Genome-Wide Characterization and Functional Validation of the ACS Gene Family in the Chestnut Reveals Its Regulatory Role in Ovule Development.

Ovule abortion significantly contributes to a reduction in chestnut yield. Therefore, an examination of the mechanisms underlying ovule abortion is crucial for increasing chestnut yield. In our previous study, we conducted a comprehensive multiomic analysis of fertile and abortive ovules and found that ACS genes in chestnuts (CmACS) play a crucial role in ovule development. Therefore, to further study the function of ACS genes, a total of seven CmACS members were identified, their gene structures, conserved structural domains, evolutionary trees, chromosomal localization, and promoter cis-acting elements were analyzed, and their subcellular localization was predicted and verified. The spatiotemporal specificity of the expression of the seven CmACS genes was confirmed via qRT-PCR analysis. Notably, CmACS7 was exclusively expressed in the floral organs, and its expression peaked during fertilization and decreased after fertilization. The ACC levels remained consistently greater in fertile ovules than in abortive ovules. The ACSase activity of CmACS7 was identified using the genetic transformation of chestnut healing tissue. Micro Solanum lycopersicum plants overexpressing CmACS7 had a significantly greater rate of seed failure than did wild-type plants. Our results suggest that ovule fertilization activates CmACS7 and increases ACC levels, whereas an overexpression of CmACS7 leads to an increase in ACC content in the ovule prior to fertilization, which can lead to abortion. In conclusion, the present study demonstrated that chestnut ovule abortion is caused by poor fertilization and not by nutritional competition. Optimization of the pollination and fertilization of female flowers is essential for increasing chestnut yield and reducing ovule abortion.

Assembly and analysis of the genome of Notholithocarpus densiflorus.

Tanoak (Notholithocarpus densiflorus) is an evergreen tree in the Fagaceae family found in California and southern Oregon. Historically, tanoak acorns were an important food source for Native American tribes, and the bark was used extensively in the leather tanning process. Long considered a disjunct relictual element of the Asian stone oaks (Lithocarpus spp.), phylogenetic analysis has determined that the tanoak is an example of convergent evolution. Tanoaks are deeply divergent from oaks (Quercus) of the Pacific Northwest and comprise a new genus with a single species. These trees are highly susceptible to "sudden oak death" (SOD), a plant pathogen (Phytophthora ramorum) that has caused widespread deaths of tanoaks. In this study, we set out to assemble the genome and perform comparative studies among a number of individuals that demonstrated varying levels of susceptibility to SOD. First, we sequenced and de novo assembled a draft reference genome of N. densiflorus using cobarcoded library processing methods and an MGI DNBSEQ-G400 sequencer. To increase the contiguity of the final assembly, we also sequenced Oxford Nanopore long reads to 30× coverage. To our knowledge, the draft genome reported here is one of the more contiguous and complete genomes of a tree species published to date, with a contig N50 of ∼1.2 Mb, a scaffold N50 of ∼2.1 Mb, and a complete gene score of 95.5% through BUSCO analysis. In addition, we sequenced 11 genetically distinct individuals and mapped these onto the draft reference genome, enabling the discovery of almost 25 million single nucleotide polymorphisms and ∼4.4 million small insertions and deletions. Finally, using cobarcoded data, we were able to generate a complete haplotype coverage of all 11 genomes.

Draft genome of Castanopsis chinensis, a dominant species safeguarding biodiversity in subtropical broadleaved evergreen forests.

OBJECTIVES: Castanopsis is the third largest genus in the Fagaceae family and is essentially tropical or subtropical in origin. The species in this genus are mainly canopy-dominant trees, and the key components of evergreen broadleaved forests play a crucial role in the maintenance of local biodiversity. Castanopsis chinensis, distributed from South China to Vietnam, is a representative species. It currently suffers from a high disturbance of human activity and climate change. Here, we present its assembled genome to facilitate its preliminary conservation and breeding on the genome level. DATA DESCRIPTION: The C. chinensis genome was assembled and annotated by Nanopore and MGI whole-genome sequencing and RNA-seq reads using leaf tissues. The assembly was 888,699,661 bp in length, consisting of 133 contigs and a contig N50 of 23,395,510 bp. A completeness assessment of the assembly with Benchmarking Universal Single-Copy Orthologs (BUSCO) indicated a score of 98.3%. Repetitive elements comprised 471,006,885 bp, accounting for 55.9% of the assembled sequences. A total of 51,406 genes that coded for 54,310 proteins were predicted. Multiple databases were used to functionally annotate the protein sequences.

Development of polymorphic microsatellite markers for Fagus pashanica (Fagaceae) using next-generation sequencing.

Fagus pashanica is an endangered and endemic tree species in China. To understand its genetic diversity and structure for effective conservation, we used next-generation sequencing data to develop a set of microsatellite markers. Twenty-three of the 68 designed loci were successfully amplified. Fifteen polymorphic loci with clear peaks were selected for further analyses in three F. pashanica populations sampled from Nanjiang, Wangcang and Pingwu counties in Sichuan Province, China. The number of alleles per locus ranged from two to 11. The levels of observed and expected heterozygosity ranged from 0.033-0.852 and 0.033-0.787, respectively. All 23 loci were also successfully amplified in F. longipetiolata and F. lucida, and 19 were successfully amplified in F. engleriana. These microsatellite markers will be useful for population genetic studies of F. pashanica and other Fagus species.

Use of retrotransposon based iPBS markers for determination of genetic relationship among some Chestnut Cultivars (Castanea sativa Mill.) in Türkiye.

BACKGROUND: The aim of this study was to reveal the genetic relationships among some economically important chestnut cultivars for Türkiye by using retrotransposon-based inter primer binding site (iPBS) markers. METHODS AND RESULTS: In this study, a total of 19 iPBS markers were used to determine the genetic relationships among 11 chestnut cultivars (Castanea sativa Mill.). In the study, chestnut cultivars named Haciömer, Osmanoglu, Sariaslama, Erfelek, Kemer, Isiklar, Sekerci, Siyah Bursa, Tülü, Bouche De Betizac and Marigoule were the preferred cultivars utilised. Using the online marker efficiency calculator (iMEC), some indices of polymorphism, such as the mean heterozygosity, polymorphism information content, marker index and discriminating power, were determined. In addition, the size ranges of alleles, number of average alleles, number of total alleles, number of polymorphic alleles, and polymorphism rate were determined at a successful level. The chestnut cultivars of Haciömer and Sekerci were determined to be the most similar cultivars with a similarity coefficient value of 0.924, and they formed a subgroup together with the chestnut cultivars Osmanoglu and Erfelek, showing close similarity with these two cultivars. CONCLUSIONS: The use of iPBS markers in chestnuts in Türkiye was carried out for the first time in this study. The power of iPBS markers to evaluate the genetic relationship for our preferred chestnut cultivars was revealed. For this reason, it has emerged that it will be useful in the molecular characterization of both genotypes in natural chestnut populations and chestnut breeding materials such as varieties and cultivars in chestnut breeding programs.

Distribution, causal agents, and infection dynamic of emerging ink disease of sweet chestnut in Southern Switzerland.

Emerging diseases caused by both native and exotic pathogens represent a main threat to forest ecosystems worldwide. The two invasive soilborne pathogens Phytophthora cinnamomi and Phytophthora × cambivora are the causal agents of ink disease, which has been threatening Castanea sativa in Europe for several centuries and seems to be re-emerging in recent years. Here, we investigated the distribution, causal agents, and infection dynamics of ink disease in southern Switzerland. A total of 25 outbreaks were identified, 19 with only P. cinnamomi, 5 with only P. × cambivora, and 1 with both species. Dendrochronological analyses showed that the disease emerged in the last 20-30 years. Infected trees either died rapidly within 5-15 years post-infection or showed a prolonged state of general decline until death. Based on a generalized linear model, the local risk of occurrence of ink disease was increased by an S-SE aspect of the chestnut stand, the presence of a pure chestnut stand, management activities, the proximity of roads and buildings, and increasing annual mean temperature and precipitation. The genetic structure of the local P. cinnamomi population suggests independent introductions and local spread of the pathogen.

Validation of an Alternative Small Stem Assay for Blight Resistance in Chestnut Seedlings and Recommendations for Broader Use.

We evaluated an alternative small stem assay (AltSSA) for blight resistance in backcross hybrid chestnut trees (Castanea dentata/mollissima). Whereas standard small stem assays (SSAs) are done by inoculating small incisions in stems, in our AltSSA, 4- to 5-mm stems are cut off, and the exposed (living) stem tips are inoculated with discs of Cryphonectria parasitica inoculum and temporarily covered with plastic sleeves. Intended primarily for forward selection, this method was designed to be easy to implement, to consistently induce cankering, and to better enable seedling recovery via the development of lateral shoots from the lower stem. After 90+ days, cankers are evaluated and removed, and seedlings are prepared for out-planting. Previous results showed that AltSSAs performed at least as well as a common SSA method in distinguishing resistant and susceptible types. In this follow-up analysis of 35 lines of backcross seedlings studied in 2020 and 2021, we showed that mean orange zone canker length (OZCL) and a multifactor principal components analysis-based blight resistance index gave results consistent with predictions derived from two methods of blight resistance phenotyping and percentage of American chestnut ancestry of the parents of each line. As expected, based upon the apparent polygenic inheritance of blight resistance in backcross chestnut trees, mean OZCL of backcross families ranged from intermediate (F1 hybrid-level) to low (wild-type American chestnut-level). Consistent with prior results, canker production was near 100%, survivorship after out-planting was very high, and postinoculation stem dieback was not apparently related to the stem tip inoculations. Altogether, these results suggest that the AltSSA is a viable method for early detection of relative blight resistance in seedlings and may enable a reduction in the numbers of trees out-planted and placed under care for long-term evaluation and breeding. Thus, the AltSSA can prevent time, resources, and orchard space from being used on susceptible trees.

Genetic Diversity and Population Structure Analysis of Castanopsis hystrix and Construction of a Core Collection Using Phenotypic Traits and Molecular Markers.

Castanopsis hystrix is a valuable native, broad-leaved, and fast-growing tree in South China. In this study, 15 phenotypic traits and 32 simple sequence repeat (SSR) markers were used to assess the genetic diversity and population structure of a natural population of C. hystrix and to construct a core germplasm collection by a set of 232 accessions. The results showed that the original population of C. hystrix had relatively high genetic diversity, with the number of alleles (Na), effective number of alleles (Ne), observed heterozygosity (Ho), expected heterozygosity (He), Shannon's information index (I), and polymorphism information content (PIC) averaging at 26.188, 11.565, 0.863, 0.897, 2.660, and 0.889, respectively. Three sub-populations were identified based on a STRUCTURE analysis, indicating a strong genetic structure. The results from the phylogenetic and population structures showed a high level of agreement, with 232 germplasms being classified into three main groups. The analysis of molecular variance (AMOVA) test indicated that 96% of the total variance was derived from within populations, which revealed a low differentiation among populations. A core collection composed of 157 germplasms was firstly constructed thereafter, of which the diversity parameters non-significantly differed from the original population. These results revealed the genetic diversity and population structure of C. hystrix germplasms, which have implications for germplasm management and genome-wide association studies on C. hystrix, as well as for core collection establishment applications in other wood-producing hardwood species.

Gene flow between wild trees and cultivated varieties shapes the genetic structure of sweet chestnut (Castanea sativa Mill.) populations.

Gene flow between cultivated and wild gene pools is common in the contact zone between agricultural lands and natural habitats and can be used to study the development of adaptations and selection of novel varieties. This is likely the case in the northern Adriatic region, where centuries-old cultivated orchards of sweet chestnut (Castanea sativa Mill.) are planted within the natural distribution area of the species. Thus, we investigated the population structure of several orchards of sweet chestnuts. Furthermore, the genetic background of three toponymous clonal varieties was explored. Six genomic simple sequence repeat (gSSR) and nine EST-derived SSR (EST-SSR) loci were utilized in this research, and both grafted and non-grafted individuals were included in this study. Five closely related clones were identified, which represent a singular, polyclonal marron variety, found in all three cultivation areas. Furthermore, many hybrids, a result of breeding between cultivated and wild chestnuts, have been found. Analyzed semi-wild orchards defined by a diverse genetic structure, represent a hotspot for further selection and could result in creation of locally adapted, high-yielding varieties.

The Use of qPCR to Detect Cryphonectria parasitica in Plants.

Cryphonectria parasitica is a fungal pathogen that causes lethal bark necrosis in chestnut. A duplex qPCR allowing detection of the pathogen and its host, Castanea sativa, is described. The method can be used for early detection of the pathogen in chestnut bark tissues with an internal control of false-negative results caused by PCR inhibitors and/or DNA extraction failure. A positive amplification control of qPCR that allows detection of any deviation from a normal qPCR run based on a control chart is also described. As C. parasitica is a regulated pathogen in Europe, the protocol also provides information on the way to collect and handle bark samples to fulfil biosecurity rules.

Frozen in time: Rangewide genomic diversity, structure, and demographic history of relict American chestnut populations.

American chestnut (Castanea dentata) was once the most economically and ecologically important hardwood species in the eastern United States. In the first half of the 20th century, an exotic fungal pathogen-Cryphonectria parasitica-decimated the species, killing billions of chestnut trees. Two approaches to developing blight-resistant American chestnut populations show promise, but both will require introduction of adaptive genomic diversity from wild germplasm to produce diverse, locally adapted restoration populations. Here we characterize population structure, demographic history, and genomic diversity in a range-wide sample of 384 wild American chestnuts to inform conservation and breeding with blight-resistant varieties. Population structure analyses suggest that the chestnut range can be roughly divided into northeast, central, and southwest populations. Within-population genomic diversity estimates revealed a clinal pattern with the highest diversity in the southwest, which likely reflects bottleneck events associated with Quaternary glaciation. Finally, we identified genomic regions under positive selection within each population, which suggests that defence against fungal pathogens is a common target of selection across all populations. Taken together, these results show that American chestnut underwent a postglacial expansion from the southern portion of its range leading to three extant genetic populations. These populations will serve as management units for breeding adaptive genetic variation into the blight-resistant tree populations for targeted reintroduction efforts.

Transcriptome Analysis Reveals the Regulatory Networks of Cytokinin in Promoting Floral Feminization in Castanea henryi.

Castanea henryi is a monoecious plant with a low female-to-male ratio, which limits its yield. The phytohormone cytokinin (CK) plays a crucial role in flower development, especially gynoecium development. Here, the feminizing effect of CK on the development of C. henryi was confirmed by the exogenous spraying of N-(2-chloro-4-pyridyl)-N'-phenylurea (CPPU). Spraying CPPU at 125 mg·L-1 thrice changed the male catkin into a pure female catkin, whereas at 5 mg·L-1 and 25 mg·L-1, only a part of the male catkin was transformed into a female catkin. A comparative transcriptome analysis of male catkins subjected to CPPU was performed to study the mechanism of the role of CKs in sex differentiation. Using Pearson's correlation analysis between hormone content and hormone synthesis gene expression, four key genes, LOG1, LOG3, LOG7 and KO, were identified in the CK and GA synthesis pathways. Moreover, a hub gene in the crosstalk between JA and the other hormone signaling pathways, MYC2, was identified, and 15 flowering-related genes were significantly differentially expressed after CPPU treatment. These results suggest that CK interacts with other phytohormones to determine the sex of C. henryi, and CK may directly target floral organ recognition genes to control flower sex.

Chromosome-scale genome assembly of Castanopsis tibetana provides a powerful comparative framework to study the evolution and adaptation of Fagaceae trees.

Fagaceae species are increasingly used as models to elucidate the process and mechanism of adaptation and speciation by integrating ecology, evolution and genomics. The genus Castanopsis belongs to the family Fagaceae and is mainly distributed across subtropical and tropical Asia. In the present study, we reported the first chromosome-scale genome assembly of Castanopsis tibetana, a common species of evergreen broadleaved forests in subtropical China. The combination of Nanopore sequencing and Hi-C technologies enabled a high-quality genome assembly. The final assembled genome size of C. tibetana was 878.6 Mb (97.6% of the estimated genome size), consisting of 477 contigs with an N50 length of 3.3 Mb. The benchmarking universal single-copy orthologue (BUSCO) assessment indicated a completeness of 93.0%. Hi-C scaffolding generated 12 pseudochromosomes, representing 98.7% of the assembled genome. Subsequently, 40,937 protein-coding genes were predicted and 90.04% of them were functionally annotated. More than 476.9 Mb of repetitive sequences (54.3% of the genome) were identified, and the percentage of the genome covered by TE elements was 39.98%. Comparative genomics analysis revealed that C. tibetana was most closely related to Castanea mollissima and diverged at 18.48 Ma, and that C. tibetana has undergone considerable gene family expansion and contraction. Evidence of positive selection was detected in 53 genes, which showed different arrangement pattern compared to Quercus robur. The chromosome-scale genome assembly of C. tibetana will expand Fagaceae genome resources across the family and provide a powerful comparative framework to study the adaptation and evolution of Fagaceae trees.

Pathogen-induced expression of a blight tolerance transgene in American chestnut.

American chestnut (Castanea dentata) is a susceptible host of the invasive necrotrophic fungus Cryphonectria parasitica, which causes chestnut blight disease. The fungal pathogen attacks chestnut stems by invading wounded tissue and secreting oxalate. This process leads to the death of infected host cells and the formation of cankers, eventually girdling stems and killing the tree above the infections. To reduce damage caused by fungal oxalate, American chestnut has been genetically engineered to express a wheat oxalate oxidase (OxO). This enzyme degrades the oxalate produced by the pathogen and confers elevated tolerance to Cryphonectria parasitica infection. We report new lines of transgenic American chestnut that have been developed with the win3.12 inducible promoter from poplar (Populus deltoides) driving OxO expression. This promoter is responsive to both wounding and pathogen infection, with a low level of baseline expression. Targeted expression of OxO to wounded and infected tissue is sought as an alternative to constitutive expression for potential metabolic resource conservation and transgene stability over the long lifetime of a tree and over successive generations of breeding. Transgenic Castanea dentata lines harbouring the win3.12-OxO construct were evaluated for transgene expression patterns and tolerance to chestnut blight infection. OxO transcript levels were low in uninfected plants, but robust infection-induced expression levels were observed, with one transgenic line reaching levels comparable to those of previously characterized CaMV35S-OxO lines. In chestnut blight infection bioassays, win3.12-OxO lines showed elevated disease tolerance similar to blight-resistant Chinese chestnut (Castanea mollissima) controls.

Evolution of Castanea in North America: restriction-site-associated DNA sequencing and ecological modeling reveal a history of radiation, range shifts, and disease.

PREMISE: Although chestnuts and chinquapins are some of the best known and most widely loved of any plants in North America, relatively little genomic sequencing has been done, and much is still unknown about their evolution. METHODS: We used double-digest restriction-site-associated DNA (ddRAD) sequencing data to infer the species-level phylogeny for Castanea and assess the phylogeography of the North American species using samples collected from populations that span the full extent of the species' ranges. We also constructed species distribution models using digitized herbarium specimens and observational data from field surveys. RESULTS: We identified strong population structure within Castanea dentata (American chestnut) that reflects a stepwise northern migration since the last glacial maximum. Our species distribution models further confirmed this scenario and matched closely with the Castanea fossil pollen record. We also found significant structure within the Castanea pumila lineage, most notably a genetic cluster that corresponds to the frequently recognized Castanea pumila var. ozarkensis. CONCLUSIONS: The two North American Castanea species have contrasting patterns of population structure, but each is typical of plant phylogeography in North America. Within the C. pumila complex, we found novel genetic structure that provides new insights about C. pumila taxonomy. Our results also identified a series of distinctive populations that will be valuable in ongoing efforts to conserve and restore chestnuts and chinquapins in North America.

Chromosome-level genome assembly of Japanese chestnut (Castanea crenata Sieb. et Zucc.) reveals conserved chromosomal segments in woody rosids.

Japanese chestnut (Castanea crenata Sieb. et Zucc.), unlike other Castanea species, is resistant to most diseases and wasps. However, genomic data of Japanese chestnut that could be used to determine its biotic stress resistance mechanisms have not been reported to date. In this study, we employed long-read sequencing and genetic mapping to generate genome sequences of Japanese chestnut at the chromosome level. Long reads (47.7 Gb; 71.6× genome coverage) were assembled into 781 contigs, with a total length of 721.2 Mb and a contig N50 length of 1.6 Mb. Genome sequences were anchored to the chestnut genetic map, comprising 14,973 single nucleotide polymorphisms (SNPs) and covering 1,807.8 cM map distance, to establish a chromosome-level genome assembly (683.8 Mb), with 69,980 potential protein-encoding genes and 425.5 Mb repetitive sequences. Furthermore, comparative genome structure analysis revealed that Japanese chestnut shares conserved chromosomal segments with woody plants, but not with herbaceous plants, of rosids. Overall, the genome sequence data of Japanese chestnut generated in this study is expected to enhance not only its genetics and genomics but also the evolutionary genomics of woody rosids.

Adaptive Evolution of Chalcone Isomerase Superfamily in Fagaceae.

Chalcone Isomerase (CHI) catalyzes the biosynthesis of flavonoids and secondary metabolism in plants. Currently, there is no systematic analysis of CHIs gene family in Fagaceae which is available. In this study, twenty-two CHI proteins were identified in five species of the Fagaceae family. The CHI superfamily in Fagaceae can be classified into three subfamilies and five groups using phylogenetic analysis, analysis of physicochemical properties, and structural prediction. Results indicated that serine (Ser) and isoleucine (Ile) residues determine the substrate preferred by active Type I Fagaceae CHI, and the chalcone isomerase-like (CHIL) of Fagaceae had active site residues. Adaptive analysis of CHIs showed that CHIs are subject to selection pressure. The active CHI gene of Fagaceae was located in the cytoplasm, and it had the typical gene structure of CHI and contains four exons. All the twenty-two identified CHIs had the conserved domain motif 3, and the different groups had their own structural characteristics. In the process of fatty acid binding protein (FAP) evolution to CHIL and CHI, the physical and chemical properties of proteins also had significant differences in addition to changes in protein functions.

Plastome Characterization and Phylogenomics of East Asian Beeches with a Special Emphasis on Fagus multinervis on Ulleung Island, Korea.

Beech trees of the genus Fagus (Fagaceae) are monoecious and distributed in the Northern Hemisphere. They represent an important component of mixed broad-leaved evergreen-deciduous forests and are an economically important source of timber. Despite their ecological and economical importance, however, little is known regarding the overall plastome evolution among Fagus species in East Asia. In particular, the taxonomic position and status of F. multinervis, a beech species endemic to Ulleung Island of Korea, remains unclear even today. Therefore, in this study, we characterized four newly completed plastomes of East Asian Fagus species (one accession each of F. crenata and F. multinervis and two accessions of F. japonica). Moreover, we performed phylogenomic analyses comparing these four plastomes with F. sylvatica (European beech) plastome. The four plastomes were highly conserved, and their size ranged from 158,163 to 158,348 base pair (bp). The overall GC content was 37.1%, and the sequence similarity ranged from 99.8% to 99.99%. Codon usage patterns were similar among species, and 7 of 77 common protein-coding genes were under positive selection. Furthermore, we identified five highly variable hotspot regions of the Fagus plastomes (ccsA/ndhD, ndhD/psaC, ndhF/rpl32, trnS-GCU/trnG-UCC, and ycf1). Phylogenetic analysis revealed the monophyly of Fagus as well as early divergence of the subgenus Fagus and monophyletic Engleriana. Finally, phylogenetic results supported the taxonomic distinction of F. multinervis from its close relatives F. engleriana and F. japonica. However, the sister species and geographic origin of F. multinervis on Ulleung Island could not be determined.

Construction of Pseudomolecules for the Chinese Chestnut (Castanea mollissima) Genome.

The Chinese chestnut (Castanea mollissima Bl.) is a woody nut crop with a high ecological value. Although many cultivars have been selected from natural seedlings, elite lines with comprehensive agronomic traits and characters remain rare. To explore genetic resources with aid of whole genome sequence will play important roles in modern breeding programs for chestnut. In this study, we generated a high-quality C. mollissima genome assembly by combining 90× Pacific Biosciences long read and 170× high-throughput chromosome conformation capture data. The assembly was 688.93 Mb in total, with a contig N50 of 2.83 Mb. Most of the assembled sequences (99.75%) were anchored onto 12 chromosomes, and 97.07% of the assemblies were accurately anchored and oriented. A total of 33,638 protein-coding genes were predicted in the C. mollissima genome. Comparative genomic and transcriptomic analyses provided insights into the genes expressed in specific tissues, as well as those associated with burr development in the Chinese chestnut. This highly contiguous assembly of the C. mollissima genome provides a valuable resource for studies aiming at identifying and characterizing agronomical-important traits, and will aid the design of breeding strategies to develop more focused, faster, and predictable improvement programs.