Improved de novo chromosome-level genome assembly of the vulnerable walnut tree Juglans mandshurica reveals gene family evolution and possible genome basis of resistance to lesion nematode.

Manchurian walnut (Juglans mandshurica Maxim.) is a synonym of J. cathayensis, a diploid, vulnerable, temperate deciduous tree valued for its wood and nut. It is also valued as a rootstock for Juglans regia because of its reported tolerance of lesion nematode. Reference genomes are available for several Juglans species, our goal was to produce a de novo, chromosome-level assembly of the J. mandshurica genome. Here, we reported an improved assembly of J. mandshurica with a contig N50 size of 6.49 Mb and a scaffold N50 size of 36.1 Mb. The total genome size was 548 Mb encoding 29,032 protein coding genes which were annotated. The collinearity analysis showed that J. mandshurica and J. regia originated from a common ancestor, with both species undergoing two WGD events. A genomic comparison showed that J. mandshurica was missing 1657 genes found in J. regia, and J. mandshurica includes 2827 genes not found in of the J. regia genome. The J. mandshurica contained 1440 unique paralogues that were highly enriched for flavonoid biosynthesis, phenylpropanoid biosynthesis, and plant-pathogen interaction. Four gene families related to disease resistance notable contraction (rapidly evolving; LEA, WAK, PPR, and PR) in J. mandshurica compared to eight species. JmaPR10 and JmaPR8 contained three orthologous gene pairs with J. regia that were highly expressed in root bark. JmaPR10 is a strong candidate gene for lesion nematodes resistance in J. mandshurica. The J. mandshurica genome should be a useful resource for study of the evolution, breeding, and genetic variation in walnuts (Juglans).

Yan F ，Xi RM ，She RX ，Chen PP ，Yan YJ ，Yang G ，Dang M ，Yue M ，Pei D ，Woeste K ，Zhao P ... -  《-》

Genome-Wide Identification and Transcriptional Expression Profiles of Transcription Factor WRKY in Common Walnut (Juglans regia L.).

Hao F ，Yang G ，Zhou H ，Yao J ，Liu D ，Zhao P ，Zhang S ... -  《Genes》

Genome-wide identification analysis of the 4-Coumarate: CoA ligase (4CL) gene family expression profiles in Juglans regia and its wild relatives J. Mandshurica resistance and salt stress.

Persian walnut (Juglans regia) and Manchurian walnut (Juglans mandshurica) belong to Juglandaceae, which are vulnerable, temperate deciduous perennial trees with high economical, ecological, and industrial values. 4-Coumarate: CoA ligase (4CL) plays an essential function in plant development, growth, and stress. Walnut production is challenged by diverse stresses, such as salinity, drought, and diseases. However, the characteristics and expression levels of 4CL gene family in Juglans species resistance and under salt stress are unknown. Here, we identified 36 Jr4CL genes and 31 Jm4CL genes, respectively. Based on phylogenetic relationship analysis, all 4CL genes were divided into three branches. WGD was the major duplication mode for 4CLs in two Juglans species. The phylogenic and collinearity analyses showed that the 4CLs were relatively conserved during evolution, but the gene structures varied widely. 4CLs promoter region contained multiply cis-acting elements related to phytohormones and stress responses. We found that Jr4CLs may be participated in the regulation of resistance to anthracnose. The expression level and some physiological of 4CLs were changed significantly after salt treatment. According to qRT-PCR results, positive regulation was found to be the main mode of regulation of 4CL genes after salt stress. Overall, J. mandshurica outperformed J. regia. Therefore, J. mandshurica can be used as a walnut rootstock to improve salt tolerance. Our results provide new understanding the potential functions of 4CL genes in stress tolerance, offer the theoretical genetic basis of walnut varieties adapted to salt stress, and provide an important reference for breeding cultivated walnuts for stress tolerance.

Ma J ，Zuo D ，Zhang X ，Li H ，Ye H ，Zhang N ，Li M ，Dang M ，Geng F ，Zhou H ，Zhao P ... -  《BMC PLANT BIOLOGY》

The Manchurian Walnut Genome: Insights into Juglone and Lipid Biosynthesis.

Manchurian walnut (Juglans mandshurica Maxim.) is a tree with multiple industrial uses and medicinal properties in the Juglandaceae family (walnuts and hickories). J. mandshurica produces juglone, which is a toxic allelopathic agent and has potential utilization value. Furthermore, the seed of J. mandshurica is rich in various unsaturated fatty acids and has high nutritive value. Here, we present a high-quality chromosome-scale reference genome assembly and annotation for J. mandshurica (n = 16) with a contig N50 of 21.4 Mb by combining PacBio high-fidelity reads with high-throughput chromosome conformation capture data. The assembled genome has an estimated sequence size of 548.7 Mb and consists of 657 contigs, 623 scaffolds, and 40,453 protein-coding genes. In total, 60.99% of the assembled genome consists of repetitive sequences. Sixteen super-scaffolds corresponding to the 16 chromosomes were assembled, with a scaffold N50 length of 33.7 Mb and a BUSCO complete gene percentage of 98.3%. J. mandshurica displays a close sequence relationship with Juglans cathayensis, with a divergence time of 13.8 million years ago. Combining the high-quality genome, transcriptome, and metabolomics data, we constructed a gene-to-metabolite network and identified 566 core and conserved differentially expressed genes, which may be involved in juglone biosynthesis. Five CYP450 genes were found that may contribute to juglone accumulation. NAC, bZip, NF-YA, and NF-YC are positively correlated with the juglone content. Some candidate regulators (e.g., FUS3, ABI3, LEC2, and WRI1 transcription factors) involved in the regulation of lipid biosynthesis were also identified. Our genomic data provide new insights into the evolution of the walnut genome and create a new platform for accelerating molecular breeding and improving the comprehensive utilization of these economically important tree species.

Li X ，Cai K ，Zhang Q ，Pei X ，Chen S ，Jiang L ，Han Z ，Zhao M ，Li Y ，Zhang X ，Li Y ，Zhang S ，Chen S ，Qu G ，Tigabu M ，Chiang VL ，Sederoff R ，Zhao X ... -  《GigaScience》

Genome-wide identification, characterization, and expression pattern of the late embryogenesis abundant (LEA) gene family in Juglans regia and its wild relatives J. mandshurica.

Late Embryogenesis Abundant (LEA) proteins are a class of proteins associated with plant stress resistance. Two Juglans species, Juglans regia and J. mandshurica, are both diploid (2n = 32), monoecious perennial economic tree species with high edible, pharmaceutical, and timber value. The identification, characterization, and expression patterns of LEA proteins in J. regia and its wild relative, J. mandshurica, would not only provide the genetic basis of this gene family, but it would also supply clues for further studies of the evolution and regulating mechanisms of LEA proteins in other tree species. In this study, we identified 25 and 20 members of the LEA gene family in Juglans regia and its wild relative, Juglans mandshurica, respectively. The results of phylogenetic analysis showed that the LEA members were divided into eight main subgroups. Predictions of their physicochemical properties showed the variable characteristics of LEA proteins, and the subcellular localization analysis indicated that most LEA proteins are localized in the nucleus. Chromosomal localization analysis and gene replication pattern prediction indicated that WGD is the predominant duplication mode of LEA genes. The results of the comparative analysis indicated a high level of collinearity between the two Juglans species. Analysis of cis-acting elements indicated that LEA genes had a relatively wide range of responses to abiotic stresses and phytohormonal processes, particularly in two phytohormones, methyl jasmonate and abscisic acid. Transcriptome profiling and qRT-PCR experiments showed that JrLEAs are commonly expressed in leaves, green husks, and male and female flowers, and most JmLEAs are more highly expressed in male flowers. We also hypothesized that JrLEAs are involved in the process of anthracnose resistance. Anthracnose-resistant varieties of JrLEAs presented relatively high expression levels at later stages. In this study, we provide a theoretical basis for the functional study of LEA genes in J. regia and J. mandshurica. Analysis of cis-acting elements and gene expression indicated that JrLEAs and JmLEAs play important roles in resistance to biotic stresses in these species.

Ma J ，Zuo D ，Ye H ，Yan Y ，Li M ，Zhao P ... -  《BMC PLANT BIOLOGY》