Review Article Genomics: cracking the mysteries of walnuts Fei Chen1*#, Junhao Chen2*, Zhengjia Wang2, Jiawei Zhang1, Meigui Lin1, Liangsheng Zhang1# 1State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops; Key Laboratory of Genetics, Breeding and Multiple Utilization of Corps (Fujian Agriculture and Forestry University), Ministry of Education, Fujian Provincial Key Laboratory of Haixia Applied Plant Systems Biology, Fujian Agriculture and Forestry University, Fuzhou 350002, China 2State Key Laboratory of Subtropical Silviculture, School of Forestry and Biotechnology, Zhejiang Agriculture and Forestry University, Hangzhou, 311300, China *Co-first author #Co-corresponding authors: Fei Chen, E-mail: [email protected]; Liangsheng Zhang, E-mail: [email protected] Abstract The Juglans plants are economically important by providing nuts, wood, and garden trees. They also play an important ecological role by supplying food for wild insects and animals. The decoding of genome sequences has fundamental values for understanding the evolution of Juglans plants and molecules, and is also a prerequisite for molecular breeding. During the last three years, the rapid development of sequencing technology has made walnut research into the genome era. Here, we reviewed the progress of genome sequencing of six Juglans species, the resequencing of four Juglans populations, as well as the genome sequencing of the closely related species Pterocarpa stenoptera. The analysis of the J. regia genome uncovers a whole genome duplication event. Based on the molecular dating of the divergence time of six Juglans species, we proposed this whole genome duplication event was associated with the cretaceous-Palaeogene (K-Pg) boundary happened ~65 million-year ago. Genomic sequences also provide clear details for understanding the evolution and development of GGT and PPO genes involved in fruit development. The decoding of these genomes has made it easier for us to understand and speed up the use of walnuts. We expect that the functional genomics research of walnut research will also develop rapidly in the near future. Keywords: walnut (Juglans spp.); genome sequencing; resequencing; divergence time; whole genome duplication; fruit development Genome sequencing advances The genus Juglans covers 7 subgenera and 59 species with worldwide distributions. Several Juglans species provide delicious nuts and hard, dense, tight-grained timber, thereby are economic valuable trees. The genome sequences of Juglans species are vital for basic researches, molecular breeding, conservation, and cultivar barcoding. In 2016, Martínez‐García and colleagues from University of California, Davis, reported the first Persian walnut genome sequence of J. regia (Martínez-García et al. 2016). In 2018, Stevens and colleagues from the same university reported the six walnut genomes, including J. regia, Juglans nigra, Juglans hindsii, Juglans microcarpa, Juglans sigillata, Juglans cathayensis, together with an outgroup, Chinese wingnut (Pterocarya stenoptera) (Juglandoideae) (Stevens et al. 2018). Luo et al. constructed two maps, including a genetic map and a bacterial artificial chromosome (BAC) based physical map for J. regia (Luo et al. 2015). Martínez‐García relied on the Illumina HiSeq2500 platform to decode the genome of J. regia (Martínez-García et al. 2016). They obtained 500 million reads of 151 bp. Besides, 1000 PacBio reads produced by PacBio RS II instrument were used for assembly validation. Stevens et al. further improved the assembly of J. regia by integrating the 6 Gb new PacBio sequencing data (~10 fold genome coverage) with the previous Illumina data. Their most recent V1.5 genome assembly was significantly improved than that of the V1.0, in terms of contiguity with scaffold N50 of 639 kbp vs 242 kbp. The other five walnut species and P. stenoptera were all sequenced using HiSeq 2500. All six walnut species are diploid with 32 chromosomes and their genome sizes varied in small sizes, from 571 Mb-594 Mb. The genome assemblies were evaluated using CEGMA and BUSCO, as high as 93.55% (J. nigra), 96.37% (J. sigillata), 94.24% (J. microcarpa), 96.18% (J. regia) (Stevens et al. 2018). These results suggested that these genomes are in good quality for further in-depth comparative analyses. Population genomics studies the genome-wide variants, thus help to improve our understanding of microevolution of walnuts. 63 individuals were sequenced, covering two nut species J. nigra (13 individulas) and J. regia (27 individuals) and two rootstock species J. hindsii (10 individuals) and J. microcarpa (12 individuals). Although genome-wide association studies are lacking at the moment, we believe these open-source data are valuable for global researchers. These data are freely available for the public (Chen et al. 2018; Wegrzyn et al. 2008). Genomic synteny analysis suggested that J. regia has undergone a whole genome duplication (WGD) event approximately 60 million-year-ago (Luo et al. 2015). Ks distributions of homeologous genes in J. regia supported that a single WGD event happened in the Juglans lineage. Van de Peer and collaborators analyzed the J. regia genome and proposed that a WGD event happened ~49- 68 MYA. Since the split of Juglans and outgroup P. stenoptera occurred ~68MYA, we suggested that the ancestor of Juglans species has experienced the WGD, happened at K-Pg boundary ~65 MYA (Fig. 1a). Nevertheless, accurate timing estimation should rely on more genomic data. Evo-devo of the nuts Gallate 1-b-glucosyltransferase (GGT) is involved in the synthesis of a precursor for synthesizing hydrolysable tannins, while polyphenol oxidase (PPO) catalyzes the oxidation of phenolic metabolites as observed in J. regia. Analysis of the J. regia genome found 130 GGT genes, and two of them were verified with transcriptome sequencing and PCR validation, as potential precursors for the synthesis of hydrolysable tannins. In pre-genomic era, only one PPO gene was identified in J. regia (Martínez-García et al. 2016). In the V1.0 genome of J. regia, two polyphenol oxidase (PPO) genes (PPO1 and PPO2) were characterized on distince scaffolds (Martínez-García et al. 2016). But since the contigs were not able to anchor on the chromosomes, the genomic locations of the two PPO genes remain unknown. Stevens et al. employed the improved V1.5 J. regia genome and comparative genomics of other five Juglans species, together with the outgroup P. stenoptera, observed that PPO1 and PPO2 are in close proximity, as well as in the same relative orientation and on the same assembly scaffold (Stevens et al. 2018). Taken together, the results of protein homology, micro-synteny, and k-mer depth, suggest that single functional copies PPO1 and PPO2 genes are in fact tandem in all Juglans species examined, consistent with an ancestral gene duplication. Future perspective We encourage the research priority to J. regia, the most important crop in the genus Juglans. Since the current V1.5 J. regia genome still consists of 4,402 genomic fragments, there’s growing demand of a haploid map of 14 chromosomes. Also, creating a pan-genome reference for J. regia to integrate and describe variations would accelerate the understanding of the micro- evolution and the utilization of interested genes. Furthermore, genome-wide association study (GWAS) of genome-wide DNA variants in different individuals will help to associate the variant(s) with one or many traits. Acknowledgement F.C. is supported by a grant from national science foundation, China (31801898), a grant from state key laboratory of ecological pest control for Fujian and Taiwan crops (SKB2017004) and a grant from natural science foundation of Fujian Province (2018J01603). References Chen F., Dong W., Zhang J., Guo X., Chen J., Wang Z., et al. 2018 The sequenced angiosperm genomes and genome databases. Front Plant Sci. 9, 418. Luo M., You F. M., Li P., Wang J., Zhu T., Dandekar A. M., et al. 2015 Synteny analysis in Rosids with a walnut physical map reveals slow genome evolution in long-lived woody perennials. BMC Genomics 16, 707. Martínez-García P. J., Crepeau M. W., Puiu D., Gonzalez-Ibeas D., Whalen J., Stevens K. A., et al. 2016 The walnut (Juglans Regia) genome sequence reveals diversity in genes coding for the biosynthesis of non- structural polyphenols.” Plant J. 87, 507–32. Stevens K. A., Woeste K., Chakraborty S., Crepeau M. W., Leslie C.A., Martínez-García P. J., et al. 2018 Genomic variation among and within six Juglans species.” G3: Genes, Genomes, Genetics 8, 2153–65. Wegrzyn J. L., Lee J. M., Tearse B. R., Neale D. B. 2008 TreeGenes: A forest tree genome database. Int. J. Plant Genomics 2008, 412875. Received 4 October 2018; revised 31 December 2018; accepted 3 January 2019 Correspondng editor. Qingpo Liu a Cretaceous–Paleogene extinction b Divergence time (MYA) 80 70 60 50 40 30 20 10 0 Asymmetric evolution of PPOs PPO1 PPO2 Juglans regia n domesticated o 22.39 y r timber & nuts a c s Juglans sigillata o i 42.11 domesticated D nuts domestication n o y Juglans cathayensis r a wild c o 45.00 ornamental i d r a C Juglans hindsii wild timber & rootstock n o 28.92 Juglans microcarpa y r WGD a wild c rootstock o s 21.58 y h Juglans nigra R wild Timber & nuts p Pterocarya stenoptera u o r wild g t ornamental & rootstock u 0.020 O Fig. 1 | Evolution of six genome sequenced Juglans species and the asymmetric evolution of POLYPHENOL OXIDASE (PPO) genes. a, A whole genome duplication (WGD) event probably happened approximately 65 million year ago. b, The domestication effect on the PPO genes show asymmetric evolution of PPO1 and PPO2 in J. regia and J. sigillata. .