Evaluation of UAV-derived multimodal remote sensing data for biomass prediction and drought tolerance assessment in bioenergy sorghum

Screening for drought tolerance is critical to ensure high biomass production of bioenergy sorghum in arid or semi-arid environments. The bottleneck in drought tolerance selection is the challenge of accurately predicting biomass for a large number of genotypes. Although biomass prediction by lowaltitude remote sensing has been widely investigated on various crops, the performance of the predictions are not consistent, especially when applied in a breeding context with hundreds of genotypes. In some cases, biomass prediction of a large group of genotypes benefited from multimodal remote sensing data;while in other cases, the benefits were not obvious. In this study, we evaluated the performance of single and multimodal data(thermal, RGB, and multispectral) derived from an unmanned aerial vehicle(UAV) for biomass prediction for drought tolerance assessments within a context of bioenergy sorghum breeding. The biomass of 360 sorghum genotypes grown under well-watered and water-stressed regimes was predicted with a series of UAV-derived canopy features, including canopy structure, spectral reflectance, and thermal radiation features. Biomass predictions using canopy features derived from the multimodal data showed comparable performance with the best results obtained with the single modal data with coefficients of determination(R2) ranging from 0.40 to 0.53 under water-stressed environment and0.11 to 0.35 under well-watered environment. The significance in biomass prediction was highest with multispectral followed by RGB and lowest with the thermal sensor. Finally, two well-recognized yieldbased drought tolerance indices were calculated from ground truth biomass data and UAV predicted biomass, respectively. Results showed that the geometric mean productivity index outperformed the yield stability index in terms of the potential for reliable predictions by the remotely sensed data.Collectively, this study demonstrated a promising strategy for the use of different UAV-based imaging sensors to quantify yield-based drought tolerance.

Capacitance Sensor for Nondestructive Determination of Total Oil Content in Peanut Kernels

In this work, attempts were made to estimate the total oil content (TOC) in single peanut kernels, using the CI meter (Chari’s Impedance meter, described below). Mature peanut kernels of selected varieties with a range of oil contents from 47% to 61% were placed one at a time, between the parallel-plate electrodes of the CI meter, and the impedance (Z) and phase angle (q) of the system were measured, and capacitance, C was computed at 1, 5 and 9 MHz. After the measurements, the TOC of each kernel was determined by Soxhlet method. Using the known TOC values, and the corresponding C, Z and q values, initially on a calibration group of kernels, calibration equations were developed. Using the model coefficients from the calibration, the TOCs of kernel samples of 31 diverse peanut genotypes grown in different environments in Australia were determined. The method predicted the TOC values of peanut kernels of 31 peanut genotypes, within 2% of the Soxhlet values, with an R2 of 0.87 (P 0.001).

Bioinformatic Game Theory and Its Application to Biological Affinity Networks

The exact evolutionary history of any set of biological taxa is unknown, and all phylogenetic reconstructions are approximations. The problem becomes harder when one must consider a mix of vertical and lateral phylogenetic signals. In this paper we propose a game theoretic approach to constructing biological networks. The key hypothesis is that evolution is driven by distinct mechanisms that seek to maximize two competing objectives, taxonomic conservation and diversity. One branch of the mathematical theory of games is brought to bear. It translates this evolutionary game hypothesis into a mathematical model in two-player zero-sum games, with the zero-sum assumption conforming to one of the fundamental constraints in nature in mass and energy conservation. We demonstrate why and how a mechanistic and localized adaptation to seek out greater information for conservation and diversity may always lead to a global Nash equilibrium in phylogenetic affinity. Our game theoretic method, referred to as bioinformatic game theory, is used to construct network clusters. As an example, we applied this method to clustering of a multidomain protein family. The protein clusters identified were consistent with known protein subfamilies, indicating that this game-theoretic approach provides a new framework in biological sequence analysis, especially in studying gene-genome and domain-protein relationships.

Economic Development through Medicinal and Aromatic Plants(MAPs) Cultivation in Hindu Kush Himalaya Mountains of District Swat, Pakistan

Poverty is pervasive in the Swat Valley in northwestern Pakistan, and most people survive by farming small landholdings. However, many supplement their meager subsistence earnings by collecting and selling plant material for use in herbal medicine. This material is wild-harvested, but collectors seem not to fully appreciate the potential value of the plant material they collect nor the longterm impact their collection has on local plant populations. A model project supported by the International Food Policy Research Institute(IFPRI)persuaded small-scale farmers in four different villages to use some of their land for cultivating traditionally wild-harvested species of medicinal and aromatic plants(MAPs) with high market value. The farmers were provided seeds or rhizomes of five MAPs and asked to monitor their germination and growth on 25 m2 plots during a 12 month period. At the end of the study, growth and yield data from the four localities were compared and economic analyses conducted to determine the profitability of the species based on yields, prevailing market prices, and costs of production. Five of the cultivated species were subsequently marketed and their value evaluated:Sesamum indicum, Linum usitatissimum, Ocimum basilicum, Nigella sativa and Viola pilosa. The MAPs V. pilosa and O. basilicum were the most profitable,whereas Nigella sativa was the least profitable because of its low germination rate. The net income from all but Nigella was higher than that would have been earned by planting the same area with the predominant cereals or tomatoes. In addition to demonstrating the feasibility and financial benefits of cultivating MAPs as a cash crop, this model study identified a number of additional steps that would increase the benefits of MAPs cultivation in this area.A combination of specialized education, market infrastructure development and a small loans program would enable farmers to increase their agricultural income without damaging the area's plant diversity.

Mutational Analyses of a Fork Head Associated Domain Protein, DAWDLE, in <i>Arabidopsis thaliana</i>

DAWDLE (DDL) gene encodes a protein that contains an N-terminal arginine-rich domain and a C-terminal Fork Head Associated (FHA) domain in Arabidopsis thaliana. DDL protein is believed to function in microRNA biogenesis by mediating the recruitment of pri-microRNA to DICER-LIKE 1 and also stabilizing the microRNA. The aim of this study was to conduct a structure-function analysis to identify the regions in DDL that are of functional significance. Targeted Induced Local Lesions in Genome screen was performed in the Columbia erecta-105 background of Arabidopsis resulting in the identification of eight point mutations spanning DDL. The mutants were characterized by phenotypic and molecular analyses based on the prior knowledge on ddl knockout mutants. Height of the plant, hypocotyl and root length, and fertility were measured for phenotypic characterization, and microRNA172 levels were measured to assess the mutation effect at the molecular level. Phenotypic and molecular analyses of the mutants revealed effects resulting in ddl phenotypes of varying degrees in different organs and each mutant displayed at least one phenotype studied. Reduction in fertility and increase in stem length were two phenotypes that most of the mutants consistently displayed. Identification and characterization of several key residues in the arginine rich region and FHA domain will serve as an important tool for elucidation of DDL signaling pathway.

Rhizobacteria facilitate physiological and biochemical drought tolerance of Halimodendron halodendron (Pall.) Voss

Growth-promoting bacteria(GPB)have shown promising effects on serving plants against environmental constraints such as drought.Nevertheless,simultaneous effects of different GPB have less been considered for arid land plants and under field conditions.We investigated the effects of single and combined application of GPB,including free-living nitrogen-fixing bacteria(NFB),phosphate solubilizing bacteria(PSB),potassium solubilizing bacteria(KSB),a combination of NFB,PSB,and KSB(NPK),and control,at three drought stress treatments.In order to better understand the interactions between drought and GPB,we measured the morphological,biochemical,and physiological plant traits.The target plant was salt tree(Halimodendron Halodendron(Pall.)Voss),a legume shrub native to arid lands of Central and West Asia.All biofertilizer treatments enhanced the growth,physiology,and biochemistry of salt tree seedlings,and there were significant differences among the treatments.KSB and PSB treatments increased photosynthetic pigments,but KSB treatment was more efficient in transpiration rate and stomatal regulation and increased the soluble carbohydrates.PSB treatment had the highest effect on root traits,such as taproot length,root volume,cumulative root length,and the ratio of root to shoot.NFB treatment enhanced root diameter and induced biomass translocation between root systems.However,only the application of mixed biofertilizer(i.e.,NPK treatment)was the most significant treatment to improve all plant morphological and physiological characteristics of salt tree under drought stress.Therefore,our results provided improvement of some specific plant traits simultaneous with application of three biofertilizers to increase growth and establishment of salt tree seedlings in the degraded arid lands.

Protease inhibitor ASP enhances freezing tolerance by inhibiting protein degradation in kumquat

Cold acclimation is a complex biological process leading to the development of freezing tolerance in plants.In this study,we demonstrated that cold-induced expression of protease inhibitor FmASP in a Citrus-relative species kumquat[Fortunella margarita(Lour.)Swingle]contributes to its freezing tolerance by minimizing protein degradation.Firstly,we found that only cold-acclimated kumquat plants,despite extensive leaf cellular damage during freezing,were able to resume their normal growth upon stress relief.To dissect the impact of cold acclimation on this anti-freezing performance,we conducted protein abundance assays and quantitative proteomic analysis of kumquat leaves subjected to cold acclimation(4◦C),freezing treatment(−10◦C)and post-freezing recovery(25◦C).FmASP(Against Serine Protease)and several non-specific proteases were identified as differentially expressed proteins induced by cold acclimation and associated with stable protein abundance throughout the course of low-temperature treatment.FmASP was further characterized as a robust inhibitor of multiple proteases.In addition,heterogeneous expression of FmASP in Arabidopsis confirmed its positive role in freezing tolerance.Finally,we proposed a working model of FmASP and illustrated how this extracellular-localized protease inhibitor protects proteins from degradation,therebymaintaining essential cellular function for post-freezing recovery.These findings revealed the important role of protease inhibition in freezing response and provide insights on how this role may help develop new strategies to enhance plant freezing tolerance.

TrG2P:A transfer-learning-based tool integrating multi-trait data for accurate prediction of crop yield

Yield prediction is the primary goal of genomic selection(GS)-assisted crop breeding.Because yield is a complex quantitative trait,making predictions from genotypic data is challenging.Transfer learning can produce an effective model for a target task by leveraging knowledge from a different,but related,source domain and is considered a great potential method for improving yield prediction by integrating multi-trait data.However,it has not previously been applied to genotype-to-phenotype prediction owing to the lack of an efficient implementation framework.We therefore developed TrG2P,a transfer-learning-based framework.TrG2P first employs convolutional neural networks(CNN)to train models using non-yield-trait phenotypic and genotypic data,thus obtaining pre-trained models.Subsequently,the convolutional layer parameters from these pre-trained models are transferred to the yield prediction task,and the fully connected layers are retrained,thus obtaining fine-tuned models.Finally,the convolutional layer and the first fully connected layer of the fine-tuned models are fused,and the last fully connected layer is trained to enhance prediction performance.We applied TrG2P to five sets of genotypic and phenotypic data from maize(Zea mays),rice(Oryza sativa),and wheat(Triticum aestivum)and compared its model precision to that of seven other popular GS tools:ridge regression best linear unbiased prediction(rrBLUP),random forest,support vector regression,light gradient boosting machine(LightGBM),CNN,DeepGS,and deep neural network for genomic prediction(DNNGP).TrG2P improved the accuracy of yield prediction by 39.9%,6.8%,and 1.8%in rice,maize,and wheat,respectively,compared with predictions generated by the best-performing comparison model.Our work therefore demonstrates that transfer learning is an effective strategy for improving yield prediction by integrating information from non-yield-trait data.We attribute its enhanced prediction accuracy to the valuable information available from traits associated with yield and to training dataset augmentation.The Python implementation of TrG2P is available at https://github.com/lijinlong1991/TrG2P.The web-based tool is available at http://trg2p.ebreed.cn:81.

湿度对田间自然热罩防治柑橘黄龙病的影响

比较加湿处理后热罩和自然条件下热罩对黄龙病的防治效果,结果表明自然条件处理后30d叶片平均病菌浓度下降了85%以上,而加湿条件下叶片平均病菌浓度仅下降3.7%,与自然条件处理间差异达到显著水平;自然条件热罩处理后90d叶片平均病菌浓度下降了97%以上,而加湿处理病树叶片平均病菌浓度下降了38%,与自然条件处理间的差异达到极显著水平。对照组叶片平均病菌浓度处理后30d和90d均有略微增加;加湿未热罩处理叶片平均病菌浓度在处理后30d和90d分别增加了4.5倍和8.7倍,但与对照组无显著差异。本研究结果表明加湿热罩处理病树显著减弱了其杀灭黄龙病菌效果,同时表明增加土壤湿度对黄龙病菌繁殖可能具有一定的促进作用。

甘蓝型油菜脂肪酸碳链延长酶BnaA.FAE1与BnaC.FAE1的底物特异性分析

研究脂肪酸碳链延长酶(FAE1)的底物特异性,旨在为改良油料种子脂肪酸提供信息,将甘蓝型油菜A、C基因组的BnaA. FAE1和BnaC. FAE1分别在亚麻荠种子中表达。其转基因种子中,山嵛酸与芥酸含量的比值(C22∶0/C22∶1)分别为0. 18和0. 88,表明BnaA. FAE1对单不饱和脂肪酸亲和力较高,而BnaC. FAE1则对饱和脂肪酸亲和力较高,这种差异在各转基因世代表现稳定。通过分析T3转基因家系种子中酰基辅酶A(Acyl-Co As)的组成,进一步证明了上述结论。但是在拟南芥中分别异源表达上述基因,并没有观察到类似的底物特异性差异。在甘蓝型油菜祖先种,白菜型油菜和甘蓝种子中调查其C22∶0/C22∶1比值,同样未发现其FAE1底物特异性存在差异。综上认为BnaA. FAE1和BnaC. FAE1在亚麻荠中存在底物特异性。

Impact of Oriental Fruit Fly Postharvest Treatments on Avocado

The detection in August 2015 of the Oriental Fruit Fly (Bactrocera dorsalis Hendel, Diptera: Tephritidae) in the Redland area in Miami-Dade County, Florida triggered a quarantine that restricted the movement of fruit fly host material in an approximately 99-square mile (256-square kilometer) area. The quarantine affected 4000 acres of fruit bearing commercial avocado groves. Approved post-harvest treatments for B. dorsalis and avocado included in the USDA Treatment Manual were acceptable for immediate certification and movement of fresh avocados from the quarantine area. However, it was unknown if Florida avocados would meet quality standards (US combination grade) after the treatments. Three post-harvest treatments that combine methyl bromide fumigation and cold storage periods were tested on six avocado varieties from Florida. The treatments differed in the durations of the fumigation and cold treatment periods. A seven day transit period at 8.3°C (47°F) was added to account for the time when the fruit leaves the packing house until it is sold by retailers. None of the six varieties had met the US combination grade after the treatments and transit period. Treated fruit exhibited both internal (pulp) and external (skin) damage. Damage was attributed to the fumigation component of the treatment, but the six varieties tolerated the cold portion of the treatment. Damage by fumigation ranged from 26% - 100%. In general, the longer the fumigation period the worse the effect. The need for alternative post-harvest treatments for Florida avocados is discussed.

Combination of genetic analysis and ancient literature survey reveals the diverge nee of traditional Brassica rapa varieties from Kyoto, Japan

Since ancient times,humans have bred several plants that we rely on today.However,little is known about the divergence of most of these plants.In the present study,we investigated the divergence of Mibuna(Brassica rapa L.subsp.nipposinica L.H.Bailey),a traditional leafy vegetable in Kyoto(Japan),by combining genetic analysis and a survey of ancient literature.Mibuna is considered to have been bred 200 years ago from Mizuna,another traditional leafy vegetable in Kyoto.Mibuna has simple spatulate leaves,whereas Mizuna has characteristic serrated leaves.The quantitative trait loci(QTL)and gene expression analyses suggested that the downregulation of BrTCP15 expression contributed to the change in the leaf shape from serrated to simple spatulate.Interestingly,the SNP analysis indicated that the genomic region containing the BrTCP15 locus was transferred to Mibuna by introgression.Furthermore,we conducted a survey of ancient literature to reveal the divergence of Mibuna and found that hybridization between Mizuna and a simple-leaved turnip might have occurred in the past.Indeed,the genomic analysis of multiple turnip cultivars showed that one of the cultivars,Murasakihime,has almost the same sequence in the BrTCP15 region as Mibuna.These results suggest that the hybridization between Mizuna and turnip has resulted in the establishment of Mibuna.

Crosstalk between Ubiquitination and Other Posttranslational Protein Modifications in Plant Immunity

Post-translational modifications(PTMs)are central to the modulation of protein activity,stability,subcellular localization,and interaction with partners.They greatly expand the diversity and functionality of the proteome and have taken the center stage as key players in regulating numerous cellular and physiological processes.Increasing evidence indicates that in addition to a single regulatory PTM,many proteins are modified by multiple different types of PTMs in an orchestrated manner to collectively modulate the biological outcome.Such PTM crosstalk creates a combinatorial explosion in the number of proteoforms in a cell and greatly improves the ability of plants to rapidly mount and fine-tune responses to different external and internal cues.While PTM crosstalk has been investigated in depth in humans,animals,and yeast,the study of interplay between different PTMs in plants is still at its infant stage.In the past decade,investigations showed that PTMs are widely involved and play critical roles in the regulation of interactions between plants and pathogens.In particular,ubiquitination has emerged as a key regulator of plant immunity.This review discusses recent studies of the crosstalk between ubiquitination and six other PTMs,i.e.,phosphorylation,SUMOylation,poly(ADP-ribosyl)ation,acetylation,redox modification,and glycosylation,in the regulation of plant immunity.The two basic ways by which PTMs communicate as well as the underlying mechanisms and diverse outcomes of the PTM crosstalk in plant immunity are highlighted.

Cell-specific pathways recruited for symbiotic nodulation in the Medicago truncatula legume

Medicago truncatula is a model legume species that has been studied for decades to understand the symbiotic relationship between legumes and soil bacteria collectively named rhizobia.This symbiosis called nodulation is initiated in roots with the infection of root hair cells by the bacteria,as well as the initiation of nodule primordia from root cortical,endodermal,and pericycle cells,leading to the development of a new root organ,the nodule,where bacteria fix and assimilate the atmospheric dinitrogen for the benefit of the plant.Here,we report the isolation and use of the nuclei from mock and rhizobia-inoculated roots for the single nuclei RNA-seq(sNucRNA-seq)profiling to gain a deeper understanding of early responses to rhizobial infection in Medicago roots.A gene expression map of the Medicago root was generated,comprising 25 clusters,which were annotated as specific cell types using 119 Medicago marker genes and orthologs to Arabidopsis cell-type marker genes.A focus on root hair,cortex,endodermis,and pericycle cell types,showing the strongest differential regulation in response to a short-term(48 h)rhizobium inoculation,revealed not only known genes and functional pathways,validating the sNucRNA-seq approach,but also numerous novel genes and pathways,allowing a comprehensive analysis of early root symbiotic responses at a cell type-specific level.

STAG-CNS： An Order-Aware Conserved Noncoding Sequences Discovery Tool for Arbitrary Numbers of Species

One method for identifying noncoding regulatory regions of a genome is to quantify rates of divergence between related species, as functional sequence will generally diverge more slowly. Most approaches to identifying these conserved noncoding sequences （CNSs） based on alignment have had relatively large minimum sequence lengths （≥15 bp） compared with the average length of known transcription factor binding sites. To circumvent this constraint, STAG-CNS that can simultaneously integrate the data from the promoters of conserved orthologous genes in three or more species was developed. Using the data from up to six grass species made it possible to identify conserved sequences as short as 9 bp with false discovery rate ≤0.05. These CNSs exhibit greater overlap with open chromatin regions identified using DNase I hypersensitivity assays, and are enriched in the promoters of genes involved in transcriptional regulation. STAG-CNS was further employed to characterize loss of conserved noncoding sequences associated with retained duplicate genes from the ancient maize polyploidy. Genes with fewer retained CNSs show lower overall expression, although this bias is more apparent in samples of complex organ systems containing many cell types, suggesting that CNS loss may correspond to a reduced number of expression contexts rather than lower expression levels across the entire ancestral expression domain.

PBS3 Protects EDS1 from Proteasome-Mediated Degradation in Plant Immunity

Plant immunity is controlled by both positive regulators such as PBS3 and EDS1 and negative regulators such as NPR3 and NPR4.However,the relationships among these important immune regulators remain elusive.In this study,we found that PBS3 interacts with EDS1 in both the cytoplasm and the nucleus,and is required for EDS1 protein accumulation?NPR3 and NPR4,which function as salicylic acid receptors and adaptors of Cullin3-based E3 ligase,interact with and mediate the degradation of EDS1 via the 26S proteasome.We further discovered that PBS3 inhibits the polyubiquitination and subsequent degradation of EDS1 by reducing the association of EDS1 with the Cullin3 adaptors NPR3 and NPR4.Furthermore,we showed that PBS3 and EDS1 also contribute to PAMP-triggered immunity in addition to effector-triggered immunity.Collectively,our study reveals a novel mechanism by which plants fine-tune defense resporises by inhibiting the degradation of a positive player in plant immunity.

A Plant Immune Receptor Degraded by Selective Autophagy

Plants recycle non-activated immune receptors to maintain a functional immune system.The Arabidopsis immune receptor kinase FLAGELLIN-SENSING 2 (FLS2)recognizes bacterial flagellin.However,the molecular mechanisms by which non-activated FLS2 and other non-activated plant PRRs are recycled remain not well understood.Here,we provide evidence showing thatArabidopsis orosomucoid (ORM)proteins,which have been known to be negative regulators of sphingoUpid biosynthesis,act as selective autophagy receptors to mediate the degradation of FLS2.Arabidopsis plants overexpressing ORM 1 or ORM2 have undetectable or greatly diminished FLS2 accumulation,nearly lack FLS2 signaling,and are more susceptible to the bacterial pathogen Pseudomonas syringae.On the other hand,ORM1/2 RNAi plants and orml or orrn2 mutants generated by the CRISPR/Cas9-mediated gene editing have increased FLS2 accumulation and enhanced FLS2 signaling,and are more resistant to P.syringae.ORM proteins interact with FLS2 and the autophagy-related protein ATG8.Interestingly,overexpression of ORM1 or ORM2 in autophagydefective mutants showed FLS2 abundance that is comparable to that in wild-type plants.Moreover, FLS2 levels were not decreased in Arabidopsis plants overexpressing ORM1/2 derivatives that do not interact with ATGS.Taken together,these results suggest that selective autophagy functions in maintaining the homeostasis of a plant immune receptor and that beyond sphingolipid metabolic regulation ORM proteins can also act as selective autophagy receptors.

A High-Throughput Phenotyping Pipeline for Image Processing and Functional Growth Curve Analysis

High-throughput phenotyping system has become more and more popular in plant science research.The data analysis for such a system typically involves two steps:plant feature extraction through image processing and statistical analysis for the extracted features.The current approach is to perform those two steps on different platforms.We develop the package“implant”in R for both robust feature extraction and functional data analysis.For image processing,the“implant”package provides methods including thresholding,hidden Markov random field model,and morphological operations.For statistical analysis,this package can produce nonparametric curve fitting with its confidence region for plant growth.A functional ANOVA model to test for the treatment and genotype effects on the plant growth dynamics is also provided.

Hyperspectral reflectance-based phenotyping for quantitative genetics in crops: Progress and challenges

Many biochemical and physiological properties of plants that are of interest to breeders and geneticists have extremely low throughput and/or can only be measured destructively.This has limited the use of information on natural variation in nutrient and metabolite abundance,as well as photosynthetic capacity in quantitative genetic contexts where it is necessary to collect data from hundreds or thousands of plants.A number of recent studies have demonstrated the potential to estimate many of these traits from hyperspectral reflectance data,primarily in ecophysiological contexts.Here,we summarize recent advances in the use of hyperspectral reflectance data for plant phenotyping,and discuss both the potential benefits and remaining challenges to its application in plant genetics contexts.The performances of previously published models in estimating six traits fromhyperspectral reflectance data in maizewere evaluated on newsample datasets,and the resulting predicted trait values shown to be heritable(e.g.,explained by genetic factors)were estimated.The adoption of hyperspectral reflectance-based phenotyping beyond its current uses may accelerate the study of genes controlling natural variation in biochemical and physiological traits.

An Exome-seq Based Tool for Mapping and Selection of Candidate Genes in Maize Deletion Mutants

Despite the large number of genomic and transcriptomic resources in maize, there is still much to learn about the function of genes in developmental and biochemical processes. Some maize mutants that were generated by gamma-irradiation showed clear segregation for the kernel phenotypes in B73 ? Mo17 F2 ears. To better understand the functional genomics of kernel development,we developed a mapping and gene identi?cation pipeline, bulked segregant exome sequencing(BSEx-seq), to map mutants with kernel phenotypes including opaque endosperm and reduced kernel size. BSEx-seq generates and compares the sequence of the exon fraction from mutant and normal plant F2 DNA pools. The comparison can derive mapping peaks, identify deletions within the mapping peak, and suggest candidate genes within the deleted regions. We then used the public kernel-speci?c expression data to narrow down the list of candidate genes/mutations and identi?ed deletions ranging from several kb to more than 1 Mb. A full deletion allele of the Opaque-2 gene was identi?ed in mutant 531, which occurs within a $200-kb deletion. Opaque mutant 1486 has a 6248-bp deletion in the mapping interval containing two candidate genes encoding RNA-directed DNA methylation 4(RdDM4) and AMP-binding protein, respectively. This study demonstrates the ef?-ciency and cost-effectiveness of BSEx-seq for causal mutation mapping and candidate gene selection,providing a new option in mapping-by-sequencing for maize functional genomics studies.