Aphids are among the most common insect pests that reduce crop production worldwide. However, the breeding of aphid-resistant crops is currently hindered by the scarcity of resistance genes. This problem is particularly significant in sorghum, for which resistant cultivars are urgently needed to control the aphid Melanaphis sorghi (MES). Here, we report map-based cloning of RMES1A and RMES1B, which encode two atypical resistance proteins that confer strong defense against MES. Analysis of knockout mutants and natural variations demonstrated that RMES1A and RMES1B are both required for sorghum resistance against MES. Both genes are induced by MES feeding, specifically in sclerenchyma cells and vascular bundles. RMES1A/RMES1B interact with the sorghum aphid protein MsEF1 to form a functional complex in sorghum cells, leading to key defense responses such as the H2O2 burst and enhanced callose deposition. Accordingly, silencing of MsEF1 expression by RNA interference disrupted RMES1A/RMES1B-mediated resistance, as evidenced by significantly greater growth and fecundity of MES aphids on a resistant cultivar. Structural modeling predicted that RMES1A/RMES1B possess a potential nucleotide-binding domain and two leucine-rich repeat domains but lack the coiled-coil or Toll/interleukin-1 receptor/resistance domains observed in typical nucleotide-binding leucine-rich repeat immune receptors (NLRs). Moreover, RMES1A/RMES1B and their homologs form a distinct cluster in a phylogenetic tree of plant NLRs and likely represent a new type of plant NLR. Our work thus reveals new resistance genes that can be used to investigate and improve immunity against insect pests in sorghum and other crops.

Thousand-seed weight (TSW) is a critical target for genetic improvement in rapeseed (Brassica napus L.). However, phenotypic selection for this trait remains challenging due to its polygenic regulation by multiple quantitative trait loci (QTL). Here, six favorable TSW QTL alleles from two donor parents were introgress into an elite restorer line, 621R, using an integrated strategy combining marker-assisted backcrossing and speed breeding protocols. Through six rounds of backcrossing and convergent crossing followed by two generations of selfing strategies, we developed 13 advanced lines with diverse TSW QTL combinations within 24 months. Field evaluations across three environments revealed that all lines exhibited significantly increased TSW in spring conditions (Minle, Gansu) and winter environments (Wuhan and Jiangling, Hubei) except for two lines which only showed increase in the spring environment. Hybridization assays using these lines as male parents crossed with two male-sterile lines (RG430A and 616A) demonstrated transgressive segregation for TSW: For RG430A-derived hybrids, all crosses significantly outperformed the original control (RG430A×621R) in Wuhan, with 8/13 and 9/13 crosses showing significant TSW increases in Minle and Jiangling, respectively. For 616A-derived hybrids, 11/13 and 10/13 crosses exhibited significant TSW enhancement in Minle and Jiangling, compared to 3/13 in Wuhan. Notably, two top-performing hybrids achieved 13.0% and 6.8% higher plot yields, respectively. Our results demonstrate that strategic pyramiding of complementary TSW QTL alleles effectively enhances seed weight in rapeseed, and these improved lines represent valuable genetic resources for developing high-yield hybrids.

The sorghum aphid (Melanaphis sorghi), a phloem-feeding insect, is a major pest affecting sorghum production. Despite advances in understanding plant resistance mechanisms, the molecular responses of aphids to resistant host plants remain poorly characterized. Here, we aimed to elucidate transcriptional changes in sorghum aphids feeding on the resistant sorghum variety HN16 and to identify key aphid regulatory genes involved in host adaptation. RNA-seq analysis identified 1,388 differentially expressed genes (DEGs) in aphids feeding on HN16. Expression profiling revealed coordinated regulation of genes involved in apoptosis and detoxification. Through weighted gene co-expression network analysis (WGCNA), 10 candidate response genes were identified. Notably, knockdown of the DEG MsCathB1, encoding a cathepsin B-like protease, significantly impaired aphid fitness on resistant plants. Functionally, MsCathB1 also suppressed cryptogein-induced plant cell death and hydrogen peroxide accumulation. These findings suggest that sorghum aphid responses to host resistance are closely linked to apoptosis-related pathways, and that MsCathB1 may function as a virulence effector modulating both aphid performance and plant immunity. This work provides new insights into aphid-host interactions and supports the development of RNAi-based strategies for aphid control.

Background Improving the quality and frying stability of rapeseed oil has long been an important goal in rapeseed quality breeding. This goal can be achieved through molecular marker-assisted selection (MAS) combined with speed breeding strategies. In this study, genomic data from the high-oleic-acid, low-linolenic-acid material L3 and the recessive genic male sterile (RGMS) line RG430A were used to identify functional variation sites underlying key quality traits: one governing oleic acid content and two controlling linolenic acid content. Additionally, Kompetitive Allele-Specific PCR markers were developed to efficiently genotype these oleic and linolenic acid-related variants. Results By integrating speed breeding with MAS and phenotypic screening, a new RGMS line was developed with high oleic acid, low linolenic acid content, and favorable agronomic performance. Compared with the original RG430A, the new line shows an approximately 15.0% increase in oleic acid content (reaching 78.0%) and an approximately 52.6% reduction in linolenic acid content (decreasing to 3.6%), with no significant alterations in major agronomic traits. Conclusions Our study offers strategic insights for rapeseed quality breeding, and the developed RGMS line represents a promising germplasm resource for future high-quality hybrid breeding.

Seed color is a critical quality trait in numerous plant species. In oilseed Brassica crops, including rapeseed and mustard, yellow seeds are distinguished by their significantly higher oil content and faster germination rates compared to black or brown counterparts. Despite the agronomic significance of the yellow seeds being a prime breeding target, the mechanisms underlying elevated oil content remain obscure. In this study, we assembled the first telomere-to-telomere (T2T) genome of B. juncea and further investigated the genetic regulation, molecular mechanism, and the evolutionary history of yellow seeds in B. juncea. Through an analysis of allelic variation in the TRANSPARENT TESTA 8 (TT8) genes across 1,002 worldwide B. juncea accessions, we traced the single origin of yellow seeds to approximately 2,300 y ago in Southwestern China. Furthermore, we discovered the MADS-box gene SEEDSTICK (STK) coevolved with TT8, and they coordinately regulated seed size, oil accumulation, and seed coat proportion in B. juncea. These findings open broad avenues for targeted breeding of yellow-seeded Brassica crops with elevated oil content.

N6-methyladenosine (m6A) modification involves the addition of a methyl group to the nitrogen atom at position six of adenine in RNA. It is the most prevalent type of dynamic internal RNA methylation modification, plays an important role in plant development and abiotic stress. The m6A modification is facilitated by m6A writers (m6A methyltransferases), m6A erasers (m6A demethylation enzymes), and m6A readers (m6A methylated reading proteins). In order to study the characterization and expression of m6A methyltransferases and demethylases in Brassica napus (rapeseed), we used five methyltransferases and two demethylases from Arabidopsis thaliana as reference sequences. A total of 34 methyltransferases and 12 demethylases were identified in B. napus, B. oleracea, and B. rapa. We analyzed the physicochemical properties, gene structures, conserved domains, chromosome localization, and expression pattern across all tissues, as well as the effects of hormone and stress treatments on B. napus. Our findings revealed that the methyltransferase BnaHAKAI was highly expressed during the late stages of seed development. It may be related to the synthesis of oil content and seed size in the later stage of seed growth. In contrast, the demethylase BnaALKBH10B exhibited high expression primarily in the petals, followed by the pods, buds. This expression pattern may be associated with flower development and the timing of flowering. Furthermore, BnaALKBH10B primarily responded to abiotic stresses such as salinity, drought, osmotic, cold, and freezing, as well as to hormones like jasmonic acid and gibberellins. The qRT-PCR results showed that BnaALKBH10B responded to freezing and salt stress. In summary, a total of 34 methyltransferases and 12 demethylases genes were identified in B. napus, B. oleracea, and B. rapa, and their phylogenetic relationships, structural domains, and expression patterns in tissues and under abiotic stress were comprehensively analyzed. This research will serve as a foundation for future studies on m6A in B. napus.

IntroductionBrassica juncea is a major oilseed crop of Brassica. The seed weight is one of yield components in oilseed Brassica crops. Research on the genetic mechanism of seed weight is not only directly related to the yield and economic value of Brassica juncea but also can provide a theory foundation for studying other Brassica crops.MethodsTo map the genes for seed weight, the parental and F2 extreme bulks derived were constructed from the cross between the heavy-seeded accession 7981 and the light-seeded one Sichuan yellow (SY) of B. juncea, and used in bulk segregant sequencing (BSA-seq). Meanwhile, RNA-sequencing (RNA-seq) was performed for both parents at six seed development stages.ResultsOur results showed that a total of thirty five SNPs were identified in thirty two genes located on chromosomes A02 and A10, while fifty eight InDels in fifty one genes located on A01, A03, A05, A07, A09, A10, B01, B02 and B04. The 7,679 differentially expressed genes were identified in developing seeds between the parents. Furthermore, integrated analysis of BSA-seq and RNA-seq data revealed a cluster of nine genes on chromosome A10 and one gene on chromosome A05 that are putative candidate genes controlling seed weight in B. juncea.DiscussionThis study provides a new reference for research on Brassica seed weight and lays a solid foundation for the examination of seed in other Brassica crops.

Despite many years of research, the molecular mechanisms underlying the activation and regulation of host plant resistance (HPR) to insects remain elusive. Recently, Guo et al. reported that a nucleotide-binding leucine-rich repeat NLR protein activates HPR through direct recognition of an insect effector and that autophagy-mediated degradation of this effector negatively regulates HPR.

Molecular characterization of resistance genes is crucial for efficiently understanding and fortifying plant immunity against insect herbivores. Here we report that RMES1A and RMES1B proteins confer resistance to the sorghum aphid Melanaphis sorghi when activated by an insect effector MsEF1. Map-based cloning plus genetic analysis of knockout mutants confirm that RMES1A and RMES1B are both required for aphid resistance. Upon aphid attack, RMES1A and RMES1B expression is elevated in the sclerenchyma cells and vascular bundles of leaves; the two proteins interact with MsEF1 in the exocysts, thus upregulating key defense processes such as reactive oxygen species burst. Structural modeling predicts that RMES1A and RMES1B each carry an ATP binding site and two leucine-rich-repeat domains but lack coiled-coil or Toll/Interleukin-1 receptor/resistance domain, thus likely representing a new type of resistance controlling proteins in plants. Our work reveals new genes and mechanisms for further deciphering and improving plant immunity to insect pests. ### Competing Interest Statement The authors have declared no competing interest.

【Objective】The genome-wide association analysis was performed to identify SNP loci significantly associated with nitrogen use efficiency (NUE) traits in Brassica juncea at seedling stage and to predict the relevant candidate genes, providing a theoretical basis for revealing the molecular mechanism of nitrogen use efficiency in rapeseed and creating nitrogen-efficient germplasm.【Method】The population of 153 Brassica juncea resources was used as the analysis population. Two treatments, low N and normal N, were established using three replicates for each treatment, and two replicated nutrient culture trials were conducted over a two-year period (2021 and 2022). The relative values of root-shoot ratio and shoot nitrogen concentration (low/normal N) were calculated and utilized as NUE traits for a genome-wide association study (GWAS) aimed at exploring candidate genes for NUE.【Result】NUE traits of Brassica juncea resources exhibited abundant variation, ranging from 0.21-2.44 with coefficients of variation of 22.92%-26.19%. The GWAS identified 45 significant SNP loci, among which 16 overlapped between the first relative root-shoot ratio (RRSR1) and the second relative root-shoot ratio (RRSR2), accounting for a phenotype variance range of 10.69%-15.39%. Additionally, 29 significant SNP loci were shared between the first relative shoot nitrogen concentration (RSNC1) and the second relative shoot nitrogen concentration (RSNC2), explaining a phenotype variance range of 13.22%-23.96%. 15 candidate genes for NUE were identified within 200 kb upstream and downstream regions of significant SNP loci, including 5 genes related to nitrate transport (BjuNPF5.8, BjuNRT2.7, BjuNPF2.3, BjuCLCb and BjuNRT1.3), 3 genes associated with nitrogen metabolism (BjuASN3, BjuGLU2 and BjuADCS), 4 genes involved in plant growth and development (BjuCOBL8, BjuPYL6, BjuSAUR72 and BjuUP3) and 3 genes participated in stress response (BjuNTP7, BjuJUB1 and BjuPYL6).【Conclusion】45 SNP loci were detected significantly associated with NUE traits and 15 candidate genes for NUE were identified in this study.

十字花科菘蓝为我国大宗常用中药材植物,其叶和根分别是大青叶和板蓝根的原料.本文总结了菘蓝的基因组结构、生物活性成分及其生物合成途径、远缘杂交及新材料创建等方面的研究进展.菘蓝(2n=2x=14)的基因组大小为300Mb,为tPCK核型,3万余蛋白编码基因.高质量的基因组测序解析了菘蓝的主要生物活性成分吲哚生物碱、苯丙烷类、萜类的合成途径及其候选基因.由于染色体消除,菘蓝(父本)与白菜及甘蓝型油菜的族间有性杂交只产生了具有少数菘蓝遗传成分的非预期杂种.菘蓝与萝卜的体细胞杂种具有双亲染色体,但自交及回交均未产生后代.菘蓝与白菜的体细胞杂种具有加倍的菘蓝染色体组,花粉部分可育而雌性不育.菘蓝与甘蓝型油菜的体细胞杂种与甘蓝型油菜连续回交后创建了全套的7个甘蓝型油菜-菘蓝附加系,一些附加系具有比菘蓝更强的广谱抗病毒效果;体细胞杂交中发生的双亲线粒体基因组重组导致甘蓝型油菜新细胞质雄性不育系的产生,菘蓝特定染色体上的育性基因导入培育了恢复系.最后讨论了这些附加系对菘蓝遗传研究的价值及开发利用前景.

Brassica juncea (L.) Czern. et Coss. was formed 8000–14,000 years ago by hybridization between B. rapa and B. nigra. Domestication and improvement of this species have resulted in diverse morphotypes, which produce seed oil, condiment, swollen root and stem vegetables and leafy vegetables. Various studies had been done in the past to investigate the genetic diversity and origin of B. juncea germplasm and varieties using morphological traits and biochemical markers. However, the origin and domestication of the ancient allotetraploid species B. juncea remain uncertain. The genome sequence of three types B. juncea has been reported, which provides an opportunity to resolve the origin, domestication and diversification of B. juncea. Genetic variation in mustard was identified by resequencing of 480 global accessions. Population genetic and phylogenetic analyses revealed evidence for three clade and six distinct genetic groups. Nuclear and organelle genomic analyses supported a monophyletic origin of B. juncea in West Asia. During mustard eastward spread following three independent migrations, new forms were evolved by gene mutations and introgressions. Sweep scan, genome-wide association study and RNA seq identified causal variants and genes for flowering time and morphological variations associated with domestication and diversification of the versatile mustard. In this chapter, we provide comprehensive insight into the diversity, origin and domestication of B. juncea.

The release of mitochondrial genome sequences provides the basis for characterizing interspecific and intraspecific variation in Brassica mitochondrial genomes. However, few B. juncea (mustard) mitochondrial genomes have been published. We assembled the mitochondrial genomes of three B. juncea subspecies and compared them with previously published genomes. The genomes were phylogenetically classified into A, B, C, and Bna clades. Two variant sites, a transversion (C → A) at nt 79,573 and a 31-bp copy-number variation between nts 65,564 and 65,596, were identified. Based on these variant sites, mitotype-specific sequence markers were developed to characterize the variation among worldwide 558 B. juncea accessions. Three mitochondrial genome types (mitotypes MT1–MT3) were identified. In terms of geographical distribution, MT1 and MT2 accessions were distributed mainly to the north and MT3 to the south of 34°N. Root mustards carried only MT1, leaf and stem mustards carried mainly MT3, and seed mustards carried all three mitotypes, implying that the mitotypes underwent selection during B. juncea domestication. A new form of oil mustard evolved by hybridization between two gene pools in southwest China.

Brassica napus inap cytoplasmic male sterility (CMS) is a novel sterile line with potential application in rapeseed hybrid breeding. Sterile cytoplasm was obtained from Isatis indigotica through somatic fusion and then recurrent backcrossing with B. napus. Previous studies have shown that inap CMS abortion occurred before the stamen primordia (stage 4–5), but the genetic mechanism of sterility needs to be studied. RNA-seq analyses were performed on the floral buds at two stages (0–5 and 6–8), before and after the formation of stamen primordium. As a result, a total of 1769 and 594 differentially expressed genes (DEGs) were detected in the CMS line compared to its maintainer line at the two stages, respectively. In accordance with the CMS phenotype, the up- and downstream regulators of the stamen identity genes AP3 and PI were up- and downregulated in the CMS line, respectively. Furthermore, isobaric tags for relative and absolute quantitation (iTRAQ) analysis showed that a total of 760 differentially abundant proteins (DAPs) were identified in flower buds at stages 0–8, and most of the proteins related to the anther development, oxidative phosphorylation, and programmed cell death (PCD) were downregulated in inap CMS. In combined transcriptomic and proteomic analysis, a total of 32 DEGs/DAPs were identified, of which 7 common DEGs/DAPs had the same expression trend at stage 0–8 of flower development. The downregulation of genes related to the energy deficiency, hormone signal transduction, and the maintenance of mitochondrial metabolic homeostasis at stage 0–5 might disturb the normal differentiation of stamen primordium, resulting in carpelloid stamen of inap CMS. The study will help provide insights into the molecular mechanism of this new male sterility.

Anthocyanins contribute to most colors of plants and play protective roles in response to abiotic stresses. Brassica napus is widely cultivated worldwide as both an oilseed and a vegetable. However, only several high anthocyanin-containing cultivars have been reported, and the mechanisms of anthocyanin accumulation have not been well-elucidated in B. napus. Here, the phenotype, comparative whole-genome identification, and gene expression analysis were performed to investigate the dynamic change of the anthocyanin content and the gene expression patterns of anthocyanin biosynthetic genes (ABGs) in B. napus. A total of 152 ABGs were identified in the B. napus reference genome. To screen out the critical genes involved in anthocyanin biosynthesis and accumulation, the RNA-seq of young leaves of two B. napus lines with purple leaves (PL) or green leaves (GL), and their F1 progeny at 41, 91, and 101 days were performed to identify the differentially expressed genes. The comparative expression analysis of these ABGs indicated that the upregulation of TT8 together with its target genes (such as DFR, ANS, UFGT, and TT19) might promote the anthocyanin accumulation in PL at the early developmental stage (41–91 days). While the downregulation of those ABGs and anthocyanin degradation at the late developmental stage (91–101 days) might result in the decrease in anthocyanin accumulation. Our results would enhance the understanding of the regulatory network of anthocyanin dynamic accumulation in B. napus.

甘蓝型油菜小孢子细胞诱导胚状体以子叶胚和非子叶胚如球形胚、心型胚和类似于胚的结构形态存在.子叶胚在成苗培养基上能够快速生长成植株,但是非子叶胚难以一次成苗,容易褐化死亡,因此减少非子叶状胚、培育高质量子叶胚是提高油菜小孢子再生成植株效率的关键步骤.为了提高甘蓝型油菜小孢子培养过程中诱导子叶胚的数目,对3个甘蓝型油菜品系分离得到的小孢子细胞在25℃下暗培养2周,之后转移到固液双层培养基上培养.通过对摇床的振荡周期、培养温度、固液双层培养基中活性炭的浓度以及6-BA不同梯度的处理,分析诱导子叶胚和非子叶胚发生的数目.结果显示,3个品系小孢子细胞在固液双层培养基上诱导得到的子叶胚数目显著高于液体培养基上诱导发生的数目.小孢子细胞在固液双层培养基上振荡培养1周所得子叶胚数目显著增加.降低小孢子培养温度至23℃,出胚总数和对照差异不显著,但子叶胚的数目显著增加.在固液双层培养基中的下层固体培养基中加入0.1％活性炭和0.25 mg/L 6-BA能够有效提高子叶胚的数目.此外,相比前人只用液体培养基培养的方法,本试验建立了两步培养方法(先液体培养后固-液双层培养)能有效获得子叶胚,从而快速生成植株,提高了甘蓝型油菜小孢子培养技术创制育种资源的效率.

Despite early domestication around 3000 BC, the evolutionary history of the ancient allotetraploid species Brassica juncea (L.) Czern & Coss remains uncertain. Here, we report a chromosome-scale de novo assembly of a yellow-seeded B. juncea genome by integrating long-read and short-read sequencing, optical mapping and Hi-C technologies. Nuclear and organelle phylogenies of 480 accessions worldwide supported that B. juncea is most likely a single origin in West Asia, 8,000–14,000 years ago, via natural interspecific hybridization. Subsequently, new crop types evolved through spontaneous gene mutations and introgressions along three independent routes of eastward expansion. Selective sweeps, genome-wide trait associations and tissue-specific RNA-sequencing analysis shed light on the domestication history of flowering time and seed weight, and on human selection for morphological diversification in this versatile species. Our data provide a comprehensive insight into the origin and domestication and a foundation for genomics-based breeding of B. juncea .

The aneuploidy chromosome addition lines with individual chromosomes of one species into the whole genome of another species developed from interspecific or intergeneric crosses are ideal for studying the gene expression regulation under asymmetrical genome interactions. Here, we chose two rapeseed-radish addition lines (2n=40, AACC+2R) with all chromosomes of rapeseed and one pair of radish chromosomes carrying 25S rDNA loci and both 5S and 25S rDNA loci, respectively. Transcriptome sequencing of these two rapeseed-radish addition lines, together with two parents (Brassica napus, 2n=38, AACC; Raphanus sativus, 2n=18, RR), was performed to assess gene expression changes due to the aneuploidy effect. Our results showed that the global gene expression perturbations in two addition lines showed asymmetric distributions between A and C subgenomes, for more downregulated genes located in the C subgenome. Moreover, several dysregulated domains occurred in the addition lines and majority of them were clustered in C subgenome, further revealing that C subgenome was more inclined to be repressed by the aneuploidy. Homoeolog expression was slightly biased toward C subgenome in the parent B. napus, but had no preference to either of A or C subgenome in the addition lines. The total number of biased genes increased sharply and most of them were shared only by two addition lines, indicating that aneuploidy could change the extent and direction of homoeolog expression. The triplicated subgenomes which were orthologs to the chromosomes of Arabidopsis thaliana also exhibited dramatic alternations in two additions. Together, our results revealed that the addition of individual radish chromosomes could induce dramatically transcriptomic disturbances and those expression changes gave asymmetric distributions between two subgenomes in B. napus. The possible mechanisms including the uniparental expression of rRNA genes in the allopolyploids and additions for the findings are discussed.

形态标记不仅在品种选育、种子生产和纯度鉴定时作为指示性状,而且与作物的产量、品质和抗性相关.综述了近年在油菜叶色、叶面蜡粉、叶型、矮秆、花色、种子颜色和多室等7个标记性状上的基因定位和克隆研究进展,发现往往有多个基因通过同一代谢途径或不同代谢途径控制同一性状,异源四倍体的油菜、芥菜控制性状的基因数目一般是其祖先亲本种白菜、甘蓝的2倍,在相同的染色体或同源区段能找到同源基因.这些研究成果既为更多性状的基因定位克隆提供了借鉴,也为通过基因编辑等手段创制具有特殊标记性状的新种质提供了可选择的靶标位点.

Novel Brassica napus cytoplasmic male sterility (CMS) with carpelloid stamens (inap CMS) was produced by intertribal somatic hybridization with Isatis indigotica (Chinese woad), but its RF (restorer of fertility) gene(s) existed in one particular woad chromosome that was carried by one fertile monosomic alien addition line (MAAL) of rapeseed. Herein, the selfed progenies of this MAAL were extensively selected and analyzed to screen the rapeseed-type plants (2n = 38) with good male fertility and to produce their doubled haploid (DH) lines by microspore culture. From the investigation of fertility restoration in the F1 hybrids with inap CMS, one DH line (RF 39) was identified to adequately restore male fertility and likely carried one dominant RF gene. Specifically, this restorer produced brown pollen grains, similar to the woad and the MAAL, suggesting that this trait is closely linked with the RF gene(s) and serves as one phenotypic marker for the restorer. This restorer contained 38 chromosomes of rapeseed and no intact chromosomes of woad, but some DNA fragments of woad origin were detected at low frequency. This restorer was much improved for pollen and seed fertility and for low glucosinolate content. The successful breeding of the restorer for inap CMS rendered this new pollination control system feasible for rapeseed hybrid production.