less glyphosate sensitive, respectively, than the S population. The resistant populations showed a lesser shikimic acid accumulation than the S population. In the latter one, 24.9% of 14C-glyphosate was translocated to the roots at 96 h after treatment; in the R1 and R2 populations only 12.9 and 15.5%, respectively, was translocated. Qualitative results confirmed the reduced 14C-glyphosate translocation in the resistant populations. The EPSPS enzyme activity of the S population was 128.4 and 8.5-fold higher than the R1 and R2 populations of glyphosate-treated plants, respectively. A single (Pro-106-Ser), and a double (Thr-102-Ile followed by Pro-106-Ser) mutations were identified in the EPSPS2 gene conferred high resistance in R1 population. Target-site mutations associated with a reduced translocation were responsible for the higher glyphosate resistance in the R1 population. The low-intermediate resistance of the R2 population was mediated by reduced translocation. This is the first glyphosate resistance case confirmed in hairy beggarticks in the world.

Cistrome Control of Hematopoieitic Stem/Progenitor Cell Function

Cis-regulatory mechanisms control chromatin structure and cellular identity. At the GATA2 locus, two cis-elements are linked to human pathologies, including a primary immunodeficiency (MonoMAC syndrome) associated with multiple complex phenotypes, myelodysplastic syndrome, and acute myeloid leukemia (AML). Mutations that disrupt the function of an intronic GATA2 +9.5 element cause MonoMAC syndrome, while an inversion that relocates the distal GATA2 -77 element to the EVI1 locus induces AML. The +9.5 and -77 cis-elements are GATA-2-occupied and confer context-dependent enhancer activities in select hematopoietic cell types in vivo. In knockout mouse models, the Gata2 +9.5 cis-element is required for hematopoietic stem cell (HSC) genesis, whereas the Gata2 -77 cis-element governs a unique sector of the myeloid progenitor cell transcriptome without impacting HSC genesis. Three other GATA-2-occupied cis-elements (-1.8, -2.8 and -3.9) were not individually required for hematopoietic development, and had relatively mild effects on Gata2 expression; the -1.8 site was required to maintain Gata2 repression in late-stage erythroblasts, the -2.8 conferred maximal Gata2 expression, and the -3.9 had no effect on Gata2 expression. We predict that additional cis-elements exist in the genome with functions resembling the +9.5 and -77, and their analysis will provide important mechanistic and biological insights. We utilized the known properties of Gata2 cis-elements as learning tools to identify prospective constituents of a hematopoietic stem/progenitor cell (HSPC) regulatory cistrome genome-wide. Using sequence attributes shared with the critically-important +9.5 element, namely a CATCTG-8bp spacer-AGATAA, we generated a list of 797 candidate cis-elements ("+9.5-like" elements). This list was prioritized using chromatin occupancy by GATA-2 and Scl/TAL-1, among others, chromatin accessibility, evolutionary conservation, and histone modifications in a multitude of biologically-relevant cell types. Gene editing was used to delete three high-ranked elements (Samd14 +2.5, Bcl2l1 +12.2, and Dapp1 +23.5), revealing their importance for transcriptional activation, GATA-2 occupancy and chromatin accessibility, while deletion of two low-ranked elements (Mrps9 +17.6 and Mgmt +182) had no effect on gene transcription. One such cis-element (Samd14 +2.5) resided in Samd14, a gene with undescribed biological function. Samd14 has a conserved sterile α-motif and coiled-coil domain, and is highly expressed in hematopoietic progenitors and differentiated progeny. Mouse knockout of the Samd14 +2.5 element dramatically lowered expression of Samd14 in hematopoietic progenitors. We conducted loss-of-function analysis to elucidate Samd14 function in lineage-depleted (Lin-) E14.5 fetal liver cells infected with control or Samd14 shRNA-expressing retrovirus. In a CFU assay, Samd14 knockdown reduced BFU-E and CFU-GM colonies 3.4-fold. Early erythroid precursor R1 (CD71low, Ter119-) and R2 (CD71high, Ter119-) cell populations decreased ~2-fold, concomitant with increases in more mature R3 and R4/5 populations (Ter119+). In R1/R2 cells, Samd14 knockdown reduced surface c-Kit expression by 1.6-fold and prevented Stem Cell Factor/c-Kit activation of AKT. Cellular deficits resulting from Samd14 knockdown could be rescued by c-Kit. In -77-/- common myeloid progenitors, Samd14 was ~20-fold downregulated. Thus, the importance of Samd14 and the Samd14 +2.5 element on progenitor function and SCF/c-Kit signaling validates our strategy for identifying cis-elements relevant for hematopoiesis. Our findings demonstrate that +9.5-like elements control cell signaling (Samd14 +2.5) and apoptosis (Bcl2l1 +12.2), and we predict that additional cistrome constituents will control these and other important HSPC processes. I will discuss the mechanistic and biological properties of additional cis-elements analyzed from a cohort of 68 GATA-2-occupied elements and general principles arising from the HSPC cistrome analysis, which provide unique insights into the control of hematopoiesis and GATA-2-linked pathologies. DisclosuresNo relevant conflicts of interest to declare.

Target-site resistance to bensulfuron-methyl in Sagittaria trifolia L. populations

Sagittaria trifolia L. is one of the most serious weeds in paddy fields in northeast of China and cannot be controlled effectively by bensulfuron-methyl in recent years. In this study, two suspected resistant S. trifolia populations (R1 and R2) were collected in Liaoning province of China. Whole-plant dose–response studies showed that R1 and R2 were highly resistant to bensulfuron-methyl, with the GR50 R/S ratios of 76.99 and 49.94 respectively. In vitro acetolactate synthase (ALS) assays revealed that resistance was due to reduced sensitivity of the ALS to bensulfuron-methyl inhibition, with I50 R/S ratios of 81.86 and 67.48 for R1 and R2, respectively. Total ALS activity was similar for the S and R2 populations, whereas the R1 population displayed significantly higher ALS activity than did the S population. The mutations Pro-197-Leu and Pro-197-Ser were identified in the ALS gene of the R1 and R2 populations, respectively. This is the first report examining bensulfuron-resistant S. trifolia in Liaoning province, China. The Pro197 mutation is likely responsible for resistance to bensulfuron-methyl in S. trifolia populations.

Identification of the first glyphosate-resistant wild radish (Raphanus raphanistrum L.) populations.

In Australia, glyphosate has been used routinely to control wild radish (Raphanus raphanistrum L.) for the past 40 years. This study focuses on two field-evolved glyphosate-resistant populations of wild radish collected from the grainbelt of Western Australia. Two wild radish biotypes were confirmed to be glyphosate resistant by comparing R/S of two suspected populations. Based on R/S from dose-response curves, the R1 and R2 populations were 2.3 and 3.2 times more resistant to glyphosate respectively. Dose response on glyphosate-selected progeny (>1080 g ha(-1)) demonstrated that the glyphosate resistance mechanism was heritable. When compared with the pooled mortality results of three known susceptible populations (S1, S2 and S3), the R1 and R2 subpopulations were 3.4-fold and 4.5-fold more resistant at the LD50 level respectively. Both populations were found to have multiple resistance to the phytoene desaturase inhibitor; diflufenican, the synthetic auxin; 2,4-D and the ALS inhibitors; chlorsulfuron, sulfometuron-methyl, imazethapyr and metosulam. This is the first report confirming glyphosate resistance evolution in wild radish and serves to re-emphasise the importance of diverse weed control strategies. Proactive and integrated measures for resistance management need to be developed to diversify control measures away from glyphosate and advance the use of non-herbicidal techniques.

Characterization and genetic mapping of a Photoperiod-sensitive dwarf 1 locus in rice (Oryza sativa L.)

Plant height is an important agronomic trait for crop architecture and yield. Most known factors determining plant height function in gibberellin or brassinosteroid biosynthesis or signal transduction. Here, we report a japonica rice (Oryza sativa ssp. japonica) dominant dwarf mutant, Photoperiod-sensitive dwarf 1 (Psd1). The Psd1 mutant showed impaired cell division and elongation, and a severe dwarf phenotype under long-day conditions, but nearly normal growth in short-day. The plant height of Psd1 mutant could not be rescued by gibberellin or brassinosteroid treatment. Genetic analysis with R1 and F2 populations determined that Psd1 phenotype was controlled by a single dominant locus. Linkage analysis with 101 tall F2 plants grown in a long-day season, which were derived from a cross between Psd1 and an indica cultivar, located Psd1 locus on chromosome 1. Further fine-mapping with 1017 tall F2 plants determined this locus on an 11.5-kb region. Sequencing analysis of this region detected a mutation site in a gene encoding a putative lipid transfer protein; the mutation produces a truncated C-terminus of the protein. This study establishes the genetic foundation for understanding the molecular mechanisms regulating plant cell division and elongation mediated by interaction between genetic and environmental factors.