Establishment of an Efficient Agrobacterium-Mediated Genetic Transformation System to Enhance the Tolerance of the Paraquat Stress in Engineering Goosegrass (Eleusine Indica L.).

Eleusine indica (goosegrass) is a problematic weed worldwide known for its multi-herbicide tolerance/resistance biotype. However, a genetic transformation method in goosegrass has not been successfully established, making a bottleneck for functional genomics studies in this species. Here, we report a successful Agrobacterium-mediated transformation method for goosegrass. Firstly, we optimized conditions for breaking seed dormancy and increasing seed germination rate. A higher callus induction rate from germinated seeds was obtained in N6 than in MS or B5 medium. Then the optimal transformation efficiency of the gus reporter gene was obtained by infection with Agrobacterium tumefaciens culture of OD600 = 0.5 for 30 min, followed by 3 days of co-cultivation with 300 µmol/L acetosyringone. Concentrations of 20 mg L-1 kanamycin and 100 mg L-1 timentin were used to select the transformed calli. The optimal rate of regeneration of the calli was generated by using 0.50 mg L-1 6-BA and 0.50 mg L-1 KT in the culture medium. Then, using this transformation method, we overexpressed the paraquat-resistant EiKCS gene into a paraquat-susceptible goosegrass biotype MZ04 and confirmed the stable inheritance of paraquat-resistance in the transgenic goosegrass lines. This approach may provide a potential mechanism for the evolution of paraquat-resistant goosegrass and a promising gene for the manipulation of paraquat-resistance plants. This study is novel and valuable in future research using similar methods for herbicide resistance.

QTL mapping and identification of candidate genes linked to red rot resistance in sugarcane.

Sugarcane (Saccharum species hybrid) is one of the most important commercial crops cultivated worldwide for products like white sugar, bagasse, ethanol, etc. Red rot is a major sugarcane disease caused by a hemi-biotrophic fungus, Colletotrichum falcatum Went., which can potentially cause a reduction in yield up to 100%. Breeding for red rot-resistant sugarcane varieties has become cumbersome due to its complex genome and frequent generation of new pathotypes of red rot fungus. In the present study, a genetic linkage map was developed using a selfed population of a popular sugarcane variety CoS 96268. A QTL linked to red rot resistance (qREDROT) was identified, which explained 26% of the total phenotypic variation for the trait. A genotype-phenotype network analysis performed to account for epistatic interactions, identified the key markers involved in red rot resistance. The differential expression of the genes located in the genomic region between the two flanking markers of the qREDROT as well as in the vicinity of the markers identified through the genotype-phenotype network analysis in a set of contrasting genotypes for red rot infection further confirmed the mapping results. Further, the expression analysis revealed that the plant defense-related gene coding 26S protease regulatory subunit is strongly associated with the red rot resistance. The findings can help in the screening of disease resistant genotypes for developing red rot-resistant varieties of sugarcane. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03481-7.

Identification of small effect quantitative trait loci of plant architectural, flowering, and early maturity traits in reciprocal interspecific introgression population in cotton.

Plant architecture, flowering time and maturity traits are important determinants of yield and fiber quality of cotton. Genetic dissection of loci determining these yield and quality components is complicated by numerous loci with alleles conferring small differences. Therefore, mapping populations segregating for smaller numbers and sizes of introgressed segments is expected to facilitate dissection of these complex quantitative traits. At an advanced stage in the development of reciprocal advanced backcross populations from crosses between elite Gossypium hirsutum cultivar 'Acala Maxxa' (GH) and G. barbadense 'Pima S6' (GB), we undertook mapping of plant architectural traits, flowering time and maturity. A total of 284 BC4F1 and BC4F2 progeny rows, 120 in GH and 164 in GB background, were evaluated for phenotype, with only 4 and 3 (of 7) traits showing significant differences among progenies. Genotyping by sequencing yielded 3,186 and 3,026 SNPs, respectively, that revealed a total of 27 QTLs in GH background and 22 in GB, for plant height, days to flowering, residual flowering at maturity and maturity. More than of 90% QTLs identified in both backgrounds had small effects (%PV < 10), supporting the merit of this population structure to reduce background noise and small effect QTLs. Germplasm developed in this study may serve as potential pre-breeding material to develop improved cotton cultivars.

Improved Upland Cotton Germplasm for Multiple Fiber Traits Mediated by Transferring and Pyramiding Novel Alleles From Ethyl Methanesulfonate-Generated Mutant Lines Into Elite Genotypes.

Ethyl methanesulfonate (EMS) mutagenesis offers important advantages for improving crops, such as cotton, with limited diversity in elite gene pools. EMS-induced point mutations are less frequently associated with deleterious traits than alleles from wild or exotic germplasm. From 157 mutant lines that have significantly improved fiber properties, we focused on nine mutant lines here. A total of eight populations were developed by crossing mutant lines in different combinations into GA230 (GA2004230) background. Multiple lines in each population were significantly improved for the fiber trait that distinguished the donor parent(s), demonstrating that an elite breeding line (GA230) could be improved for fiber qualities using the mutant lines. Genotypes improved for multiple fiber traits of interest suggesting that allele pyramiding is possible. Compared to midparent values, individual progeny in the population conferred fiber quality improvements of as much as 31.7% (in population O) for micronaire (MIC), 16.1% (in population P) for length, 22.4% (in population K) for strength, 4.1% (in population Q) for uniformity, 45.8% (in population N) for elongation, and 13.9% (in population O) for lint percentage (lint%). While further testing for stability of the phenotype and estimation of yield potential is necessary, mutation breeding shows promise as an approach to reduce the problem of the genetic bottleneck of upland cotton. The populations developed here may also contribute to identifying candidate genes and causal mutations for fiber quality improvement.

Insights into the Genomic Architecture of Seed and Pod Quality Traits in the U.S. Peanut Mini-Core Diversity Panel.

Traits such as seed weight, shelling percent, percent sound mature kernels, and seed dormancy determines the quality of peanut seed. Few QTL (quantitative trait loci) studies using biparental mapping populations have identified QTL for seed dormancy and seed grade traits. Here, we report a genome-wide association study (GWAS) to detect marker-trait associations for seed germination, dormancy, and seed grading traits in peanut. A total of 120 accessions from the U.S. peanut mini-core collection were evaluated for seed quality traits and genotyped using Axiom SNP (single nucleotide polymorphism) array for peanut. We observed significant variation in seed quality traits in different accessions and different botanical varieties. Through GWAS, we were able to identify multiple regions associated with sound mature kernels, seed weight, shelling percent, seed germination, and dormancy. Some of the genomic regions that were SNP associated with these traits aligned with previously known QTLs. For instance, QTL for seed dormancy has been reported on chromosome A05, and we also found SNP on the same chromosome associated with seed dormancy, explaining around 20% of phenotypic variation. In addition, we found novel genomic regions associated with seed grading, seed germination, and dormancy traits. SNP markers associated with seed quality and dormancy identified here can accelerate the selection process. Further, exploring the function of candidate genes identified in the vicinity of the associated marker will assist in understanding the complex genetic network that governs seed quality.

Detection of subgenome bias using an anchored syntenic approach in Eleusine coracana (finger millet).

BACKGROUND: Finger millet (Eleusine coracana 2n = 4x = 36) is a hardy, nutraceutical, climate change tolerant, orphan crop that is consumed throughout eastern Africa and India. Its genome has been sequenced multiple times, but A and B subgenomes could not be separated because no published genome for E. indica existed. The classification of A and B subgenomes is important for understanding the evolution of this crop and provide a means to improve current and future breeding programs. RESULTS: We produced subgenome calls for 704 syntenic blocks and inferred A or B subgenomic identity for 59,377 genes 81% of the annotated genes. Phylogenetic analysis of a super matrix containing 455 genes shows high support for A and B divergence within the Eleusine genus. Synonymous substitution rates between A and B genes support A and B calls. The repetitive content on highly supported B contigs is higher than that on similar A contigs. Analysis of syntenic singletons showed evidence of biased fractionation showed a pattern of A genome dominance, with 61% A, 37% B and 1% unassigned, and was further supported by the pattern of loss observed among cyto-nuclear interacting genes. CONCLUSION: The evidence of individual gene calls within each syntenic block, provides a powerful tool for inference for subgenome classification. Our results show the utility of a draft genome in resolving A and B subgenomes calls, primarily it allows for the proper polarization of A and B syntenic blocks. There have been multiple calls for the use of phylogenetic inference in subgenome classification, our use of synteny is a practical application in a system that has only one parental genome available.

The Ligon lintless -2 Short Fiber Mutation Is Located within a Terminal Deletion of Chromosome 18 in Cotton.

Extreme elongation distinguishes about one-fourth of cotton (Gossypium sp.) seed epidermal cells as "lint" fibers, useful for the textile industry, from "fuzz" fibers (<5 mm). Ligon lintless-2 (Li 2 ), a dominant mutation that results in no lint fiber but normal fuzz fiber, offers insight into pathways and mechanisms that differentiate spinnable cotton from its progenitors. A genetic map developed using 1,545 F2 plants showed that marker CISP15 was 0.4 cM from Li 2 , and "dominant" simple sequence repeat (SSR) markers (i.e. with null alleles in the Li 2 genotype) SSR7 and SSR18 showed complete linkage with Li 2 Nonrandom distribution of markers with null alleles suggests that the Li 2 phenotype results from a 176- to 221-kb deletion of the terminal region of chromosome 18 that may have been masked in prior pooled-sample mapping strategies. The deletion includes 10 genes with putative roles in fiber development. Two Glycosyltransferase Family 1 genes showed striking expression differences during elongation of wild-type versus Li 2 fiber, and virus-induced silencing of these genes in the wild type induced Li 2 -like phenotypes. Further, at least 7 of the 10 putative fiber development genes in the deletion region showed higher expression in the wild type than in Li 2 mutants during fiber development stages, suggesting coordinated regulation of processes in cell wall development and cell elongation, consistent with the hypothesis that some fiber-related quantitative trait loci comprise closely spaced groups of functionally diverse but coordinately regulated genes.

A novel mutation A212T in chloroplast Protoporphyrinogen oxidase (PPO1) confers resistance to PPO inhibitor Oxadiazon in Eleusine indica.

BACKGROUND: Protoporphyrinogen oxidase (PPO) with two isoforms, chloroplast-targeted (PPO1) and mitochondrial-targeted (PPO2), catalyzes a step in the biosynthesis of chlorophyll and heme. PPO1 and PPO2 are herbicide target sites of PPO-inhibiting herbicides. Target-site mutations conferring resistance to PPO inhibitors have all thus far been in PPO2. Oxadiazon is a unique PPO inhibitor utilized for preemergence Eleusine indica control. In this research, we evaluated the response of two previously confirmed oxadiazon-resistant and susceptible E. indica biotypes to other PPO inhibitors and identified the resistance mechanism in two oxadiazon-resistant E. indica biotypes. RESULTS: Two E. indica biotypes were resistant to oxadiazon, but not to other structurally unrelated PPO inhibitors, such as lactofen, flumioxazin and sulfentrazone. A novel mutation A212T was identified in the chloroplast-targeted PPO1, conferring resistance to oxadiazon in a heterologous expression system. Computational structural modeling provided a mechanistic explanation for reduced herbicide binding to the variant protein: the presence of a methyl group of threonine 212 changes the PPO1 active site and produces repulsive electrostatic interactions that repel oxadiazon from the binding pocket. CONCLUSION: The novel A212T mutation in PPO1 conferring resistance specifically to PPO inhibitor oxadiazon was characterized. This is the first evidence of the direct role of PPO1 in the PPO mode of action, and the first evidence of evolved resistance in PPO1. © 2019 Society of Chemical Industry.

Genetic Analysis of Gossypium Fiber Quality Traits in Reciprocal Advanced Backcross Populations.

In mapping populations segregating for many loci, the large amount of variation among genotypes often masks small-effect quantitative trait loci (QTL). This problem can be reduced by development of populations with fewer chromosome segments segregating. Here, we report early QTL detection in reciprocal advanced backcross populations from crosses between elite Gossypium hirsutum L. 'Acala Maxxa' (GH) and G. barbadense L. 'Pima S6' (GB). A total of 297 BCF and BCF progeny rows-127 segregating for GB chromosome segments in GH background and 170 segregating for GH chromosome segments in GB background-were evaluated in three environments. Totals of 3186 and 3026 polymorphic single-nucleotide polymorphisms (SNPs) in GH and GB backgrounds, respectively, were identified and used for trait mapping. Small-effect QTL (<10% variance explained) made up 87 and 100% of QTL in GH and GB backgrounds, respectively. In both species, favorable alleles were found with effects being masked or neutralized by unfavorable alleles, with greater scope for improvement of GH than GB by introgressive breeding. A total of three stable QTL-two in GH background for fiber elongation (ELO) and micronaire (MIC) and one in GB background for upper-half mean length (UHM)-were identified in two out of three environments. Curiously, only four QTL-three for UHM and one for ELO-showed the expected opposite effects in reciprocal backgrounds, perhaps reflecting the combined consequences of epistasis, small phenotypic effects, and low coverage of some genomic regions. Along with new information for marker-assisted breeding, this study adds to knowledge that can be used to unravel complex genetic networks governing fiber quality traits.

Identification of putative candidate genes for red rot resistance in sugarcane (Saccharum species hybrid) using LD-based association mapping.

Red rot is a serious disease of sugarcane caused by the fungus Colletotrichum falcatum that has a colossal damage potential. The fungus, prevalent mainly in the Indian sub-continent, keeps on producing new pathogenic strains leading to breakdown of resistance in newly released varieties and hence the deployment of linked markers for marker-assisted selection for resistance to this disease can fine tune the breeding programme. This study based on a panel of 119 sugarcane genotypes fingerprinted for 944 SSR alleles was undertaken with an aim to identify marker-trait associations (MTAs) for resistance to red rot. Mixed linear model containing population structure and kinship as co-factor detected four MTAs that were able to explain 10-16 % of the trait variation, individually. Among the four MTAs, EST sequences diagnostic of three could be BLAST searched to the sorghum genome with significant sequence homology. Several genes encoding important plant defence related proteins, viz., cytochrome P450, Glycerol-3-phosphate transporter-1, MAP Kinase-4, Serine/threonine-protein kinase, Ring finger domain protein and others were localized to the vicinity of these MTAs. These positional candidate genes are worth of further investigation and possibly these could contribute directly to red rot resistance, and may find a potential application in marker-assisted sugarcane breeding.

Alleles conferring improved fiber quality from EMS mutagenesis of elite cotton genotypes.

KEY MESSAGE: Genetic improvements for many fiber traits are obtained by mutagenesis of elite cottons, mitigating genetic uniformity in this inbred polyploid by contributing novel alleles important to ongoing crop improvement. The elite gene pool of cotton (Gossypium spp.) has less diversity than those of most other major crops, making identification of novel alleles important to ongoing crop improvement. A total of 3,164 M5 lines resulting from ethyl methanesulfonate (EMS) mutagenesis of two G. hirsutum breeding lines, TAM 94L-25 and Acala 1517-99, were characterized for basic components of fiber quality and selected yield components. Across all measured traits, the ranges of phenotypic values among the mutant lines were consistently larger than could be explained by chance (5.27-10.1 for TAM 94 L-25 and 5.29-7.94 standard deviations for Acala 1517-99-derived lines). Multi-year replicated studies confirmed a genetic basis for these differences, showing significant correlations between lines across years and environments. A subset of 157 lines selected for superior fiber qualities, including fiber elongation (22 lines), length (22), lint percent (17), fineness (23), Rd value (21), strength (19), uniformity (21) and multiple attributes in a selection index (26) were compared to 55 control lines in replicated trials in both Texas and Georgia. For all traits, mutant lines showing substantial and statistically significant improvements over control lines were found, in most cases from each of the two genetic backgrounds. This indicates that genetic improvements for a wide range of fiber traits may be obtained from mutagenesis of elite cottons. Indeed, lines selected for one fiber trait sometimes conferred additional attributes, suggesting pleiotropic effects of some mutations and offering multiple benefits for the incorporation of some alleles into mainstream breeding programs.