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2024 Vol. 23 No. 10 Previous Issue   

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Special Focus: Three decades and beyond: Breeding, biotech breakthroughs and future of China’s GM insect-resistant cotton
Section 1: Cotton functional genomics
Section 2: Cotton biotechnology
Section 3: Cotton molecular design breeding
Horticulture
Plant Protection
Agro-ecosystem & Environment


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Cotton, a crucial textile fiber in China’s economy, faced challenges in the 1990s due to cotton bollworm infestations and excessive pesticide use causing environmental and health issues. To address this, a genetically modified insect-resistant cotton program was launched in China, leading to successful development in 1994. Over the past 30 years, more than 200 new insect-resistant cotton varieties have been cultivated, reducing pesticide use by over 650,000 tons and increasing output value by 65 billion CNY. This success has driven innovation in genetically modified cotton and advanced research in cotton genomics, biotechnology, and molecular breeding. This special issue commemorates the 30th anniversary of China’s insect-resistant cotton development. The cover photo showcases three important members of China’s insect-resistant cotton: monogenic insect-resistant cotton, digenic insect-resistant cotton, and tri-line insect-resistant cotton, highlighting the achievements in China’s insect-resistant cotton research and the growth of China’s cotton industry. Dr. Chengzhen Liang from the Biotechnology Research Institute of the Chinese Academy of Agricultural Sciences provided the cover image. See pages 3243–3505 for more details.




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Special Focus: Three decades and beyond: Breeding, biotech breakthroughs and future of China’s GM insect-resistant cotton

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Editorial – Three decades and beyond: Breeding, biotech breakthroughs and future of China’s GM insect-resistant cotton Chengzhen Liang, Shuangxia Jin 2024, 23(10): 3243-3249.  DOI: 10.1016/j.jia.2024.07.041 Abstract ( )   PDF in ScienceDirect   Cotton (Gossypium spp.) is a pivotal crop in the global textile industry, providing essential natural fibers. Additionally, cottonseed offers significant value as a source of oil and as feed for livestock (Huang et al. 2021; Wen et al. 2023). The sector, dependent on cotton, features a comprehensive value chain extending from the processing of fibers to the production of finished textiles, and it employs tens of millions of individuals (Dorward et al. 1970). The vitality of the cotton industry is essential for the economic prosperity of a wide array of interconnected industries. It directly influences the livelihoods of millions of households (Chapman et al. 1972). Therefore, the cotton industry plays a pivotal role in accelerating agriculture modernization. The cotton bollworm, Helicoverpa armigera, represents a significant agricultural pest with a global distribution (Wu 2007a). During the 1990s, China experienced annual outbreaks of this pest, substantially threatening key crop production, including cotton, maize, and various vegetables (Wu 2007b). Statistical data from 1992 indicate that the cotton bollworm affected an aggregate area of about 21.9 million hectares across different crop types within China, resulting in direct economic losses exceeding 10 billion CNY (Wu 2007b). The widespread infestation of the cotton bollworm precipitated a multitude of economic, social, and environmental challenges. These encompassed diminished profitability of cotton cultivation, escalated pesticide pollution, and acute poisoning incidents among humans and livestock (Lu 2016). These challenges significantly impeded the advancement of cotton production and rural economic development in China. To mitigate the devastating impact of the cotton bollworm, the Chinese government initiated a crucial research project to develop transgenic insect-resistant cotton (Jia et al. 2001). Prof. Sandui Guo, along with his team, developed a novel insect-resistant gene through the fusion of two Bacillus thuringiensis (Bt) genes, Cry1Ab and Cry1Ac, utilizing DNA synthesis techniques (Jia et al. 2001; Guo et al. 2015). This innovation, GFM Cry1Ab/c, is characterized by its independent intellectual property rights. The team successfully developed a monogenic Bt insect-resistant cotton germplasm, demonstrating an insecticidal efficiency exceeding 80% (Cui and Guo 1996). This achievement positioned China as the second nation, following the United States, to hold independent intellectual property rights for insect-resistant cotton. In response to the observed decline in the resistance of monogenic insect-resistant cotton over time, the researchers advanced their work by creating digenic insect-resistant cotton, achieved by integrating the GFM Cry1Ab/c and CpTI genes. The breakthrough resulted in an about 35% increase in the corrected mortality rate of cotton bollworm during the later growth stages, thereby establishing China as a leader in insect-resistant cotton research globally (Guo et al. 1999). To concurrently enhance cotton’s insect resistance and yield, a three-line hybrid insect-resistant cotton breeding system was developed. This system improved seed production efficiency by about 20%, reduced costs by approximately 60%, and increased the yield of hybrid cotton by around 10% (Zhang et al. 2005). This development marked a significant breakthrough in cotton hybrid breeding. The opening and sharing of China’s insect-resistant cotton germplasm resources have expedited the industrial application of these cultivars within the Chinese market. Leveraging the comprehensive benefits of diverse varieties, superior adaptability, and competitive pricing, the market share of China’s insect-resistant cotton surged from 10% in 1999 to 50% in 2003, subsequently reaching over 99% (Guo et al. 2015). Researchers have developed over 200 new insect-resistant cotton varieties using this germplasm, cumulatively extending to an area exceeding about 35 million ha. This development has reduced pesticide usage by over 650,000 tons and generated an incremental output value of about 65 billion RMB (Guo et al. 2015). The adoption of insect-resistant cotton in China has effectively mitigated cotton bollworm damage, substantially increasing cotton yield potential. Consequently, China’s cotton yield has surpassed 1,000 kilograms per hectare, establishing the country as a global leader in this domain. The development of insect-resistant cotton in China represents a significant milestone in agricultural biotechnology. These insect-resistant cotton varieties have been successfully deployed and protected by stringent intellectual property rights, promoting an ecosystem conducive to innovation and exclusivity (Guo et al. 2015; Lu 2016). The strategic and methodical approach adopted in the research, development, and commercialization processes of these transgenic insect-resistant cotton varieties has substantially enhanced China’s proficiency in achieving autonomy in the innovation of transgenic cotton technologies. Recent progress in cotton research, encompassing areas such as multi-genome assembly, precise genome editing techniques, elucidation of fiber formation mechanisms, comprehensive metabolite profiling, and advanced genetic breeding strategies, has markedly propelled the field of cotton science forward (Yang et al. 2020; Huang et al. 2021; Long Y et al. 2023). These advancements underscore the substantial progress achieved and establish a solid theoretical foundation that will underpin future innovations in cotton research and development, thereby contributing to the sustainability and productivity of cotton agriculture. This special issue publishes 17 articles, organized into three thematic sections. The initial section presents research articles on cotton functional genomics, addressing areas such as developing fibers and seeds, resistance to Verticillium wilt (VW), and responses to abiotic stresses and nutritional factors. The subsequent section highlights progress in cotton biotechnology, specifically in transgenic methodologies and genome editing technologies. The final section concentrates on utilizing biotechnological strategies in the molecular design breeding of cotton. Section 1: Cotton functional genomics The quality of cotton fiber is a crucial factor in determining textile product quality precise quantification of critical fiber characteristics, such as maturity, fineness, and neps, using accurate instruments is vital for understanding the molecular determinants of fiber quality and guiding genetic enhancement efforts. Li H G et al. (2024) conducted a comprehensive study on 383 G. hirsutum accessions, utilizing the Advanced Fiber Information System (AFIS) for 12 single fiber quality traits and the High Volume Instrument (HVI) for eight conventional traits. Their genome-wide association study (GWAS) analyses revealed key genes linked to fiber quality. They identified the pleiotropic locus FL_D11 affecting fiber length, and novel loci FM_A03, FF_A05, and FN_A07 governing fiber maturity, fineness, and neps, respectively. Significant genes, including GhKRP6 (fiber length), GhMAP8 (maturity), and GhDFR (fineness), were pinpointed. Deng et al. (2024) performed a comparative analysis of the PEL gene family within 10 Malvaceae genomes, identifying 14 PEL genes in Gossypium barbadense linked to superior fiber length and strength. Additionally, six genes (GhPEL1-25, GhPEL4-09, GhPEL5b-01, GbPEL1-29, GbPEL5b-01, and GbPEL5b-02) were identified as key influencers of fiber characteristics, corroborated by SNP associations and expression analyses. These findings provide valuable insights for breeders who use molecular methods to improve agronomic traits. Lint percentage (LP) is a critical determinant of cotton fiber yield. Wang W W et al. (2024) identified the qLPA01.1 locus through map-based cloning in an F2 population derived from a cross between G. hirsutum cultivar CCRI35 and the chromosome segment substitution line HT_390. Further analysis revealed S-acyltransferase protein 24 (GoPAT24) as the candidate gene for qLPA01.1, providing a crucial genetic marker for marker-assisted selection in breeding programs targeting the LP trait. VW, caused by Verticillium dahliae Kleb., significantly reduces cotton yield and fiber quality, leading to losses of up to 80% (Wei et al. 2015). Termed “the cancer of cotton”, it affects 2.5 million hectares or 50% of China’s cotton areas annually, causing economic losses between 250–310 million USD (Li et al. 2015). Managing this disease is challenging due to V. dahliae’s soil persistence and the lack of effective treatments for G. hirsutum. Efforts to breed resistant cultivars are limited by the scarcity of genetic resources and target genes for genetic engineering (Cheng and Jia 2001; Zhang et al. 2015). Liu J et al. (2024) identified GhMYB3D5, a novel R2R3-MYB transcription factor, as a specific responder to V. dahliae infection in cotton. This factor directly upregulates GhADH1 transcription, subsequently increasing the expression of genes involved in lignin biosynthesis, including PAL, C4H, 4CL, and POD/LAC. The resultant defense-induced lignin accumulation significantly improves cotton’s resistance to VW. Yu et al. (2024) found that overexpressing GhGRPL increases lignin production, thereby reinforcing the secondary cell wall and enhancing resistance to VW. Ethylene has been shown to bolster resistance against VW. Aini et al. (2024) identified GhERF91, an ethylene response factor, as a critical protein in combating V. dahliae, using transcriptomic analyses and weighted gene co-expression network analysis. GhERF91 plays a vital role in the ethylene signaling pathway, enhancing the plant’s defense against V. dahliae infection. Chai et al. (2024) discovered that GhPR6-5b, a key PR6 gene, enhances resistance to V. dahliae. This gene is positively regulated by GhWRKY75, a critical component of cotton’s defense against VW, through direct binding to the W-box TTGAC(T/C). The aforementioned study provides genetic resources for breeding cotton resistant to V. dahlia. Another strategy for VW control employs host-induced gene silencing (HIGS) to introduce dsRNA targeting V. dahliae pathogenic genes into host plants. Wang Q et al. (2024) engineered two transgenic cotton lines, VdThit-RNAi-1 and VdThit-RNAi-2, by introducing dsRNA against the VdThit, a thiamine transporter protein gene. These lines demonstrated enhanced resistance to VW and significantly improved yields in field trials. Recently, China’s cotton cultivation has increasingly concentrated in Xinjiang, a region where abiotic stresses, notably drought and salinity, substantially limit production. Identifying and cloning genes pivotal for stress adaptation, and integrating them into cotton breeding to enhance abiotic stress resilience, are key to ensuring stable yields. Yu et al. (2024) showed that the expression of GhGRPL positively affected both plant growth and elevated salt stress resilience in saline environments. Wang C X et al. (2024) observed that the expression of the atypical protein kinase genes, GhABC1K2-A05 and GhABC1K12-A07, was induced by various abiotic stress treatments in cotton. Furthermore, the knockdown of GhABC1K2-A05 and GhABC1K12-A07 resulted in increased sensitivity of cotton to salt and drought stress. It has long been established that the morphological traits and developmental dynamics of plant roots are crucial for water acquisition in plants, significantly contributing to adaptation under various environmental stresses. Zhu et al. (2024) showed that root drenching with exogenous melatonin significantly boosts cotton yield through enhanced root growth and reduced drought-related damage, establishing a basis for melatonin use in agriculture via this method. Wu et al. (2024) examined six root traits, including main root length (MRL), root fresh weight (RFW), total root length (TRL), root surface area (RSA), root volume (RV), and root average diameter (AvgD), across 242 upland cotton accessions, identifying 41 elite loci and 17 candidate genes associated with root development by GWAS. Furthermore, they provided experimental validation of GhWPP2’s positive effect on root development. Section 2: Cotton biotechnology Over the past three decades, insect-resistant cotton developed in China has emerged as one of the most successful applications of genetic engineering technology in plant breeding. The widespread adoption of Bt cotton in China has effectively reduced infestations of cotton bollworms. However, several studies have demonstrated that the expression of insecticidal proteins varies significantly among different cotton tissues, with the lowest expression observed in cotton bolls during their formation stage. Additionally, abiotic stressors, particularly high temperatures, have been shown to significantly reduce the levels of these proteins, thereby challenging the control of cotton bollworm populations. Liu Z Y et al. (2024) demonstrated that through the synergistic regulation of amino acids and ethylene diamine tetraacetic acid (EDTA), the Bt protein content was significantly enhanced in both cotton bolls and their subtending leaves, with increases of 67.5 and 21.7%, respectively. Furthermore, the discrepancy in Bt protein levels between cotton bolls and their subtending leaves was reduced by 31.2%. This effect is attributed to the elevation of soluble protein content and transaminase activity, coupled with a reduction in catabolic enzyme activity, thereby facilitating an increase in Bt protein content. The advent of transgenic insect-resistant cotton has provided critical insights into overcoming cotton production challenges. Weed infestations, a significant threat, compete for vital resources and exacerbate disease and pest issues, negatively impacting cotton yield and quality. Herbicide-resistant crops now account for about 80% of the global genetically modified (GM) crop area, with these cotton varieties representing 90% in the United States. China has made notable progress in developing herbicide-resistant cotton germplasms (Liang et al. 2017), particularly with the certification of the highly herbicide-resistant cotton strain GGK2 by the Ministry of Agriculture and Rural Affairs of China. This certification marks a significant step forward in expanding the available germplasm resources for the swift development and application of herbicide-resistant cotton varieties. Interestingly, Yan et al. (2024) discovered a stable dwarf phenotype, DHR1, in EPSPS-overexpressing cotton, attributing DHR1 dwarfism to the EPSPS gene. This inheritable phenotype exhibited elevated flavonoid metabolites and reduced lignin metabolites in DHR1 lines, modifying auxin signaling gene expression, thereby influencing auxin response and cell elongation. This study advances understanding of the biological functions of EPSPS in cotton. Beyond expressing exogenous genes like Bt and EPSPS for insect resistance or herbicide tolerance, researchers are leveraging transgenic technology to precisely control the expression of several endogenous genes in cotton, aiming to attain specific trait enhancements. Chong et al. (2024) showed that overexpressing the G. barbadense LMI1 gene (GbLMI1) significantly boosts vegetative growth in cotton, leading to increased leaf size and dry weight and, consequently, enhanced biomass. Gene editing, an advanced biotechnological method, enables precise genetic modifications by inserting, deleting, or replacing DNA sequences, facilitating targeted phenotypic improvements in organisms (Manghwar et al. 2019). In cotton research, the rapid advancement of gene editing technologies, particularly the development of various CRISPR/Cas systems by Chinese scientists, has been notable (Yang et al. 2024). These systems, including CRISPR/Cas9, Cas12a, Cas12b, cytosine base editors (CBE), adenine base editors (ABE), ABE8e, Cas13a/13b/13d, CasRX, dCas9-TV, and targeted RNA methylation/demethylation editors (TME/TDE), offer high editing efficiency, specificity, and reduced off-target effects (Gao et al. 2017; Long et al. 2018; Wang et al. 2018, 2020, 2022; Zhu et al. 2018; Li B et al. 2019; Qin et al. 2020; Li et al. 2022; Yu et al. 2023). They enable various genetic interventions, from gene knockouts and knock-ins to base editing, point mutations, RNA editing, transcriptional activation, and epigenetic modifications. A rigorous framework for evaluating the off-target effects of these CRISPR/Cas systems in cotton has been established, utilizing whole-genome high-throughput resequencing (Li J et al. 2019). Utilizing these gene editing tools, Li T W et al. (2024) generated the ghpdct mutant in cotton, which disrupts GhPDCT1/2 and influences the conversion of phosphatidylcholine to diacylglycerol in cottonseeds, resulting in changes to oleic acid, linoleic acid, palmitic acid, and stearic acid levels. This new germplasm with elevated oleic acid content in cottonseed oil could enhance the economic and nutritional value of cotton as an oil crop, promoting the industrial progress of cotton. In addition, a mutant library of over 5,000 genes has been created, addressing traits such as fiber and seed quality, anther development, plant architecture, disease and pest resistance, drought tolerance, and haploid induction. This has led to the development of novel cotton germplasms with enhanced traits, including increased oleic acid in seeds, gossypol elimination, herbicide resistance, heat tolerance, desirable plant architecture, early maturity, and efficient haploid induction (Gao et al. 2020; Chen et al. 2021; Li et al. 2021; Long L et al. 2023; Yu et al. 2023; Wang G et al. 2024). The deployment and broad application of gene editing in cotton have significantly advanced cotton functional genomics and biological breeding, promising to play a crucial role in future research and development efforts. Section 3: Cotton molecular design breeding Introducing Chinese transgenic insect-resistant cotton marks a new phase in biotechnological breeding. The completion and release of the cotton genome sequence have significantly accelerated advancements in cotton molecular design breeding. Enhanced by the proliferation of multi-omics data, such as genomics, phenomics, and transcriptomics, the process of identifying gene loci linked to essential traits has been expedited. Moreover, this data richness has enabled the identification of loci controlling complex cotton traits, which were difficult to detect using conventional approaches. In this special issue, Li H G et al. (2024) combined AFIS and HVI fiber phenotyping with GWAS to identify the pleiotropic locus FL_D11, regulating fiber length, and three novel loci (FM_A03, FF_A05, and FN_A07) influencing fiber maturity, fineness, and neps, offering key markers for breeding improved fiber quality. Similarly, Wu et al. (2024) utilized the CottonSNP80K array to analyze 56,010 SNPs for GWAS on six root traits, identifying 41 QTLs: MRL (9), RFW (6), TRL (9), RSA (12), RV (12), and AvgD (2), offering critical markers for enhancing root traits in cotton breeding. Focusing on key breeding objectives beyond yield, quality, and stress tolerance, developing early-maturing cotton varieties aims to shorten growth duration, enhance cropping efficiency, and alleviate the competition between grain and cotton cultivation in China. Ma et al. (2024) investigated five early maturity traits in cotton: whole growth period, flowering timing, node of the first fruiting branch, height of the first fruiting branch, and plant height, utilizing BSA-seq and QTL mapping. They identified a significant gene on chromosome D03 associated with these traits, providing essential markers for breeding early-maturing cotton varieties. Improving seed germination rates is essential for uniform cotton seedling emergence, directly impacting yield and profitability. Pei et al. (2024) showed that GhPMEI53 and related genes modify cell wall mechanical properties, affecting the endosperm or testa’s mechanical resistance. Additionally, they regulate phytohormone pathways, notably ABA and GA, essential for seed germination. This study lays a crucial theoretical basis for breeding cotton seeds with increased vigor. Over the preceding three decades, advancements in the domains of cotton functional genomics, biotechnology, molecular breeding, and the deployment of insect-resistant cotton varieties, have exerted substantial direct and indirect influences across diverse agricultural sectors. Specifically, in crop genomics, the generation of high-resolution reference genomes for various Gossypium species has facilitated a deeper understanding of the evolutionary trajectories of cotton germplasm (Yang et al. 2020; Long Y et al. 2023). From an agroecological perspective, empirical investigations into the modulatory effects of insect-resistant cotton on the evolutionary dynamics of pest populations have underscored that the extensive adoption of Bt cotton, in conjunction with reduced pesticide application, augments the efficacy of biological pest management strategies within agroecosystems (Wu et al. 2008; Lu et al. 2012). Unfortunately, due to space constraints, this special issue could not cover all relevant agricultural research areas or include many noteworthy cotton studies. Nonetheless, we aim to enhance our understanding of transgenic Bt cotton’s development and contributions in China through this publication, hoping it offers valuable insights to our readers. We thank Dr. Fuguang Li (Institute of Cotton Research, Chinese Academy of Agricultural Sciences (CAAS)), Dr. Hezhong Dong (Institute of Industrial Crops, Shandong Academy of Agricultural Sciences), Dr. Yongjun Zhang (Institute of Plant Protection of CAAS), and Dr. Quanjia Chen (College of Agronomy, Xinjiang Agricultural University) for serving as co-guest editors and organizing this special issue. We thank the Editorial Department and Editor-in-Chief of the Journal of Integrative Agriculture (JIA) for their collaboration in organizing this special issue, inviting the peer reviewers and eventually assembling the accepted manuscripts into this work.

Section 1: Cotton functional genomics

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Genetic dissection and origin of pleiotropic loci underlying multi-level fiber quality traits in upland cotton (Gossypium hirsutum L.) Hongge Li, Shurong Tang, Zhen Peng, Guoyong Fu, Yinhua Jia, Shoujun Wei, Baojun Chen, Muhammad Shahid Iqbal, Shoupu He, Xiongming Du 2024, 23(10): 3250-3263.  DOI: 10.1016/j.jia.2023.07.030 Abstract ( )   PDF in ScienceDirect   Cotton fiber quality is a persistent concern that determines planting benefits and the quality of finished textile products.  However, the limitations of measurement instruments have hindered the accurate evaluation of some important fiber characteristics such as fiber maturity, fineness, and neps, which in turn has impeded the genetic improvement and industrial utilization of cotton fiber.  Here, 12 single fiber quality traits were measured using Advanced Fiber Information System (AFIS) equipment among 383 accessions of upland cotton (Gossypium hirsutum L.).  In addition, eight conventional fiber quality traits were assessed by the High Volume Instrument (HVI) System.  Genome-wide association study (GWAS), linkage disequilibrium (LD) block genotyping and functional identification were conducted sequentially to uncover the associated elite loci and candidate genes of fiber quality traits.  As a result, the previously reported pleiotropic locus FL_D11 regulating fiber length-related traits was identified in this study.  More importantly, three novel pleiotropic loci (FM_A03, FF_A05, and FN_A07) regulating fiber maturity, fineness and neps, respectively, were detected based on AFIS traits.  Numerous highly promising candidate genes were screened out by integrating RNA-seq and qRT-PCR analyses, including the reported GhKRP6 for fiber length, the newly identified GhMAP8 for maturity and GhDFR for fineness.  The origin and evolutionary analysis of pleiotropic loci indicated that the selection pressure on FL_D11, FM_A03 and FF_A05 increased as the breeding period approached the present and the origins of FM_A03 and FF_A05 were traced back to cotton landraces.  These findings reveal the genetic basis underlying fiber quality and provide insight into the genetic improvement and textile utilization of fiber in G. hirsutum.

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Genome-wide identification of the pectate lyase (PEL) gene family members in Malvaceae, and their contribution to cotton fiber quality Qian Deng, Zeyu Dong, Zequan Chen, Zhuolin Shi, Ting Zhao, Xueying Guan, Yan Hu, Lei Fang 2024, 23(10): 3264-3282.  DOI: 10.1016/j.jia.2024.06.011 Abstract ( )   PDF in ScienceDirect   Pectin is a major constituent of the plant cell wall.  Pectate lyase (PEL, EC 4.2.2.2) uses anti-β-elimination chemistry to cleave the α-1,4 glycosidic linkage in the homogalacturonan region of pectin.  However, limited information is available on the comprehensive and evolutionary analysis of PELs in the Malvaceae.  In this study, we identified 597 PEL genes from 10 Malvaceae species.  Phylogenetic and motif analyses revealed that these PELs are classified into six subfamilies: Clades I, II, III, IV, Va, and Vb.  The two largest subfamilies, Clades I and II, contained 237 and 222 PEL members, respectively.  The members of Clades Va and Vb only contained four or five motifs, far fewer than the other subfamilies.  Gene duplication analysis showed that segmental duplication played a crucial role in the expansion of the PEL gene family in Gossypium species.  The PELs from Clades I, IV, Va, and Vb were expressed during the fiber elongation stage, but nearly all PEL genes from Clades II and III showed no expression in any of the investigated fiber developmental stages.  We further performed single-gene haplotype association analysis in 2,001 G. hirsutum accessions and 229 G. barbadense accessions.  Interestingly, 14 PELs were significantly associated with fiber length and strength traits in G. barbadense with superior fiber quality, while only eight GhPEL genes were found to be significantly associated with fiber quality traits in G. hirsutum.  Our findings provide important information for further evolutionary and functional research on the PEL gene family members and their potential use for fiber quality improvement in cotton.

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Map-based cloning of qLPA01.1, a favorable allele from Gossypium tomentosum chromosome segment line Wenwen Wang, Lei Chen, Yan Wu, Xin Guo, Jinming Yang, Dexin Liu, Xueying Liu, Kai Guo, Dajun Liu, Zhonghua Teng, Yuehua Xiao, Zhengsheng Zhang 2024, 23(10): 3283-3293.  DOI: 10.1016/j.jia.2024.02.011 Abstract ( )   PDF in ScienceDirect   Cotton is an important natural fiber crop worldwide which plays a vital role in our daily life.  High yield is a constant goal of cotton breeding, and lint percentage (LP) is one of the important components of cotton fiber yield.  A stable QTL controlling LP, qLPA01.1, was identified on chromosome A01 from Gossypium hirsutum introgressed lines with G. tomentosum chromosome segments in a previous study.  To fine-map qLPA01.1, an F2 population with 986 individuals was established by crossing G. hirsutum cultivar CCRI35 with the chromosome segment substitution line HT_390.  A high-resolution genetic map including 47 loci and spanning 56.98 cM was constructed in the QTL region, and qLPA01.1 was ultimately mapped into an interval corresponding to an ~80 kb genome region of chromosome A01 in the reference genome, which contained six annotated genes.  Transcriptome data and sequence analysis revealed that S-acyltransferase protein 24 (GoPAT24) might be the target gene of qLPA01.1.  This result provides the basis for cotton fiber yield improvement via marker-assisted selection (MAS) and further studies on the mechanism of cotton fiber development.

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Expression analysis of the R2R3-MYB gene family in upland cotton and functional study of GhMYB3D5 in regulating Verticillium wilt resistance Jie Liu, Zhicheng Wang, Bin Chen, Guoning Wang, Huifeng Ke, Jin Zhang, Mengjia Jiao, Yan Wang, Meixia Xie, Yanbin Li, Dongmei Zhang, Xingyi Wang, Qishen Gu, Zhengwen Sun, Liqiang Wu, Xingfen Wang, Zhiying Ma, Yan Zhang 2024, 23(10): 3294-3310.  DOI: 10.1016/j.jia.2024.07.040 Abstract ( )   PDF in ScienceDirect   Improving plant resistance to Verticillium wilt (VW), which causes massive losses in Gossypium hirsutum, is a global challenge.  Crop plants need to efficiently allocate their limited energy resources to maintain a balance between growth and defense.  However, few transcriptional regulators specifically respond to Verticillium dahliae and the underlying mechanism has not been identified in cotton.  In this study, we found that the that expression of most R2R3-MYB members in cotton is significantly changed by V. dahliae infection relative to the other MYB types.  One novel R2R3-MYB transcription factor (TF) that specifically responds to V. dahliae, GhMYB3D5, was identified.  GhMYB3D5 was not expressed in 15 cotton tissues under normal conditions, but it was dramatically induced by V. dahliae stress.  We functionally characterized its positive role and underlying mechanism in VW resistance.  Upon V. dahliae infection, the up-regulated GhMYB3D5 bound to the GhADH1 promoter and activated GhADH1 expression.  In addition, GhMYB3D5 physically interacted with GhADH1 and further enhanced the transcriptional activation of GhADH1.  Consequently, the transcriptional regulatory module GhMYB3D5-GhADH1 then promoted lignin accumulation by improving the transcriptional levels of genes related to lignin biosynthesis (GhPAL, GhC4H, Gh4CL, and GhPOD/GhLAC) in cotton, thereby enhancing cotton VW resistance.  Our results demonstrated that the GhMYB3D5 promotes defense-induced lignin accumulation, which can be regarded as an effective way to orchestrate plant immunity and growth.

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Upregulation of the glycine-rich protein-encoding gene GhGRPL enhances plant tolerance to abiotic and biotic stressors by promoting secondary cell wall development Wanting Yu, Yonglu Dai, Junmin Chen, Aimin Liang, Yiping Wu, Qingwei Suo, Zhong Chen, Xingying Yan, Chuannan Wang, Hanyan Lai, Fanlong Wang, Jingyi Zhang, Qinzhao Liu, Yi Wang, Yaohua Li, Lingfang Ran, Jie Xiang, Zhiwu Pei, Yuehua Xiao, Jianyan Zeng 2024, 23(10): 3311-3327.  DOI: 10.1016/j.jia.2024.05.025 Abstract ( )   PDF in ScienceDirect   Abiotic and biotic stressors adversely affect plant survival, biomass generation, and crop yields.  As the global availability of arable land declines and the impacts of global warming intensify, such stressors may have increasingly pronounced effects on agricultural productivity.  Currently, researchers face the overarching challenge of comprehensively enhancing plant resilience to abiotic and biotic stressors.  The secondary cell wall plays a crucial role in bolstering the stress resistance of plants.  To increase plant resistance to stress through genetic manipulation of the secondary cell wall, we cloned a cell wall protein designated glycine-rich protein-like (GhGRPL) from cotton fibers, and found that it is specifically expressed during the period of secondary cell wall biosynthesis.  Notably, this protein differs from its Arabidopsis homolog, AtGRP, since its glycine-rich domain is deficient in glycine residues.  GhGRPL is involved in secondary cell wall deposition.  Upregulation of GhGRPL enhances lignin accumulation and, consequently, the thickness of the secondary cell walls, thereby increasing the plant’s resistance to abiotic stressors, such as drought and salinity, and biotic threats, including Verticillium dahliae infection.  Conversely, interference with GhGRPL expression in cotton reduces lignin accumulation and compromises that resistance.  Taken together, our findings elucidate the role of GhGRPL in regulating secondary cell wall development through its influence on lignin deposition, which, in turn, reinforces cell wall robustness and impermeability.  These findings highlight the promising near-future prospect of adopting GhGRPL as a viable, effective approach for enhancing plant resilience to abiotic and biotic stress factors.

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Cotton ethylene response factor GhERF91 is involved in the defense against Verticillium dahliae Nurimanguli Aini, Yuanlong Wu, Zhenyuan Pan, Yizan Ma, Qiushuang An, Guangling Shui, Panxia Shao, Dingyi Yang, Hairong Lin, Binghui Tang, Xin Wei, Chunyuan You, Longfu Zhu, Dawei Zhang, Zhongxu Lin, Xinhui Nie 2024, 23(10): 3328-3342.  DOI: 10.1016/j.jia.2023.07.022 Abstract ( )   PDF in ScienceDirect   Verticillium dahliae causes significant losses in cotton production.  To reveal the mechanism of the defense response to V. dahliae in cotton, transcriptomic analyses were performed using cotton cultivars M138 (V. dahliae-resistant) and P2 (V. dahliae-susceptible).  The results revealed 11,076 and 6,640 differentially expressed genes (DEGs) in response to V. dahliae, respectively.  The weighted gene co-expression network analysis of 4,633 transcription factors (TFs) indicated a “MEblue” module containing 654 TFs that strongly correlate with resistance to V. dahliae.  Among these TFs, the ethylene response factor Ghi_A05G10166 (GhERF91) was identified as a putative hub gene with a defense response against V. dahliae.  A virus-induced gene silencing assay and exogenous application of ethephon showed that GhERF91 is activated by ethylene and positively regulates the response to V. dahliae exposure in cotton.  This study provides fundamental transcriptome data and a putative causal gene (GhERF91) associated with resistance to V. dahliae, as well as genetic resources for breeding V. dahliae-resistant cotton.

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GhWRKY75 positively regulates GhPR6-5b via binding to a W-box TTGAC (C/T) to orchestrate cotton resistance to Verticillium dahliae  Qichao Chai, Meina Zheng, Yanli Li, Mingwei Gao, Yongcui Wang, Xiuli Wang, Chao Zhang, Hui Jiang, Ying Chen, Jiabao Wang, Junsheng Zhao 2024, 23(10): 3343-3357.  DOI: 10.1016/j.jia.2024.05.017 Abstract ( )   PDF in ScienceDirect   Verticillium dahliae is an important fungal pathogen affecting cotton yield and quality.  Therefore, the mining of V. dahlia-resistance genes is urgently needed.  Proteases and protease inhibitors play crucial roles in plant defense responses.  However, the functions and regulatory mechanisms of the protease inhibitor PR6 gene family remain largely unknown.  This study provides a comprehensive analysis of the PR6 gene family in the cotton genome. We performed genome-wide identification and functional characterization of the cotton GhPR6 gene family, which belongs to the potato protease inhibitor I family of inhibitors.  Thirty-nine PR6s were identified in Gossypium arboreum, G. raimondii, G. barbadense, and G. hirsutum, and they were clustered into four groups.  Based on the analysis of pathogen-induced and Ghlmm transcriptome data, GhPR6-5b was identified as the key gene for V. dahliae resistance. Virus-induced gene silencing experiments revealed that cotton was more sensitive to V. dahliae V991 after PR6-5b silencing.  The present study established that GhWRKY75 plays an important role in resistance to Verticillium wilt in cotton by positively regulating GhPR6-5b expression by directly binding to the W-box TTGAC(T/C).  Our findings established that GhWRKY75 is a potential candidate for improving cotton resistance to V. dahliae, and provide primary information for further investigations and the development of specific strategies to bolster the defense mechanisms of cotton against V. dahliae.

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Host-induced gene silencing of the Verticillium dahliae thiamine transporter protein gene (VdThit) confers resistance to Verticillium wilt in cotton Qi Wang, Guoqiang Pan, Xingfen Wang, Zhengwen Sun, Huiming Guo, Xiaofeng Su, Hongmei Cheng 2024, 23(10): 3358-3369.  DOI: 10.1016/j.jia.2024.03.024 Abstract ( )   PDF in ScienceDirect   Verticillium wilt (VW), induced by the soil-borne fungus Verticillium dahliae (Vd), poses a substantial threat to a diverse array of plant species.  Employing molecular breeding technology for the development of cotton varieties with heightened resistance to VW stands out as one of the most efficacious protective measures.  In this study, we successfully generated two stable transgenic lines of cotton (Gossypium hirsutum L.), VdThit-RNAi-1 and VdThit-RNAi-2, using host-induced gene silencing (HIGS) technology to introduce double-stranded RNA (dsRNA) targeting the thiamine transporter protein gene (VdThit).  Southern blot analysis confirmed the presence of a single-copy insertion in each line.  Microscopic examination showed marked reductions in the colonization and spread of Vd-mCherry in the roots of VdThit-RNAi cotton compared to wild type (WT).  The corresponding disease index and fungal biomass of VdThit-RNAi-1/2 also exhibited significant reductions.  Real-time quantitative PCR (qRT-PCR) analysis demonstrated a substantial inhibition of VdThit expression following prolonged inoculation of VdThit-RNAi cotton.  Small RNA sequencing (sRNA-Seq) analysis revealed the generation of a substantial number of VdThit-specific siRNAs in the VdThit-RNAi transgenic lines.  Additionally, the silencing of VdThit by the siVdThit produced by VdThit-RNAi-1/2 resulted in the elevated expression of multiple genes involved in the thiamine biosynthesis pathway in Vd.  Under field conditions, VdThit-RNAi transgenic cotton exhibited significantly enhanced disease resistance and yield compared with WT.  In summary, our findings underscore the efficacy of HIGS targeting VdThit in restraining the infection and spread of Vd in cotton, thereby potentially enabling the development of cotton breeding as a promising strategy for managing VW.

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Knockdown of the atypical protein kinase genes GhABC1K2-A05 and GhABC1K12-A07 make cotton more sensitive to salt and PEG stress Caixiang Wang, Meili Li, Dingguo Zhang, Xueli Zhang, Juanjuan Liu, Junji Su 2024, 23(10): 3370-3386.  DOI: 10.1016/j.jia.2024.01.035 Abstract ( )   PDF in ScienceDirect   Activity of bc1 complex kinase (ABC1K) is an atypical protein kinase (aPK) that plays a crucial role in plant mitochondrial and plastid stress responses, but little is known about the responses of ABC1Ks to stress in cotton (Gossypium spp.).  Here, we identified 40 ABC1Ks in upland cotton (Gossypium hirsutum L.) and found that the GhABC1Ks were unevenly distributed across 17 chromosomes.  The GhABC1K family members included 35 paralogous gene pairs and were expanded by segmental duplication.  The GhABC1K promoter sequences contained diverse cis-acting regulatory elements relevant to hormone or stress responses.  The qRT-PCR results revealed that most GhABC1Ks were upregulated by exposure to different stresses.  GhABC1K2-A05 and GhABC1K12-A07 expression levels were upregulated by at least three stress treatments.  These genes were further functionally characterized by virus-induced gene silencing (VIGS).  Compared with the controls, the GhABC1K2-A05- and GhABC1K12-A07-silenced cotton lines exhibited higher malondialdehyde (MDA) contents, lower catalase (CAT), peroxidase (POD) and superoxide dismutase (SOD) activities and reduced chlorophyll and soluble sugar contents under NaCl and PEG stress.  In addition, the expression levels of six stress marker genes (GhDREB2A, GhSOS1, GhCIPK6, GhSOS2, GhWRKY33, and GhRD29A) were significantly downregulated after stress in the GhABC1K2-A05- and GhABC1K12-A07-silenced lines.  The results indicate that knockdown of GhABC1K2-A05 and GhABC1K12-A07 make cotton more sensitive to salt and PEG stress.  These findings can provide valuable information for intensive studies of GhABC1Ks in the responses and resistance of cotton to abiotic stresses.

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Exogenous melatonin improves cotton yield under drought stress by enhancing root development and reducing root damage Lingxiao Zhu, Hongchun Sun, Ranran Wang, Congcong Guo, Liantao Liu, Yongjiang Zhang, Ke Zhang, Zhiying Bai, Anchang Li, Jiehua Zhu, Cundong Li 2024, 23(10): 3387-3405.  DOI: 10.1016/j.jia.2024.04.011 Abstract ( )   PDF in ScienceDirect   The exogenous application of melatonin by the root drenching method is an effective way to improve crop drought resistance.  However, the optimal concentration of melatonin by root drenching and the physiological mechanisms underlying melatonin-induced drought tolerance in cotton (Gossypium hirsutum L.) roots remain elusive.  This study determined the optimal concentration of melatonin by root drenching and explored the protective effects of melatonin on cotton roots.  The results showed that 50 μmol L–1 melatonin was optimal and significantly mitigated the inhibitory effect of drought on cotton seedling growth.  Exogenous melatonin promoted root development in drought-stressed cotton plants by remarkably increasing the root length, projected area, surface area, volume, diameter, and biomass.  Melatonin also mitigated the drought-weakened photosynthetic capacity of cotton and regulated the endogenous hormone contents by regulating the relative expression levels of hormone-synthesis genes under drought stress.  Melatonin-treated cotton seedlings maintained optimal enzymatic and non-enzymatic antioxidant capacities, and produced relatively lower levels of reactive oxygen species and malondialdehyde, thus reducing the drought stress damage to cotton roots (such as mitochondrial damage).  Moreover, melatonin alleviated the yield and fiber length declines caused by drought stress.  Taken together, these findings show that root drenching with exogenous melatonin increases the cotton yield by enhancing root development and reducing the root damage induced by drought stress.  In summary, these results provide a foundation for the application of melatonin in the field by the root drenching method.

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Mining elite loci and candidate genes for root morphology-related traits at the seedling stage by genome-wide association studies in upland cotton (Gossypium hirsutum L.)  Huaxiang Wu, Xiaohui Song, Muhammad Waqas-Amjid, Chuan Chen, Dayong Zhang, Wangzhen Guo 2024, 23(10): 3406-3418.  DOI: 10.1016/j.jia.2024.03.037 Abstract ( )   PDF in ScienceDirect   Root system architecture plays an essential role in water and nutrient acquisition in plants, and it is significantly involved in plant adaptations to various environmental stresses.  In this study, a panel of 242 cotton accessions was collected to investigate six root morphological traits at the seedling stage, including main root length (MRL), root fresh weight (RFW), total root length (TRL), root surface area (RSA), root volume (RV), and root average diameter (AvgD).  The correlation analysis of the six root morphological traits revealed strong positive correlations of TRL with RSA, as well as RV with RSA and AvgD, whereas a significant negative correlation was found between TRL and AvgD.  Subsequently, a genome-wide association study (GWAS) was performed using the root phenotypic and genotypic data reported previously for the 242 accessions using 56,010 single nucleotide polymorphisms (SNPs) from the CottonSNP80K array.  A total of 41 quantitative trait loci (QTLs) were identified, including nine for MRL, six for RFW, nine for TRL, 12 for RSA, 12 for RV and two for AvgD.  Among them, eight QTLs were repeatedly detected in two or more traits.  Integrating these results with a transcriptome analysis, we identified 17 candidate genes with high transcript values of transcripts per million (TPM)≥30 in the roots.  Furthermore, we functionally verified the candidate gene GH_D05G2106, which encodes a WPP domain protein 2 in root development.  A virus-induced gene silencing (VIGS) assay showed that knocking down GH_D05G2106 significantly inhibited root development in cotton, indicating its positive role in root system architecture formation.  Collectively, these results provide a theoretical basis and candidate genes for future studies on cotton root developmental biology and root-related cotton breeding.

Section 2: Cotton biotechnology

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Optimizing the Bacillus thuringiensis (Bt) protein concentration in cotton: Coordinated application of exogenous amino acids and EDTA to reduce spatiotemporal variability in boll and leaf toxins Zhenyu Liu, Shu Dong, Yuting Liu, Hanjia Li, Fuqin Zhou, Junfeng Ding, Zixu Zhao, Yinglong Chen, Xiang Zhang, Yuan Chen, Dehua Chen 2024, 23(10): 3419-3436.  DOI: 10.1016/j.jia.2024.03.029 Abstract ( )   PDF in ScienceDirect   During the boll formation stage, cotton bolls exhibit the lowest expression of Bacillus thuringiensis (Bt) insecticidal proteins.  Resistance to insects varies notably among different organs, which poses challenges for controlling cotton bollworms.  Consequently, an experimental strategy was designed in the 2020–2021 cotton growing season to coordinate the enhancement of protein synthesis and the attenuation of degradation.  Two Bt cultivars of Gossypium hirsutum, namely the hybrid Sikang 3 and the conventional Sikang 1, were used as test materials.  Three treatments were applied at the peak flowering period: CK (the control), T1 (amino acids), and T2 (amino acids and EDTA).  The results show that, in comparison to the CK group, the Bt protein contents were significantly increased in both cotton bolls and their subtending leaves under the T1 and T2 treatments.  The maximum levels of increase observed were 67.5% in cotton bolls and 21.7% in leaves.  Moreover, the disparity in Bt protein content between cotton bolls and their subtending leaves notably decreased by 31.2%.  Correlation analysis suggested that the primary physiological mechanisms for augmenting Bt protein content involve increased protein synthesis and reduced protein catabolism, which are independent of Bt gene expression levels.  Stepwise regression and path analysis revealed that elevating the soluble protein content and transaminase activity, while reducing the catabolic enzyme activities, are instrumental in enhancing the Bt protein content.  Consequently, the coordinated application of amino acids and EDTA emerges as a strategy that can improve the overall resistance of Bt cotton and mitigate the spatiotemporal variations in Bt toxin concentrations in both cotton bolls and leaves.

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EPSPS regulates cell elongation by disrupting the balance of lignin and flavonoid biosynthesis in cotton Qingdi Yan, Wei Hu, Chenxu Gao, Lan Yang, Jiaxian Yang, Renju Liu, Masum Billah, Yongjun Lin, Ji Liu, Pengfei Miao, Zhaoen Yang, Fuguang Li, Wenqiang Qin 2024, 23(10): 3437-3456.  DOI: 10.1016/j.jia.2023.11.002 Abstract ( )   PDF in ScienceDirect   EPSPS is a key gene in the shikimic acid synthesis pathway that has been widely used in breeding crops with herbicide resistance.  However, its role in regulating cell elongation is poorly understood.  Through the overexpression of EPSPS genes, we generated lines resistant to glyphosate that exhibit an unexpected dwarf phenotype.  A representative line, DHR1, exhibits a stable dwarf phenotype throughout its entire growth period.  Except for plant height, the other agronomic traits of DHR1 are similar to its transgenic explants ZM24.  Paraffin section observations showed that DHR1 internodes are shortened due to reduced elongation and division of the internode cells.  Exogenous hormones confirmed that DHR1 is not a classical brassinolide (BR)- or gibberellin (GA)-related dwarfing mutant.  Hybridization analysis and fine mapping confirmed that the EPSPS gene is the causal gene for dwarfism, and the phenotype can be inherited in different genotypes.  Transcriptome and metabolome analyses showed that genes associated with the phenylpropanoid synthesis pathway are enriched in DHR1 compared with ZM24.  Flavonoid metabolites are enriched in DHR1, whereas lignin metabolites are reduced.  The enhancement of flavonoids likely results in differential expression of auxin signal pathway genes and alters the auxin response, subsequently affecting cell elongation.  This study provides a new strategy for generating dwarfs and will accelerate advancements in light simplification in the cultivation and mechanized harvesting of cotton.

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GbLMI1 over-expression improves cotton aboveground vegetative growth Zhili Chong, Yunxiao Wei, Kaili Li, Muhammad Aneeq Ur Rahman, Chengzhen Liang, Zhigang Meng, Yuan Wang, Sandui Guo, Liangrong He, Rui Zhang 2024, 23(10): 3457-3467.  DOI: 10.1016/j.jia.2023.05.037 Abstract ( )   PDF in ScienceDirect   Leaves are the main organ for photosynthesis and organic synthesis in cotton.  Leaf shape has important effects on photosynthetic efficiency and canopy formation, thereby affecting cotton yield.  Previous studies have shown that LMI1 (LATE MERISTEM IDENTITY1) is the main gene regulating leaf shape.  In this study, the LMI1 gene was inserted into the 35S promoter expression vector, and cotton plants overexpressing LMI1 (OE) were obtained through genetic transformation.  Statistical analysis of the biological traits of the T1 and T2 populations showed that compared to the wild type (WT), OE plants had significantly larger leaves, thicker stems and significantly greater dry weight.  Furthermore, plant sections of the main vein and petiole showed that the numbers of cells in those tissues of OE plants were significantly greater.  In addition, RNA-seq analysis revealed the differential expression of genes related to gibberellin synthesis and NAC gene family (genes containing the NAC domain) between the OE and WT plants, suggesting that LMI1 is involved in secondary wall formation and cell proliferation, which promotes stem thickening.  Moreover, Gene Ontology (GO) analysis revealed enrichment in the terms of calcium ion binding, and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed enrichment in the terms of fatty acid degradation, phosphatidylinositol signal transduction system, and cAMP (cyclic adenosine monophosphate) signal pathway.  These results suggested that LMI1 OE plants are responsive to gibberellin hormone signals, and have altered messenger signals (cAMP, Ca2+) which amplify this function, to promote stronger aboveground vegetative growth.  This study found the LMI1 greatly increased the vegetative growth in cotton, which is the basic requirement for higher yield.

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Knock-out of GhPDCT with the CRISPR/Cas9 system increases the oleic acid content in cottonseed oil Tingwan Li, Lu Long, Yingchao Tang, Zhongping Xu, Guanying Wang, Man Jiang, Shuangxia Jin, Wei Gao 2024, 23(10): 3468-3471.  DOI: 10.1016/j.jia.2024.07.030 Abstract ( )   PDF in ScienceDirect   Cotton is a pivotal economic crop for natural textile fibers that also serves as an important source of edible oil (Long et al. 2023). Cottonseed oil contains approximately 14% oleic acid and 59% linoleic acid. An increase in monounsaturated fatty acids, particularly oleic acid, enhances the oxidative stability and nutritional value of edible oil (Chen et al. 2021). Currently, the demand for edible oil in China is increasing in terms of both production and nutrition. Improving cottonseed oil’s storability and nutritional value is crucial for the comprehensive utilization of cotton. However, cottonseed has long been regarded as a by-product in the cotton industry, so research on improving the content and quality of cottonseed oil has lagged compared to other crop attributes. Phosphatidylcholine: diacylglycerol cholinephospho-transferase (PDCT) is the gate-keeping enzyme for the conversion between phosphatidylcholine and diacylglycerol (Lu et al. 2009). Studies in multiple plants have revealed increases in monounsaturated fatty acids in seeds with PDCT knock-out. To clone the PDCTs of upland cotton (Gossypium hirsutum), the protein sequences of PDCT from Arabidopsis (Lu et al. 2009), oilseed rape (Brassica napus; Bai et al. 2020), soybean (Glycine max; Li et al. 2023), peanut (Arachis hypogaea), and sesame (Sesamum indicum) were used as references for BLAST searches in CottonMD (https://yanglab.hzau.edu.cn/CottonMD; Yang et al. 2023). Four PDCT homologs in cotton were obtained and named GhPDCT1 (Gh_D06G1990), GhPDCT2 (Gh_A06G1621), GhPDCT3 (Gh_A05G3864), and GhPDCT4 (Gh_D05G1178) (Fig. 1-A). The sequence similarities between the four GhPDCTs and AtPDCT are 58.47, 60.13, 45.18, and 58.61%, respectively. Further, the phylogenetic analysis revealed that the GhPDCTs are clustered with the PDCTs of Brassica napus (Fig. 1-A). The heatmap of GhPDCTs in cotton tissues was built using released transcriptome data. The results showed that GhPDCT3 and GhPDCT4 had very little expression in all tissues (Fig. 1-B). GhPDCT2 was expressed in roots, stems, leaves and ovules at different developmental stages, but at relatively low levels. GhPDCT1 shared similar basal expression with GhPDCT2, but the transcript level of GhPDCT1 in ovules was significantly higher than that of GhPDCT2. Notably, the expression of GhPDCT1 was sharply up-regulated in ovules at 20 and 25 days post anthesis (DPA). The expression pattern of GhPDCT1 was further verified by RT-qPCR, which indicated that GhPDCT1 was up-regulated in the late stage of ovule development and peaked around 25 DPA. Previous reports highlighted the rapid accumulation of oil content in cotton seeds at 20–30 DPA (Zhao et al. 2018). Therefore, GhPDCT1 is considered the key candidate for regulating the seed oil content of cotton (Fig. 1-B). Sequence analysis showed that GhPDCT1/2 and GmPDCT1/2 contain similar conserved motifs, as well as a C-terminal PAP2_3 domain (Fig. 1-C). The GmPDCT1 and GmPDCT2 in soybean were both found to be located in the cytosol (Li et al. 2023). To study the subcellular localization of GhPDCT, a GFP-PDCT1 fusion protein was expressed in the protoplasts of cotton cotyledons (Hu et al. 2022), and the RFP-labeled transcription factor GoPGF (Zhang et al. 2024) was co-expressed to mark the nucleus. Observations with a laser scanning confocal microscope showed the green fluorescence of GFP-PDCT1 expressed in the cytoplasm (Fig. 1-D). Knock-out of GhPDCT was achieved with the optimized CRISPR/Cas9 system of cotton (Wang et al. 2018). Due to the high similarity (94.2%) of the coding sequences of GhPDCT1 and GhPDCT2, two sgRNAs respectively targeting two different sites of the 1st exon were designed for the simultaneous mutagenesis of GhPDCT1 and GhPDCT2 (Fig. 1-E). The G. hirsutum L. line ‘Jin668’ was used to produce the GhPDCT1/2 mutant of cotton (ghpdct) with Agrobacterium-mediated transformation (Zhu et al. 2023). The DNA of the ghpdct mutant was extracted for Hi-TOM sequencing, and the offspring of ghpdct-5 with the full mutation were planted for further studies. As shown in Fig. 1-F, ghpdct-5 has a 1 nt deletion at target 2 of GhPDCT2 (A subgenome). In addition, two types of mutations were found in GhPDCT1 (D subgenome), one with a 1 nt insertion at target 1, and the other with a 1 nt insertion and a 2 nt deletion at target 1. The wild type (WT) and ghpdct were planted in the field and a phenotypic study was conducted during the whole growing period. No obvious differences in plant growth were observed between WT and ghpdct. For example, the plant height, fiber length, seed weight of WT and ghpdct showed no statistically significant differences (Fig. 1-G–I). The fatty acids in seeds of WT and ghpdct were measured by gas chromatography-mass spectrometry (GC-MS) (Fig. 1-J). Oleic acid (OA, C18:1) accounted for an average of 14.46% of the total fatty acids in seeds of WT, and 16.49% in seeds of ghpdct, which indicates the up-regulation of oleic acid in the ghpdct mutant. Conversely, linoleic acid (LA, C18:2) was reduced in seeds of ghpdct (52.83%) compared to seeds of WT (59.98%). In addition, knockout of GhPDCT increased the seed content of palmitic acid (PA, C16:0) from 21.24% in WT to 25.85% in ghpdct, and the content of stearic acid (SA, C18:0) increased from 1.70% in WT seeds to 2.39% in ghpdct seeds. These results indicated that the GhPDCT mutation alters the balance of monounsaturated and polyunsaturated fatty acids in cotton seeds, with minimal impacts on growth and development beyond seed oil metabolism. In conclusion, we have produced the ghpdct mutant of cotton using the CRISPR/Cas9 system. Knock-out of GhPDCT1/2 affects the conversion between phosphatidylcholine and diacylglycerol in cottonseeds, and changes the contents of oleic acid, linoleic acid, palmitic acid, and stearic acid. We obtained a new germplasm with a higher oleic acid content in cottonseed oil, which can be applied to enhance the economic and nutritional value of cotton as an oil crop, thereby contributing to the industrial upgrading of cotton.

Section 3: Cotton molecular design breeding

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Identification of candidate genes for early-maturity traits by combining BSA-seq and QTL mapping in upland cotton (Gossypium hirsutum L.) Liang Ma, Tingli Hu, Meng Kang, Xiaokang Fu, Pengyun Chen, Fei Wei, Hongliang Jian, Xiaoyan Lü, Meng Zhang, Yonglin Yang 2024, 23(10): 3472-3486.  DOI: 10.1016/j.jia.2024.04.024 Abstract ( )   PDF in ScienceDirect   Cotton breeding for the development of early-maturing varieties is an effective way to improve multiple cropping indexes and alleviate the conflict between grains and cotton in the cultivated fields in China.  In the present study, we aimed to identify upland cotton quantitative trait loci (QTLs) and candidate genes related to early-maturity traits, including whole growth period (WGP), flowering timing (FT), node of the first fruiting branch (NFFB), height of the node of the first fruiting branch (HNFFB), and plant height (PH).  An early-maturing variety, CCRI50, and a late-maturing variety, Guoxinmian 11, were crossed to obtain biparental populations.  These populations were used to map QTLs for the early-maturity traits for two years (2020 and 2021).  With BSA-seq analysis based on the data of population 2020, the candidate regions related to early maturity were found to be located on chromosome D03.  We then developed 22 polymorphic insertions or deletions (InDel) markers to further narrow down the candidate regions, resulting in the detection of five and four QTLs in the 2020 and 2021 populations, respectively.  According to the results of QTL mapping, two candidate regions (InDel_G286-InDel_G144 and InDel_G24-InDel_G43) were detected.  In these regions, three genes (GH_D03G0451, GH_D03G0649, and GH_D03G1180) have non-synonymous mutations in their exons and one gene (GH_D03G0450) has SNP variations in the upstream sequence between CCRI50 and Guoxinmian 11.  These four genes also showed dominant expression in the floral organs.  The expression levels of GH_D03G0451, GH_D03G0649 and GH_D03G1180 were significantly higher in CCRI50 than in Guoxinmian 11 during the bud differentiation stages, while GH_D03G0450 showed the opposite trend.  Further functional verification of GH_D03G0451 indicated that the GH_D03G0451-silenced plants showed a delay in the flowering time.  The results suggest that these are the candidate genes for cotton early maturity, and they may be used for breeding early-maturity cotton varieties.

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Pectin methylesterase inhibitors GhPMEI53 and AtPMEI19 improve seed germination by modulating cell wall plasticity in cotton and Arabidopsis Yayue Pei, Yakong Wang, Zhenzhen Wei, Ji Liu, Yonghui Li, Shuya Ma, Ye Wang, Fuguang Li, Jun Peng, Zhi Wang 2024, 23(10): 3487-3505.  DOI: 10.1016/j.jia.2024.03.036 Abstract ( )   PDF in ScienceDirect   The germination process of seeds is influenced by the interplay between two opposing factors, pectin methylesterase (PME) and pectin methylesterase inhibitor (PMEI), which collectively regulate patterns of pectin methylesterification.  Despite the recognized importance of pectin methylesterification in seed germination, the specific mechanisms that govern this process remain unclear.  In this study, we demonstrated that the overexpression of GhPMEI53 is associated with a decrease in PME activity and an increase in pectin methylesterification.  This leads to seed cell wall softening, which positively regulates cotton seed germination.  AtPMEI19, the homologue in Arabidopsis thaliana, plays a similar role in seed germination to GhPMEI53, indicating a conserved function and mechanism of PMEI in seed germination regulation.  Further studies revealed that GhPMEI53 and AtPMEI19 directly contribute to promoting radicle protrusion and seed germination by inducing cell wall softening and reducing mechanical strength.  Additionally, the pathways of abscicic acid (ABA) and gibberellin (GA) in the transgenic materials showed significant changes, suggesting that GhPMEI53/AtPMEI19-mediated pectin methylesterification serves as a regulatory signal for the related phytohormones involved in seed germination.  In summary, GhPMEI53 and its homologs alter the mechanical properties of cell walls, which influence the mechanical resistance of the endosperm or testa.  Moreover, they impact cellular phytohormone pathways (e.g., ABA and GA) to regulate seed germination.  These findings enhance our understanding of pectin methylesterification in cellular morphological dynamics and signaling transduction, and contribute to a more comprehensive understanding of the PME/PMEI gene superfamily in plants.

Horticulture

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Enhancer of Shoot Regeneration 2 (ESR2) regulates pollen maturation and vitality in watermelon (Citrullus lanatus) Hu Wang, Lihong Cao, Yalu Guo, Zheng Li, Huanhuan Niu 2024, 23(10): 3506-3521.  DOI: 10.1016/j.jia.2024.05.032 Abstract ( )   PDF in ScienceDirect   Watermelon (Citrullus lanatus) holds global significance as a fruit with high economic and nutritional value.  Exploring the regulatory network of watermelon male reproductive development is crucial for developing male sterile materials and facilitating cross-breeding.  Despite its importance, there is a lack of research on the regulation mechanism of male reproductive development in watermelon.  In this study, we identified that ClESR2, a VIIIb subclass member in the APETALA2/Ethylene Responsive Factor (AP2/ERF) superfamily, was a key factor in pollen development.  RNA in situ hybridization confirmed significant ClESR2 expression in the tapetum and pollen during the later stage of anther development.  The pollens of transgenic plants showed major defects in morphology and vitality at the late development stage.  The RNA-seq and protein interaction assay confirmed that ClESR2 regulates pollen morphology and fertility by interacting with key genes involved in pollen development at both transcriptional and protein levels.  These suggest that Enhancer of Shoot Regeneration 2 (ESR2) plays an important role in pollen maturation and vitality.  This study helps understand the male reproductive development of watermelon, providing a theoretical foundation for developing male sterile materials.

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An allelic variation in the promoter of the LRR-RLK gene, qSS6.1, is associated with melon seed size Xiaoxue Liang, Jiyu Wang, Lei Cao, Xuanyu Du, Junhao Qiang, Wenlong Li, Panqiao Wang, Juan Hou, Xiang Li, Wenwen Mao, Huayu Zhu, Luming Yang, Qiong Li, Jianbin Hu 2024, 23(10): 3522-3536.  DOI: 10.1016/j.jia.2024.07.012 Abstract ( )   PDF in ScienceDirect   Seed size is an important agronomic trait in melons that directly affects seed germination and subsequent seedling growth.  However, the genetic mechanism underlying seed size in melon remains unclear.  In the present study, we employed Bulked-Segregant Analysis sequencing (BSA-seq) to identify a candidate region (~1.35 Mb) on chromosome 6 that corresponds to seed size.  This interval was confirmed by QTL mapping of three seed size-related traits from an F2 population across three environments.  This mapping region represented nine QTLs that shared an overlapping region on chromosome 6, collectively referred to as qSS6.1.  New InDel markers were developed in the qSS6.1 region, narrowing it down to a 68.35 kb interval that contains eight annotated genes.  Sequence variation analysis of the eight genes identified a SNP with a C to T transition mutation in the promoter region of MELO3C014002, a leucine-rich repeat receptor-like kinase (LRR-RLK) gene.  This mutation affected the promoter activity of the MELO3C014002 gene and was successfully used to differentiate the large-seeded accessions (C-allele) from the small-seeded accessions (T-allele).  qRT-PCR revealed differential expression of MELO3C014002 between the two parental lines.  Its predicted protein has typical LRR-RLK family domains, and phylogenetic analyses reveled its similarity with the homologs in several plant species.  Altogether, these findings suggest MELO3C014002 as the most likely candidate gene involved in melon seed size regulation.  Our results will be helpful for better understanding the genetic mechanism regulating seed size in melons and for genetically improving this important trait through molecular breeding pathways.

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The Clausena lansium genome provides new insights into alkaloid diversity and the evolution of the methyltransferase family Yongzan Wei, Yi Wang, Fuchu Hu, Wei Wang, Changbin Wei, Bingqiang Xu, Liqin Liu, Huayang Li, Can Wang, Hongna Zhang, Zhenchang Liang, Jianghui Xie 2024, 23(10): 3537-3553.  DOI: 10.1016/j.jia.2024.07.043 Abstract ( )   PDF in ScienceDirect   Wampee (Clausena lansium) is an important evergreen fruit tree native to southern China that has a long history of use for medicinal purposes.  Here, a chromosome-level genome of C. lansium was constructed with a genome size of 282.9 Mb and scaffold N50 of 30.75 Mb.  The assembled genome contains 48.70% repetitive elements and 24,381 protein-coding genes.  Comparative genomic analysis showed that C. lansium diverged from Aurantioideae 15.91–24.95 million years ago.  Additionally, some expansive and specific gene families related to methyltransferase activity and S-adenosylmethionine-dependent methyltransferase activity were also identified.  Further analysis indicated that N-methyltransferase (NMT) is mainly involved in alkaloid biosynthesis and O-methyltransferase (OMT) participates in the regulation of coumarin accumulation in wampee.  This suggested that wampee’s richness in alkaloids and coumarins might be due to the gene expansions of NMT and OMT.  The tandem repeat event was one of the major reasons for the NMT expansion.  Hence, the reference genome of C. lansium will facilitate the identification of some useful medicinal compounds from wampee resources and reveal their biosynthetic pathways.

Plant Protection

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Identification of banana leaf disease based on KVA and GR-ARNet Jinsheng Deng, Weiqi Huang, Guoxiong Zhou, Yahui Hu, Liujun Li, Yanfeng Wang 2024, 23(10): 3554-3575.  DOI: 10.1016/j.jia.2023.11.037 Abstract ( )   PDF in ScienceDirect   Banana is a significant crop, and three banana leaf diseases, including Sigatoka, Cordana and Pestalotiopsis, have the potential to have a serious impact on banana production.  Existing studies are insufficient to provide a reliable method for accurately identifying banana leaf diseases.  Therefore, this paper proposes a novel method to identify banana leaf diseases.  First, a new algorithm called K-scale VisuShrink algorithm (KVA) is proposed to denoise banana leaf images.  The proposed algorithm introduces a new decomposition scale K based on the semi-soft and middle course thresholds, the ideal threshold solution is obtained and substituted with the newly established threshold function to obtain a less noisy banana leaf image.  Then, this paper proposes a novel network for image identification called Ghost ResNeSt-Attention RReLU-Swish Net (GR-ARNet) based on Resnet50.  In this, the Ghost Module is implemented to improve the network’s effectiveness in extracting deep feature information on banana leaf diseases and the identification speed; the ResNeSt Module adjusts the weight of each channel, increasing the ability of banana disease feature extraction and effectively reducing the error rate of similar disease identification; the model’s computational speed is increased using the hybrid activation function of RReLU and Swish.  Our model achieves an average accuracy of 96.98% and a precision of 89.31% applied to 13,021 images, demonstrating that the proposed method can effectively identify banana leaf diseases.

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Machine learning ensemble model prediction of northward shift in potato cyst nematodes (Globodera rostochiensis and G. pallida) distribution under climate change conditions Yitong He, Guanjin Wang, Yonglin Ren, Shan Gao, Dong Chu, Simon J. Mckirdy 2024, 23(10): 3576-3591.  DOI: 10.1016/j.jia.2024.08.001 Abstract ( )   PDF in ScienceDirect   Potato cyst nematodes (PCNs) are a significant threat to potato production, having caused substantial damage in many countries.  Predicting the future distribution of PCN species is crucial to implementing effective biosecurity strategies, especially given the impact of climate change on pest species invasion and distribution.  Machine learning (ML), specifically ensemble models, has emerged as a powerful tool in predicting species distributions due to its ability to learn and make predictions based on complex data sets.  Thus, this research utilised advanced machine learning techniques to predict the distribution of PCN species under climate change conditions, providing the initial element for invasion risk assessment.  We first used Global Climate Models to generate homogeneous climate predictors to mitigate the variation among predictors.  Then, five machine learning models were employed to build two groups of ensembles, single-algorithm ensembles (ESA) and multi-algorithm ensembles (EMA), and compared their performances.  In this research, the EMA did not always perform better than the ESA, and the ESA of Artificial Neural Network gave the highest performance while being cost-effective.  Prediction results indicated that the distribution range of PCNs would shift northward with a decrease in tropical zones and an increase in northern latitudes.  However, the total area of suitable regions will not change significantly, occupying 16–20% of the total land surface (18% under current conditions).  This research alerts policymakers and practitioners to the risk of PCNs’ incursion into new regions.  Additionally, this ML process offers the capability to track changes in the distribution of various species and provides scientifically grounded evidence for formulating long-term biosecurity plans for their control.

Agro-ecosystem & Environment

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The microbial community, nutrient supply and crop yields differ along a potassium fertilizer gradient under wheat–maize double-cropping systems Zeli Li, Fuli Fang, Liang Wu, Feng Gao, Mingyang Li, Benhang Li, Kaidi Wu, Xiaomin Hu, Shuo Wang, Zhanbo Wei , Qi Chen, Min Zhang, Zhiguang Liu 2024, 23(10): 3592-3609.  DOI: 10.1016/j.jia.2024.01.031 Abstract ( )   PDF in ScienceDirect   Soil microorganisms play critical roles in ecosystem function.  However, the relative impact of the potassium (K) fertilizer gradient on the microbial community in wheat‒maize double-cropping systems remains unclear.  In this long-term field experiment (2008–2019), we researched bacterial and fungal diversity, composition, and community assemblage in the soil along a K fertilizer gradient in the wheat season (K0, no K fertilizer; K1, 45 kg ha−1 K2O; K2, 90 kg ha−1 K2O; K3, 135 kg ha−1 K2O) and in the maize season (K0, no K fertilizer; K1, 150 kg ha−1 K2O; K2, 300 kg ha−1 K2O; K3, 450 kg ha−1 K2O) using bacterial 16S rRNA and fungal internally transcribed spacer (ITS) data.  We observed that environmental variables, such as mean annual soil temperature (MAT) and precipitation, available K, ammonium, nitrate, and organic matter, impacted the soil bacterial and fungal communities, and their impacts varied with fertilizer treatments and crop species.  Furthermore, the relative abundance of bacteria involved in soil nutrient transformation (phylum Actinobacteria and class Alphaproteobacteria) in the wheat season was significantly increased compared to the maize season, and the optimal K fertilizer dosage (K2 treatment) boosted the relative bacterial abundance of soil nutrient transformation (genus Lactobacillus) and soil denitrification (phylum Proteobacteria) bacteria in the wheat season.  The abundance of the soil bacterial community promoting root growth and nutrient absorption (genus Herbaspirillum) in the maize season was improved compared to the wheat season, and the K2 treatment enhanced the bacterial abundance of soil nutrient transformation (genus MND1) and soil nitrogen cycling (genus Nitrospira) genera in the maize season.  The results indicated that the bacterial and fungal communities in the double-cropping system exhibited variable sensitivities and assembly mechanisms along a K fertilizer gradient, and microhabitats explained the largest amount of the variation in crop yields, and improved wheat‒maize yields by 11.2–22.6 and 9.2–23.8% with K addition, respectively.  These modes are shaped contemporaneously by the different meteorological factors and soil nutrient changes in the K fertilizer gradients.

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Water and nitrogen footprint assessment of integrated agronomic practice management in a summer maize cropping system Ningning Yu, Bingshuo Wang, Baizhao Ren, Bin Zhao, Peng Liu, Jiwang Zhang 2024, 23(10): 3610-3621.  DOI: 10.1016/j.jia.2024.03.061 Abstract ( )   PDF in ScienceDirect   The footprints of water and nitrogen (WF and NF) provide a comprehensive overview of the type and quantity of water consumption and reactive nitrogen (Nr) loss in crop production.  In this study, a field experiment over two years (2019 and 2020) compared three integrated agronomic practice management (IAPM) systems: An improved management system (T2), a high-yield production system (T3), and an integrated soil–crop management system (ISCM) using a local smallholder farmer’s practice system (T1) as control, to investigate the responses of WF, Nr losses, water use efficiency (WUE), and nitrogen use efficiency (NUE) to IAPM.  The results showed that IAPM optimized water distribution and promoted water use by summer maize.  The evapotranspiration over the whole maize growth period of IAPM increased, but yield increased more, leading to a significant increase in WUE.  The WUE of the T2, T3, and ISCM treatments was significantly greater than in the T1 treatment, in 2019 and 2020 respectively, by 19.8–21.5, 31.8–40.6, and 34.4–44.6%.  The lowest WF was found in the ISCM treatment, which was 31.0% lower than that of the T1 treatment.  In addition, the ISCM treatment optimized soil total nitrogen (TN) distribution and significantly increased TN in the cultivated layer.  Excessive nitrogen fertilizer was applied in treatment T3, producing the highest maize yield, and resulting in the highest Nr losses.  In contrast, the ISCM treatment used a reduced nitrogen fertilizer rate, sacrificing grain yield partly, which reduced Nr losses and eventually led to a significant increase in nitrogen use efficiency and nitrogen recovery.  The Nr level in the ISCM treatment was 34.8% lower than in the T1 treatment while NUE was significantly higher than in the T1 treatment by 56.8–63.1% in 2019 and 2020, respectively.  Considering yield, WUE, NUE, WF, and NF together, ISCM should be used as a more sustainable and clean system for sustainable production of summer maize.