Citrus fruits are one of the most important fruits in the world, and their seedless character is favored by consumers. WRKY is a plant-specific transcription factor family involved in all aspects of plant growth and development. However, the molecular mechanism of seedless fruit formation in citrus and the role of the WRKY gene family in seed abortion are still poorly understood. In this study, we identified 47 WRKY family genes in the citrus fruit Citrus reticulata and comprehensively characterized the WRKY gene family through gene structure and evolutionary relationships. The expression patterns and protein interaction networks of the WRKY gene family were analyzed based on citrus seed abortion transcriptome data, and several WRKY genes that may be involved in the seed abortion regulation were excavated. Furthermore, CrWRKY48 was verified to regulate seed abortion positively in Arabidopsis thaliana, and the rate of seed abortion caused by overexpression of CrWRKY48 reached 45.48%. Using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling assays, DNA affinity purification sequencing and yeast-one-hybrid assays, we found that CrWRKY48 activated excessive programmed cell death by regulating the expression of programmed cell death-related genes such as SOBIR1. Our results show the potential regulation of the WRKY gene family for citrus seed abortion and provide novel insights into the role of CrWRKY48 in mediating citrus seed abortion by activating programmed cell death.

The pollen morphological characteristics of 16 pummelos and 4 other citrus germplasms from Vietnam, Thailand, and China were observed using scanning electron microscopy (SEM). Observations included equatorial view, polar view, and exine ornamentation. The results showed that the pollen of all tested materials was monads, prolate, or subprolate. The average polar axis length of the pollen ranged from 29.121 to 37.043 µm, and that of the equatorial axis ranged from 19.861 to 25.911 µm. A t-test revealed that the polar axis of pollen from Chinese pummelo germplasms was significantly longer than that of varieties from Southeast Asia, indicating certain geographical differentiation. The apertures were all colporate type, predominantly with four to five colpi (75% of grains); only four colpi were observed in the remainder (25%). The pollen exine ornamentation of pummelo germplasms was perforated, whereas that of other citrus types was reticulate. Pummelos exhibited a smaller pori diameter (0.264–0.673 µm) and wider distance between pori (0.581–1.118 µm), while other citrus species had larger lumina (1.253–1.684 µm) and narrower muri (0.443–0.664 µm). Principal component analysis (PCA) and cluster analysis were performed based on pollen traits, and two principal component factors were extracted. The pummelo germplasms were divided into two subgroups: sweet pummelo and red pummelo, which demonstrated a correlation among their pollen morphology, flesh color, and flesh flavor. The phenotypic diversity of pollen among different pummelo germplasms may provide a valuable auxiliary reference for the identification and systematic classification of pummelos.

Natural flavonoids give citrus its unique color and flavor, and directly affect its bitterness. However, the variations in bitterness and the underlying mechanisms among different citrus varieties remain poorly understood. In this study, three pummelo varieties with distinct bitter flavor profiles were selected for metabolomic and transcriptomic analyses to investigate the accumulation of bitter compounds in citrus. A total of 439 reliable metabolites were identified, with pathway analysis revealing significant enrichment of "Flavone and flavonol biosynthesis" and "Flavonoid biosynthesis" pathways in both bitter and non-bitter pummelo varieties. Comparative analysis identified 18 differentially expressed flavonoids, and HPLC analysis confirmed that naringin and neohesperidin are the primary contributors to bitterness. All 8658 differentially expressed genes (DEGs) were clustered into eight groups using K-means analysis. Cluster 5 exhibited an expression trend consistent with the bitter taste accumulation pattern across the three pummelo varieties. Pathway enrichment analysis indicated that DEGs in cluster 5 were significantly associated with the phenylalanine metabolic pathway. Using the enhanced 'GFAnno' flavonoid gene annotation tool, four 7-Glct, five 1,2-Rhat, and five 1,6-Rhat flavone-related genes, along with phenylalanine-related genes, were identified. Expression analysis and RT-qPCR results demonstrated that key genes PAL, C4H, and 4CL in the upstream phenylpropanoid pathway of flavonoid biosynthesis were highly expressed in the two bitter pummelo cultivars, but low in the non-bitter pummelo. Joint analysis further revealed that the transcription factors MUTE/bHLH (XGF149920) and NID1/MYB-like (XGF105200) were co-expressed with naringin and neohesperidin, suggesting their involvement in regulating bitter flavanone metabolism.

Loose-skin mandarins (LSMs) are among the oldest domesticated horticultural crops, yet their domestication history and the genetic basis underlying the formation of key selected traits remain unclear. We provide a chromosome-scale and haplotype-resolved assembly for the ancient Chinese citrus variety Nanfengmiju tangerine. Through the integration of 77 resequenced and 114 published citrus germplasm genomes, we categorize LSMs into 12 distinct groups based on population genomic analyses. We infer that the ancestors of modern cultivated mandarins diverged from wild mandarins in Daoxian approximately 500,000 years ago, when they entered the Yangtze and Pearl River Basins. There, they were domesticated into four ancient cultivation groups, forming the cornerstone of modern Chinese LSM cultivation. We identify selective sweeps underlying quantitative trait loci and genes related to important fruit quality traits, including sweetness and size. We reveal that the co-selection of sugar transporter and metabolism genes are associated with increased fruit sweetness. Significant alterations in the auxin and gibberellin signaling networks may contribute to the enlargement of LSM fruits. We also provide a comprehensive, high-spatiotemporal-resolution atlas of allelic gene expression during citrus fruit development. We detect 5890 allele pairs showing specific expression patterns and a significant increase in variation levels. Our study provides valuable genomic resources and further revises the origin and domestication history of LSMs, offering insights for genetic improvement of citrus plants.

Members of the microRNA156 (miR156) family play essential roles in various biological processes; yet, their involvement in regulating fruit ripening in citrus remains poorly understood. In this study, we determined that the miR156k-SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE 2 (SPL2) module plays an important role in regulating citrus fruit ripening. Heterologous expression of csi-miR156k in Mini-Citrus Hongkong kumquat (Fortunella hindsii) significantly promoted fruit ripening, with the same phenotype observed when SPL2 was transiently silenced. Phenotypic analysis of F. hindsii transgenic lines revealed that SPL2 is the key target gene through which miR156k regulates fruit ripening in citrus. We used DNA affinity purification sequencing and interaction assays to identify PYRABACTIN RESISTANCE LIKE 2 (PYL2) as the downstream target gene of SPL2, showing that PYL2 is negatively regulated by SPL2. Transient expression or repression of PYL2 in F. hindsii fruit greatly altered the duration of fruit color break. Moreover, screening of a yeast 2-hybrid library revealed that caffeoyl CoA methyltransferase 7 (CCoAOMT7) directly interacts with SPL2. CCoAOMT7 recruits S-adenosyl-L-homocysteine hydrolase 1, leading to alterations in the DNA and histone methylation levels of the PYL2 promoter. Overall, this study reveals a previously uncharacterized mechanism by which the miR156k-SPL2 module regulates citrus fruit ripening by modulating PYL2 through epigenetic regulation.

The function of the pollen tube in higher plants is to deliver two male gametes to the embryo sac to ensure successful double fertilization. During this process, many interactions occur among pollen tubes and pistil cells and tissues. Calcium ion (Ca2 +) dynamics mediate these interactions to ensure that the pollen tube grows with correct polarity and orientation to reach its target, the embryo sac. The pistil tissue, which contains abundant Ca2+, attracts the growing pollen tube via changes in Ca2+. Recent studies have shed light on the mechanisms of vacuole biogenesis and the Ca2+ action pathway in the growing pollen tube. This successfully explains the distribution characteristics of high Ca2+ at the tip of the pollen tube and the physiological mechanism of Ca2+ controlling pollen tube growth. In last stage of pollen tube growth, some studies indicated that the cessation of pollen tube growth and breaking of the tube in embryo sac maybe related to Ca2+ dynamic, which finishes its complex journey of pollen tube in vivo.

Achieving seedlessness in citrus varieties is one of the important objectives of citrus breeding. Male sterility associated with abnormal pollen development is an important factor in seedlessness. However, our understanding of the regulatory mechanism underlying the seedlessness phenotype in citrus is still limited. Here, we determined that the miR159a-DUO1 module played an important role in regulating pollen development in citrus, which further indirectly modulated seed development and fruit size. Both the overexpression of csi-miR159a and the knocking out of DUO1 in Hong Kong kumquat (Fortunella hindsii) resulted in small and seedless fruit phenotypes. Moreover, pollen was severely aborted in both transgenic lines, with arrested pollen mitotic I and abnormal pollen starch metabolism. Through additional cross-pollination experiments, DUO1 was proven to be the key target gene for miR159a to regulate male sterility in citrus. Based on DNA affinity purification sequencing (DAP-seq), RNA-seq, and verified interaction assays, YUC2/YUC6, SS4 and STP8 were identified as downstream target genes of DUO1, those were all positively regulated by DUO1. In transgenic F. hindsii lines, the miR159a-DUO1 module down-regulated the expression of YUC2/YUC6, which decreased indoleacetic acid (IAA) levels and modulated auxin signaling to repress pollen mitotic I. The miR159a-DUO1 module reduced the expression of the starch synthesis gene SS4 and sugar transport gene STP8 to disrupt starch metabolism in pollen. Overall, this work reveals a new mechanism by which the miR159a-DUO1 module regulates pollen development and elucidates the molecular regulatory network underlying male sterility in citrus.

Alkaline soil is one of the major problems affecting the development of the citrus industry. Ziyang xiangcheng (Citrus junos Sieb. ex Tanaka) (Cj) is an alkalinity stress-tolerant citrus rootstock. In this study, two citrus rootstocks (Cj and Poncirus trifoliata (L.) Raf.) (Pt)) and one navel orange scion ('Lane Late' navel orange (Citrus sinensis (L.) Osb.) (LL)) were used. The root, stem and leaf tissues of grafted materials Cj + LL and Pt + LL grown in calcareous soil were used to identify genes and pathways that are responsive to alkalinity stress using comparative transcriptomics. Seedlings of Cj and Pt cultured in nutrient solutions at three pH grades were used to perform a supplemental experiment. A comprehensive analysis of the RNA-seq data, physiology, biochemistry, agronomic traits and mineral elements of the Cj + LL, Pt + LL, Cj and Pt materials revealed several candidate pathways and genes that were highly regulated under alkalinity stress. The data suggest that the jasmonate pathway may play crucial roles in tolerance to alkalinity stress in Cj by interacting with other plant hormones, lignin biosynthesis and the ROS scavenging system. These results provide new clues for future investigations of the mechanisms underlying the tolerance of citrus to alkalinity stress.

MYB family transcription factors (TFs) play essential roles in various biological processes, yet their involvement in regulating fruit ripening and fruit size in citrus remains poorly understood. In this study, we have established that the R2R3-MYB TF, CsMYB77, exerts a negative regulatory influence on fruit ripening in both citrus and tomato (Solanum lycopersicum), while also playing a role in modulating fruit size in citrus. The overexpression of CsMYB77 in tomato and Hongkong kumquat (Fortunella hindsii) led to notably delayed fruit ripening phenotypes. Moreover, the fruit size of Hongkong kumquat transgenic lines was largely reduced. Based on DNA affinity purification sequencing and verified interaction assays, SEVEN IN ABSENTIA OF ARABIDOPSIS THALIANA4 (SINAT4) and PIN-FORMED PROTEIN5 (PIN5) were identified as downstream target genes of CsMYB77. CsMYB77 inhibited the expression of SINAT4 to modulate abscisic acid (ABA) signaling, which delayed fruit ripening in transgenic tomato and Hongkong kumquat lines. The expression of PIN5 was activated by CsMYB77, which promoted free indole-3-acetic acid decline and modulated auxin signaling in the fruits of transgenic Hongkong kumquat lines. Taken together, our findings revealed a fruit development and ripening regulation module (MYB77-SINAT4/PIN5-ABA/auxin) in citrus, which enriches the understanding of the molecular regulatory network underlying fruit ripening and size.

为了探明产地对赣南早脐橙果实品质的影响,进而为赣南早脐橙的合理区划提供参考,在信丰、兴山和秭归3个产区采集赣南早脐橙果实样品进行品质分析,利用气相色谱质谱联用(GC-MS)检测果实挥发性物质成分,并用主成分分析法进行香气品质评价.结果表明,3个产区的赣南早脐橙果实品质没有明显差异,但果皮和果肉中挥发性物质的种类和含量均存在一定的差异.兴山赣南早脐橙果皮挥发性物质总量(4 220.37 μg/g)和果肉挥发性物质总量(40.87μg/g)均最高.D-柠檬烯是赣南早脐橙果皮和果肉中含量最高的挥发性物质.2-甲氧基-4-乙烯基苯酚、呋喃酮、苯甲酸和5-羟甲基糠醛仅在兴山赣南早脐橙果实中被检测到.单萜类及烯萜类氧化衍生物挥发性成分的差异,形成不同产地赣南早脐橙的香味特征.通过主成分分析,能将不同产区的赣南早脐橙区分开.

[目的]通过对马家柚配置不同授粉组合,探究花粉直感效应对马家柚果实品质的影响.[方法]以江西省上饶市地方优良品种马家柚为杂交母本,以信木柚、四倍体葡萄柚为杂交父本,通过人工授粉进行杂交,比较不同处理下马家柚果实品质差异.[结果]信木柚与四倍体葡萄柚花粉活力分别为94.34%和93.75%,经过异花授粉能显著提高马家柚坐果率,四倍体葡萄柚高于信木柚.在常规品质方面,信木柚授粉能提高果实平均单果质量、横径、纵径、种子质量、果皮质量和固酸比;四倍体葡萄柚授粉能提高果实平均可食率,降低果实平均单果质量、果皮厚度、种子质量和果皮质量,且授粉后种子呈瘪籽状.在代谢物方面,果肉中主要的初生代谢物为氨基酸类、有机酸类、糖类和醇类,不同授粉品种之间初生代谢物含量有明显差异,但与亲本之间没有明显规律.另外在授粉后的果实与亲本果实中共统计到85种挥发性物质,其中醛类11种,萜烯类28种,酯类16种,醇类14种,酮类14种,烷烃类化合物5种和7种酚、羧酸类化合物,不同果实中均以柠檬烯含量最高,含量占比在87.48%～92.57%之间,不同授粉品种的挥发性物质含量存在差异,且信木柚授粉组合中有21种物质表现出明显的花粉直感效应.[结论]异花授粉可以提高马家柚坐果率,可以通过花粉直感效应在一定程度上快速改善马家柚的综合品质,为马家柚实际生产提供理论依据和调控技术支撑.

Although revisiting the discoveries and implications of genetic variations using phased genomics is critical, such efforts are still lacking. Somatic mutations represent a crucial source of genetic diversity for breeding and are especially remarkable in heterozygous perennial and asexual crops. In this study, we focused on a diploid sweet orange (Citrus sinensis) and constructed a haplotype-resolved genome using high fidelity (HiFi) reads, which revealed 10.6% new sequences. Based on the phased genome, we elucidate significant genetic admixtures and haplotype differences. We developed a somatic detection strategy that reveals hidden somatic mutations overlooked in a single reference genome. We generated a phased somatic variation map by combining high-depth whole-genome sequencing (WGS) data from 87 sweet orange somatic varieties. Notably, we found twice as many somatic mutations relative to a single reference genome. Using these hidden somatic mutations, we separated sweet oranges into seven major clades and provide insight into unprecedented genetic mosaicism and strong positive selection. Furthermore, these phased genomics data indicate that genomic heterozygous variations contribute to allele-specific expression during fruit development. By integrating allelic expression differences and somatic mutations, we identified a somatic mutation that induces increases in fruit size. Applications of phased genomics will lead to powerful approaches for discovering genetic variations and uncovering their effects in highly heterozygous plants. Our data provide insight into the hidden somatic mutation landscape in the sweet orange genome, which will facilitate citrus breeding.

Seed abortion is an important process in the formation of seedless characteristics in citrus fruits. However, the molecular regulatory mechanism underlying citrus seed abortion is poorly understood. Laser capture microdissection-based RNA-seq combined with Pacbio-seq was used to profile seed development in the Ponkan cultivars 'Huagan No. 4' (seedless Ponkan) (Citrus reticulata) and 'E'gan No. 1' (seeded Ponkan) (C. reticulata) in two types of seed tissue across three developmental stages. Through comparative transcriptome and dynamic phytohormone analyses, plant hormone signal, cell division and nutrient metabolism-related processes were revealed to play critical roles in the seed abortion of 'Huagan No. 4'. Moreover, several genes may play indispensable roles in seed abortion of 'Huagan No. 4', such as CrWRKY74, CrWRKY48 and CrMYB3R4. Overexpression of CrWRKY74 in Arabidopsis resulted in severe seed abortion. By analyzing the downstream regulatory network, we further determined that CrWRKY74 participated in seed abortion regulation by inducing abnormal programmed cell death. Of particular importance is that a preliminary model was proposed to depict the regulatory networks underlying seed abortion in citrus. The results of this study provide novel insights into the molecular mechanism across citrus seed development, and reveal the master role of CrWRKY74 in seed abortion of 'Huagan No. 4'.

“华夷1号”是从“尾张”温州蜜柑芽变中选育出来的新品种。2021年3月通过农业农村部非主要农作物品种登记。成熟时果实果皮色泽呈黄橙色，果皮较光滑，易剥皮，果皮紧致不易浮皮，果实扁圆，果形指数0.81，单果重108 g，含可溶性固形物11.1%，可滴定酸0.59%，维生素C 311.6 mg/kg,

华柑4号是从靖安椪柑(Citrus reticulata Blanco.)芽变中选育出来的无核柑橘新品种.果实扁圆形,果形指数0.80,平均果皮厚度2.0 mm,平均单果质量124.81 g,成熟后果皮黄橙色.可溶性固形物含量(w,后同)为13.2％,可滴定酸含量1.17％,维生素C含量为30.77 mg·100 g-1,平均单果种子数为0.19粒,混栽或单独栽培均接近无籽;品质上.树势强,结果母枝主要以春梢和早秋梢为主,花芽分化能力强,花量中等,自然授粉坐果率5％～8％,丰产稳产.在湖北武汉地区,12月中下旬成熟.

为探究马家柚及原产地周边主要柚类株系遗传关系和来源并筛选出适宜授粉株系,利用SSR分子标记技术对江西省广丰县及周边地区共46份柚资源进行遗传关系鉴定,并对不同杂交授粉组合的马家柚进行连续2a的果实品质相关指标分析.结果表明:马家柚在长期实生繁殖与人工选择过程中发生了明显变异,极有可能由广丰及周边地区的本地土柚经长期自然与人为的双向选择衍变而来;不同杂交授粉组合中以土柚4号对马家柚果实品质提升效果最佳,为目前马家柚较为适宜的授粉父本株系.

“华柑4号”是从靖安椪柑（Citrus reticulata Blanco.）芽变中选育出来的无核柑桔新品种。2021年8月通过非主要农作物品种登记，登记编号为GPD柑橘（2021）420013。该品种果实成熟时果皮色泽呈黄橙色，果皮较薄、较光滑，果皮厚度2.0 mm，易剥皮，油胞较小，果形指数0.80，果实扁圆形，单果重124.81 g，可溶性固形物含量13.2%，可滴定酸含量1.17%，维生素C含量为30.77 mg/100 g，平均单果种子数为0.19粒，混栽或单独栽培均接近无核。

60Co-γ射线是目前辐射诱变育种中最为常见的一种辐射源.综述了60Co-γ射线在果树育种中的应用,包括诱变后的表型、辐射材料、辐射剂量、育种成果、突变体机理研究等,探讨了60Co-γ射线在果树育种中存在的问题,并对60Co-γ辐射技术与杂交育种、植物组织培养、鉴定技术、功能基因组学结合提升育种和分子机制研究效率进行了展望.

This review summarized the research progress of plant epigenetics and fruit tree bud sports selection, and analyzed the classic cases of bud sports selection promoting the high-quality and efficient development of the word's apple and citrus industries. The main results were summarized as follows: (1) Epigenetics caused by DNA methylation and histone modification, etc., was widely involved in various plant growth and development processes and adversity stress responses; (2) The selection of fruit tree bud sports was the best among the best. It had six characteristics, such as high efficiency, reproducibility, stability, diversity and pleiotropy, epigenetic characteristics, and practical effects. At present, more than 600 varieties of fruit tree bud sports have been selected, such as apples and citrus; (3) In response to the problems of the four varieties of Fuji, Delicious, Navel Oranges and Satsuma Mandarin, a series of new varieties have been bred using continuous multi-generation bud sports selection technology, forming a huge variety group and promoting the high-quality and efficient development of the industry. Therefore, two aspects should be further studied in the future: one is to further strengthen the mechanism of fruit tree bud sports and to promote the innovation of breeding technology; The second is to further enhance the awareness of the innovation of fruit tree bud sports selection and to further strengthen the understanding of the importance of the fruit tree industry, and the technical route of combining Mendelian inheritance and epigenetic inheritance should be adopt, combining conventional and molecular technology as well as hybrid breeding and bud sports selection, so as to increase the research efforts of fruit tree bud sports selection and new varieties breeding, and provide varieties support for the high-quality development of the fruit tree industry.

对我国柑橘品种发展现状及变化特点进行了分析,并对产区形成特色与品种定位进行了探讨,同时,对部分热门品种的发展及定位提出了建议.