Phased telomere-to-telomere reference genome and pangenome reveal an expansion of resistance genes during apple domestication.

The cultivated apple (Malus domestica Borkh.) is a cross-pollinated perennial fruit tree of great economic importance. Earlier versions of apple reference genomes were unphased, fragmented, and lacked comprehensive insights into the apple's highly heterozygous genome, which impeded advances in genetic studies and breeding programs. In this study, we assembled a haplotype-resolved telomere-to-telomere (T2T) reference genome for the diploid apple cultivar Golden Delicious. Subsequently, we constructed a pangenome based on 12 assemblies from wild and cultivated species to investigate the dynamic changes of functional genes. Our results revealed the gene gain and loss events during apple domestication. Compared with cultivated species, more gene families in wild species were significantly enriched in oxidative phosphorylation, pentose metabolic process, responses to salt, and abscisic acid biosynthesis process. Our analyses also demonstrated a higher prevalence of different types of resistance gene analogs (RGAs) in cultivars than their wild relatives, partially attributed to segmental and tandem duplication events in certain RGAs classes. Structural variations, mainly deletions and insertions, have affected the presence and absence of TIR-NB-ARC-LRR, NB-ARC-LRR, and CC-NB-ARC-LRR genes. Additionally, hybridization/introgression from wild species has also contributed to the expansion of resistance genes in domesticated apples. Our haplotype-resolved T2T genome and pangenome provide important resources for genetic studies of apples, emphasizing the need to study the evolutionary mechanisms of resistance genes in apple breeding.

New high-quality peach (Prunus persica L. Batsch) genome assembly to analyze the molecular evolutionary mechanism of volatile compounds in peach fruits.

Peach (Prunus persica L. Batsch) is an economically important fruit crop worldwide. Although a high-quality peach genome has previously been published, Sanger sequencing was used for its assembly, which generated short contigs. Here, we report a chromosome-level genome assembly and sequence analysis of Chinese Cling, an important founder cultivar for peach breeding programs worldwide. The assembled genome contained 247.33 Mb with a contig N50 of 4.13 Mb and a scaffold N50 of 29.68 Mb, representing 99.8% of the estimated genome. Comparisons between this genome and the recently published one (Lovell peach) uncovered 685 407 single nucleotide polymorphisms, 162 655 insertions and deletions, and 16 248 structural variants. Gene family analysis highlighted the contraction of the gene families involved in flavone, flavonol, flavonoid, and monoterpenoid biosynthesis. Subsequently, the volatile compounds of 256 peach varieties were quantitated in mature fruits in 2015 and 2016 to perform a genome-wide association analysis. A comparison with the identified domestication genomic regions allowed us to identify 25 quantitative trait loci, associated with seven volatile compounds, in the domestication region, which is consistent with the differences in volatile compounds between wild and cultivated peaches. Finally, a gene encoding terpene synthase, located within a previously reported quantitative trait loci region, was identified to be associated with linalool synthesis. Such findings highlight the importance of this new assembly for the analysis of evolutionary mechanisms and gene identification in peach species. Furthermore, this high-quality peach genome provides valuable information for future fruit improvement.

Small RNA and mRNA Sequencing Reveal the Roles of microRNAs Involved in Pomegranate Female Sterility.

Female sterility is a key factor restricting plant reproduction. Our previous studies have revealed that pomegranate female sterility mainly arose from the abnormality of ovule development. MicroRNAs (miRNAs) play important roles in ovule development. However, little is known about the roles of miRNAs in female sterility. In this study, a combined high-throughput sequencing approach was used to investigate the miRNAs and their targeted transcripts involved in female development. A total of 103 conserved and 58 novel miRNAs were identified. Comparative profiling indicated that the expression of 43 known miRNAs and 14 novel miRNAs were differentially expressed between functional male flowers (FMFs) and bisexual flowers (BFs), 30 known miRNAs and nine novel miRNAs showed significant differences among different stages of BFs, and 20 known miRNAs and 18 novel miRNAs exhibited remarkable expression differences among different stages of FMFs. Gene ontology (GO) analyses of 144 predicted targets of differentially expressed miRNAs indicated that the "reproduction process" and "floral whorl development" processes were significantly enriched. The miRNA-mRNA interaction analyses revealed six pairs of candidate miRNAs and their targets associated with female sterility. Interestingly, pg-miR166a-3p was accumulated, whereas its predicted targets (Gglean012177.1 and Gglean013966.1) were repressed in functional male flowers (FMFs), and the interaction between pg-miR166a-3p and its targets (Gglean012177.1 and Gglean013966.1) were confirmed by transient assay. A. thaliana transformed with 35S-pre-pg-miR166a-3p verified the role of pg-miR166a-3p in ovule development, which indicated pg-miR166a-3p's potential role in pomegranate female sterility. The results provide new insights into molecular mechanisms underlying the female sterility at the miRNA level.

Irregular xylem 7 (IRX7) is required for anchoring seed coat mucilage in Arabidopsis.

Large quantities of mucilage are synthesized in seed coat epidermis cells during seed coat differentiation. This process is an ideal model system for the study of plant cell wall biosynthesis and modifications. In this study, we show that mutation in Irregular Xylem 7 (IRX7) results in a defect in mucilage adherence due to reduced xylan biosynthesis. IRX7 was expressed in the seeds from 4 days post-anthesis (DPA) to 13 DPA, with the peak of expression at 13 DPA. The seed coat epidermis cells of irx7 displayed no aberrant morphology during differentiation, and these cells synthesized and deposited the same amount of mucilage as did wild type (WT) cells. However, the distribution of the water-soluble vs. adherent mucilage layers was significantly altered in irx7 compared to the WT. Both the amount of xylose and the extent of glycosyl linkages of xylan was dramatically decreased in irx7 water-soluble and adherent mucilage compared to the WT. The polymeric structure of water-soluble mucilage was altered in irx7, with a total loss of the higher molecular weight polymer components present in the WT. Correspondingly, whole-seed immunolabeling assays and dot-immunoassays of extracted mucilage indicated dramatic changes in rhamnogalacturonan I (RG I) and xylan epitopes in irx7 mucilage. Furthermore, the crystalline cellulose content was significantly reduced in irx7 mucilage. Taken together, these results indicate that xylan synthesized by IRX7 plays an essential role in maintaining the adhesive property of seed coat mucilage, and its structural role is potentially implemented through its interaction with cellulose.

Functional conservation and divergence of Miscanthus lutarioriparius GT43 gene family in xylan biosynthesis.

BACKGROUND: Xylan is the most abundant un-cellulosic polysaccharides of plant cell walls. Much progress in xylan biosynthesis has been gained in the model plant species Arabidopsis. Two homologous pairs Irregular Xylem 9 (IRX9)/9L and IRX14/14L from glycosyltransferase (GT) family 43 have been proved to play crucial roles in xylan backbone biosynthesis. However, xylan biosynthesis in grass such as Miscanthus remains poorly understood. RESULTS: We characterized seven GT43 members in M. lutarioriparius, a promising bioenergy crop. Quantitative real-time RT-PCR (qRT-PCR) analysis revealed that the expression of MlGT43 genes was ubiquitously detected in the tissues examined. In-situ hybridization demonstrated that MlGT43A-B and MlGT43F-G were specifically expressed in sclerenchyma, while MlGT43C-E were expressed in both sclerenchyma and parenchyma. All seven MlGT43 proteins were localized to Golgi apparatus. Overexpression of MlGT43A-E but not MlGT43F and MlGT43G in Arabidopsis irx9 fully or partially rescued the mutant defects, including morphological changes, collapsed xylem and increased xylan contents, whereas overexpression of MlGT43F and MlGT43G but not MlGT43A-E complemented the defects of irx14, indicating that MlGT43A-E are functional orthologues of IRX9, while MlGT43F and MlGT43G are functional orthologues of IRX14. However, overexpression of all seven MlGT43 genes could not rescue the mucilage defects of irx14 seeds. Furthermore, transient transactivation analyses of MlGT43A-E reporters demonstrated that MlGT43A and MlGT43B but not MlGT43C-E were differentially activated by MlSND1, MlMYB46 or MlVND7. CONCLUSION: The results demonstrated that all seven MlGT43s are functionally conserved in xylan biosynthesis during secondary cell wall formation but diversify in seed coat mucilage xylan biosynthesis. The results obtained provide deeper insight into xylan biosynthesis in grass, which lay the foundation for genetic modification of grass cell wall components and structure to better suit for next-generation biofuel production.

Xylan synthesized by Irregular Xylem 14 (IRX14) maintains the structure of seed coat mucilage in Arabidopsis.

During differentiation, the Arabidopsis seed coat epidermal cells synthesize and secrete large quantities of pectinaceous mucilage into the apoplast, which is then released to encapsulate the seed upon imbibition. In this study, we showed that mutation in Irregular Xylem 14 (IRX14) led to a mucilage cohesiveness defect due to a reduced xylan content. Expression of IRX14 was detected specifically in the seed coat epidermal cells, reaching peak expression at 13 days post-anthesis (DPA) when the accumulation of mucilage polysaccharides has ceased. Sectioning of the irx14-1 seed coat revealed no visible structural change in mucilage secretory cell morphology. Although the total amount of mucilage was comparable with the wild type (WT), the partition between water-soluble and adherent layers was significantly altered in irx14-1, with redistribution from the adherent layer to the water-soluble layer. The monosaccharide composition analysis revealed that xylose content was significantly reduced in irx14-1 water-soluble and adherent mucilage compared with the WT. The macromolecular characteristics of the water-soluble mucilage were modified in irx14-1 with a loss of the larger polymeric components. In accordance, glycome profiling and dot immunoblotting of seed mucilage using antibodies specific for rhamnogalacturonan I (RG I) and xylan confirmed the ultra-structural alterations in the irx14-1 mucilage. Meanwhile, the crystalline cellulose content was reduced in the irx14-1 mucilage. These results demonstrated that IRX14 was required for the biosynthesis of seed mucilage xylan, which plays an essential role in maintaining mucilage architecture potentially through altering the crystallization and organization of cellulose.

Overexpression of a Miscanthus lutarioriparius NAC gene MlNAC5 confers enhanced drought and cold tolerance in Arabidopsis.

KEY MESSAGE: MLNAC5 functions as a stress-responsive NAC transcription factor gene and enhances drought and cold stress tolerance in transgenic Arabidopsis via the ABA-dependent signaling pathway. NAC transcription factors (TFs) play crucial roles in plant responses to abiotic stress. Miscanthus lutarioriparius is one of Miscanthus species native to East Asia. It has attracted much attention as a bioenergy crop because of its superior biomass productivity as well as wide adaptability to different environments. However, the functions of stress-related NAC TFs remain to be elucidated in M. lutarioriparius. In this study, a detailed functional characterization of MlNAC5 was carried out. MlNAC5 was a member of ATAF subfamily and it showed the highest sequence identity to ATAF1. Subcellular localization of MlNAC5-YFP fusion protein in tobacco leaves indicated that MlNAC5 is a nuclear protein. Transactivation assay in yeast cells demonstrated that MlNAC5 functions as a transcription activator and its activation domain is located in the C-terminus. Overexpression of MlNAC5 in Arabidopsis had impacts on plant development including dwarfism, leaf senescence, leaf morphology, and late flowering under normal growth conditions. Furthermore, MlNAC5 overexpression lines in Arabidopsis exhibited hypersensitivity to abscisic acid (ABA) and NaCl. Moreover, overexpression of MlNAC5 in Arabidopsis significantly enhanced drought and cold tolerance by transcriptionally regulating some stress-responsive marker genes. Collectively, our results indicated that MlNAC5 functions as an important regulator during the process of plant development and responses to salinity, drought and cold stresses.

Molecular cloning and expression analysis of 13 NAC transcription factors in Miscanthus lutarioriparius.

KEY MESSAGE: The 13 MlNAC genes could respond to various abiotic stresses, suggesting their crucial roles in stress response. Overexpression of MlNAC2 in Arabidopsis led to improved drought tolerance. NAC (NAM, ATAF1/2 and CUC2) proteins are plant-specific transcription factors that play crucial roles in plant development, growth and stress responses. In this study, 13 stress-responsive NAC genes were identified from Miscanthus lutarioriparius. Full-length cDNA sequences were obtained for 11 MlNAC genes, which were phylogenetically classified into six subfamilies. Sequence alignment revealed the highly conserved NAC domain in the N-terminus of these MlNACs, while the C-terminus was highly divergent. We performed quantitative real-time RT-PCR to examine the expression profiles of MlNAC genes in different tissues including root, rhizome, mature stem, young stem, leaf and sheath. The 13 MlNAC genes displayed distinct tissue-specific patterns in six tissues examined. To gain further insight into their roles in response to abiotic stresses, expressions of MlNAC genes were analyzed under different stresses and hormone treatments including salt, drought, cold, wounding, abscisic acid, Methyl jasmonate and salicylic acid. The 13 MlNAC genes could respond to at least five stress treatments, and over 100-fold variations in transcript levels of MlNAC1, MlNAC2, MlNAC4, and MlNAC12 were observed in salt, drought and MeJA treatments, which indicated that MlNACs play crucial roles in stress response. Crosstalk among various abiotic stress and hormone responses was also discussed based on the expression of MlNAC genes. Overexpression of MlNAC2 in Arabidopsis (Col-0) led to improved drought tolerance. The water loss rate was significantly lower, and the recovery rate after a 60-min dehydration stress treatment was significantly higher in the MlNAC2 overexpression lines than the control.

Comparative population genomics reveals convergent and divergent selection in the apricot-peach-plum-mei complex.

The economically significant genus Prunus includes fruit and nut crops that have been domesticated for shared and specific agronomic traits; however, the genomic signals of convergent and divergent selection have not been elucidated. In this study, we aimed to detect genomic signatures of convergent and divergent selection by conducting comparative population genomic analyses of the apricot-peach-plum-mei (APPM) complex, utilizing a haplotype-resolved telomere-to-telomere (T2T) genome assembly and population resequencing data. The haplotype-resolved T2T reference genome for the plum cultivar was assembled through HiFi and Hi-C reads, resulting in two haplotypes 251.25 and 251.29 Mb in size, respectively. Comparative genomics reveals a chromosomal translocation of ~1.17 Mb in the apricot genomes compared with peach, plum, and mei. Notably, the translocation involves the D locus, significantly impacting titratable acidity (TA), pH, and sugar content. Population genetic analysis detected substantial gene flow between plum and apricot, with introgression regions enriched in post-embryonic development and pollen germination processes. Comparative population genetic analyses revealed convergent selection for stress tolerance, flower development, and fruit ripening, along with divergent selection shaping specific crop, such as somatic embryogenesis in plum, pollen germination in mei, and hormone regulation in peach. Notably, selective sweeps on chromosome 7 coincide with a chromosomal collinearity from the comparative genomics, impacting key fruit-softening genes such as PG, regulated by ERF and RMA1H1. Overall, this study provides insights into the genetic diversity, evolutionary history, and domestication of the APPM complex, offering valuable implications for genetic studies and breeding programs of Prunus crops.

Evolutionary genomics of climatic adaptation and resilience to climate change in alfalfa.

Given the escalating impact of climate change on agriculture and food security, gaining insights into the evolutionary dynamics of climatic adaptation and uncovering climate-adapted variation can empower the breeding of climate-resilient crops to face future climate change. Alfalfa (Medicago sativa subsp. sativa), the queen of forages, shows remarkable adaptability across diverse global environments, making it an excellent model for investigating species responses to climate change. In this study, we performed population genomic analyses using genome resequencing data from 702 accessions of 24 Medicago species to unravel alfalfa's climatic adaptation and genetic susceptibility to future climate change. We found that interspecific genetic exchange has contributed to the gene pool of alfalfa, particularly enriching defense and stress-response genes. Intersubspecific introgression between M. sativa subsp. falcata (subsp. falcata) and alfalfa not only aids alfalfa's climatic adaptation but also introduces genetic burden. A total of 1671 genes were associated with climatic adaptation, and 5.7% of them were introgressions from subsp. falcata. By integrating climate-associated variants and climate data, we identified populations that are vulnerable to future climate change, particularly in higher latitudes of the Northern Hemisphere. These findings serve as a clarion call for targeted conservation initiatives and breeding efforts. We also identified pre-adaptive populations that demonstrate heightened resilience to climate fluctuations, illuminating a pathway for future breeding strategies. Collectively, this study enhances our understanding about the local adaptation mechanisms of alfalfa and facilitates the breeding of climate-resilient alfalfa cultivars, contributing to effective agricultural strategies for facing future climate change.

Identification of key gene networks controlling anthocyanin biosynthesis in peach flower.

Flavonoids, particularly anthocyanin is the main pigment that determined the red color of peach flowers, and help the plant to attract pollinators, protect the reproductive organs of flower from photo-oxidative effects of light and various non-communicable diseases. Through weightage gene coexpression network analysis (WGCNA) we identified a network of 15 hub genes that co-expressed throughout peach flower development including 5 genes coded for the key enzymes (CHI, F3'H, DFR, LAR and UFGT) of flavonoid biosynthetic pathway and 1 gene Prupe.1G111700 identified as R2R3 family transcription factor MYB108. Over expression of PpMYB108 significantly increased anthocyanin biosynthesis in Tobacco flowers. Moreover, the expression correlation between PpMYB108 and PpDFR, suggests that PpMYB108 play the role of transcriptional activator for PpDFR. This was further supported by a 6 bp insertion of MYB biding site in the core promoter region of PpDFR in red flower. The positive interaction of PpMYB108 with PpDFR promoter from red flower was confirmed in yeast one hybrid assay. These findings may be helpful in peach breeding programs as well as in identifying anthocyanin related genes in other species.

Multi-omics approaches identify a key gene, PpTST1, for organic acid accumulation in peach.

Organic acid content in fruit is an important determinant of peach organoleptic quality, which undergoes considerable variations during development and maturation. However, its molecular mechanism remains largely unclear. In this study, an integrative approach of genome-wide association studies and comparative transcriptome analysis were applied to identify candidate genes involved in organic acid accumulation in peach. A key gene PpTST1, encoding tonoplast sugar transporter, was identified and the genotype of PpTST1 with a single-base transversion (G1584T) in the third exon which leads to a single amino acid substitution (Q528H) was associated with low level of organic acid content in peach. Overexpression of PpTST1His resulted in reduced organic acid content along with increased sugar content both in peach and tomato fruits, suggesting its dual function in sugar accumulation and organic acid content reduction. Two V-type proton ATPases interact with PpTST1 in yeast two-hybridization assay. In addition, the G1584T transversion appeared and gradually accumulated during domestication and improvement, which indicated that PpTST1 was under selection. The identification and characterization of PpTST1 would facilitate the improvement of peach fruit quality.

Characterization of a NAC transcription factor involved in the regulation of pomegranate seed hardness (Punica granatum L.).

The pomegranate, Punica granatum L., which has been cultivated since antiquity, is known to be a superfruit, possessing an array of functional anti-oxidants and various other health benefits. The hardness of pomegranate seeds is an important indicator of fruit quality, which in turn affects economic value and market demand. However, the molecular mechanism underlying pomegranate seed hardness remains to be fully understood. In this study, we found a positive correlation between seed hardness and lignin content in two pomegranate varieties: "Tunisia" and "Sanbai". Specifically, genes associated with lignin biosynthesis were differentially expressed in soft-seed and hard-seed pomegranate varieties. Among these differential genes, we cloned and characterized the NAC transcription factor PgSND1-like. Sequence alignment found a single base replacement at the 166-bp position of CDS in the PgSND1-like gene from "Tunisia" and "Sanbai". Both PgSND1-like (Sanbai) and PgSND1-like (Tunisia) proteins are localized in the cell nucleus and have a transcription activation domain in the C-terminus. Yeast two-hybrid analysis indicated that PgSND1-like protein interacts with itself to form a homodimer. Overexpression of PgSND1-like (Sanbai) in Arabidopsis showed a higher lignin content in inflorescence stem and mature seed compared with wild-type Arabidopsis. Accordingly, the expression levels of several lignin biosynthesis-associated genes were upregulated in stem cells and mature seeds of transgenic plants. However, PgSND1-like (Tunisia) transgenic Arabidopsis showed no phenotypic differences with wild-type Arabidopsis. Taken together, we suggest that PgSND1-like may regulate at least two different functions in two pomegranate varieties, promoting lignin biosynthesis and seed hardness of pomegranate.

Miscanthus NAC transcription factor MlNAC12 positively mediates abiotic stress tolerance in transgenic Arabidopsis.

NAC (NAM, ATAF1/2 and CUC2) transcription factors play critical roles in plant abiotic stress responses. However, knowledge regarding the functional roles of NACs in abiotic stress tolerance and its underlying mechanisms is relatively limited in Miscanthus. In this study, we functionally characterized a novel Miscanthus NAC gene MlNAC12 by ectopic expression in Arabidopsis. MlNAC12 was localized in the nucleus. It could specifically binds to the NAC recognition sequence (NACRS) and has a transactivation activity in the C-terminus. Overexpression of MlNAC12 in Arabidopsis conferred hypersensitivity to exogenous Abscisic acid (ABA) at seed germination and root elongation stages. In addition, MlNAC12 overexpression enhanced germination and root growth under salt (NaCl) stress. Furthermore, MlNAC12 overexpression lines exhibited significantly enhanced drought stress tolerance, which was evidenced by a higher survival rate and a lower water loss rate compared to the wild type (WT). Accordingly, the stomata aperture was remarkably reduced in MlNAC12 overexpression lines in comparison to the WT under drought stress. Furthermore, the accumulation of the reactive oxygen species (ROS) and malondialdehyde (MDA) under abiotic stresses was significantly decreased, accompanied by dramatically enhanced activities of several antioxidant enzymes including superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) in the transgenic plants. Correspondingly, the expression of six stress-responsive genes was significantly up-regulated in MlNAC12 overexpression lines. Together, our results indicate that MlNAC12 is a positive regulator of drought and salt stress tolerance through activating ROS scavenging enzymes.

The Miscanthus NAC transcription factor MlNAC9 enhances abiotic stress tolerance in transgenic Arabidopsis.

NAC (NAM, ATAF1/2, and CUC2) transcription factors are known to play important roles in responses to abiotic stresses in plants. Currently, little information regarding the functional roles of NAC genes in stress tolerance is available in Miscanthus lutarioriparius, a promising bioenergy plant for cellulosic ethanol production. In this study, we carried out the functional characterization of MlNAC9 in abiotic stresses. MlNAC9 was shown to act as a nuclear localized transcription activator with the activation domain in its C-terminus. The overexpression of MlNAC9 in Arabidopsis conferred hypersensitivity to abscisic acid (ABA) at seed germination and root elongation stages. In addition, the overexpression of MlNAC9 led to increased seed germination rate and root growth under salt (NaCl) treatment. Meanwhile, the transgenic Arabidopsis overexpressing MlNAC9 showed enhanced tolerance to drought and cold stresses. The expression of stress-responsive marker genes was significantly increased in MlNAC9 overexpression lines compared to that of WT under ABA, drought, salt, and cold stresses. Correspondingly, the activities of antioxidant enzymes superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) were significantly increased and the malondialdehyde (MDA) content was lower accumulated in MlNAC9 overexpression lines under drought and salt treatments. These results indicated that the overexpression of MlNAC9 improved the tolerance to abiotic stresses via an ABA-dependent pathway, and the enhanced tolerance of transgenic plants was mainly attributed to the increased expression of stress-responsive genes and the enhanced scavenging capability of reactive oxygen species (ROS).