3D characterization of walnut morphological traits using X-ray computed tomography.

BACKGROUND: Walnuts are grown worldwide in temperate areas and producers are facing an increasing demand. In a climate change context, the industry also needs cultivars that provide fruits of quality. This quality includes satisfactory filling ratio, thicker shell, ease of cracking, smooth shell and round-shaped walnut, and larger nut size. These desirable traits have been analysed so far using calipers or micrometers, but it takes a lot of time and requires the destruction of the sample. A challenge to take up is to develop an accurate, fast and non-destructive method for quality-related and morphometric trait measurements of walnuts, that are used to characterize new cultivars or collections in any germplasm management process. RESULTS: In this study, we develop a method to measure different morphological traits on several walnuts simultaneously such as morphometric traits (nut length, nut face and profile diameters), traits that previously required opening the nut (shell thickness, kernel volume and filling kernel/nut ratio) and traits that previously were difficult to quantify (shell rugosity, nut sphericity, nut surface area and nut shape). These measurements were obtained from reconstructed 3D images acquired by X-ray computed tomography (CT). A workflow was created including several steps: noise elimination, walnut individualization, properties extraction and quantification of the different parts of the fruit. This method was applied to characterize 50 walnuts of a part of the INRAE walnut germplasm collection made of 161 unique accessions, obtained from the 2018 harvest. Our results indicate that 50 walnuts are sufficient to phenotype the fruit quality of one accession using X-ray CT and to find correlations between the morphometric traits. Our imaging workflow is suitable for any walnut size or shape and provides new and more accurate measurements. CONCLUSIONS: The fast and accurate measurement of quantitative traits is of utmost importance to conduct quantitative genetic analyses or cultivar characterization. Our imaging workflow is well adapted for accurate phenotypic characterization of a various range of traits and could be easily applied to other important nut crops.

Association and linkage mapping to unravel genetic architecture of phenological traits and lateral bearing in Persian walnut (Juglans regia L.).

BACKGROUND: Unravelling the genetic architecture of agronomic traits in walnut such as budbreak date and bearing habit, is crucial for climate change adaptation and yield improvement. A Genome-Wide Association Study (GWAS) using multi-locus models was conducted in a panel of 170 walnut accessions genotyped using the Axiom™ J. regia 700 K SNP array, with phenological data from 2018, 2019 and legacy data. These accessions come from the INRAE walnut germplasm collection which is the result of important prospecting work performed in many countries around the world. In parallel, an F1 progeny of 78 individuals segregating for phenology-related traits, was genotyped with the same array and phenotyped for the same traits, to construct linkage maps and perform Quantitative Trait Loci (QTLs) detection. RESULTS: Using GWAS, we found strong associations of SNPs located at the beginning of chromosome 1 with both budbreak and female flowering dates. These findings were supported by QTLs detected in the same genomic region. Highly significant associated SNPs were also detected using GWAS for heterodichogamy and lateral bearing habit, both on chromosome 11. We developed a Kompetitive Allele Specific PCR (KASP) marker for budbreak date in walnut, and validated it using plant material from the Walnut Improvement Program of the University of California, Davis, demonstrating its effectiveness for marker-assisted selection in Persian walnut. We found several candidate genes involved in flowering events in walnut, including a gene related to heterodichogamy encoding a sugar catabolism enzyme and a cell division related gene linked to female flowering date. CONCLUSIONS: This study enhances knowledge of the genetic architecture of important agronomic traits related to male and female flowering processes and lateral bearing in walnut. The new marker available for budbreak date, one of the most important traits for good fruiting, will facilitate the selection and development of new walnut cultivars suitable for specific climates.

Genome-Wide Association Study Reveals Candidate Genes Involved in Fruit Trait Variation in Persian Walnut (Juglans regia L.).

Elucidating the genetic determinants of fruit quality traits in walnut is essential to breed new cultivars meeting the producers and consumers' needs. We conducted a genome-wide association study (GWAS) using multi-locus models in a panel of 170 accessions of Juglans regia from the INRAE walnut germplasm collection, previously genotyped using the AxiomTM J. regia 700K SNP array. We phenotyped the panel for 25 fruit traits related to morphometrics, shape, volume, weight, ease of cracking, and nutritional composition. We found more than 60 marker-trait associations (MTAs), including a highly significant SNP associated with nut face diameter, nut volume and kernel volume on chromosome 14, and 5 additional associations were detected for walnut weight. We proposed several candidate genes involved in nut characteristics, such as a gene coding for a beta-galactosidase linked to several size-related traits and known to be involved in fruit development in other species. We also confirmed associations on chromosomes 5 and 11 with nut suture strength, recently reported by the University of California, Davis. Our results enhance knowledge of the genetic control of important agronomic traits related to fruit quality in walnut, and pave the way for the development of molecular markers for future assisted selection.

The walnut genetic resources of INRA: chronological phenotypic data and ontology.

OBJECTIVES: Persian walnut (Juglans regia L.), the walnut species cultivated for nut production, is grown worldwide in temperate areas. In this work, chronological phenotypic data have been collected regarding a part of the walnut genetic resources of the French National Institute for Agricultural Research (INRA) of Bordeaux. Using a well described ontology, these data have been collected in order to assess the phenotypic variations among the accessions, and to better manage the germplasm collection. These data can also be helpful for any breeding program as they provide a clear phenotypic characterization of the main cultivars. DATA DESCRIPTION: This paper introduces a dataset collected for 150 J. regia accessions for a period from 1965 to 2016, and for 3 observation sites, released as comma separated value spreadsheet. It includes observations about phenological traits (e.g. flowering dates), traits related to in-shell walnut (e.g. weight and size), and traits related to kernel (e.g. color). It can be used by other researchers particularly for multi-site phenological studies in the context of climate change since climate data files are also available. In addition, a complete walnut ontology was deposited in this repository and can assist to standardize the management of any walnut germplasm collection.

Analysis of genetic diversity and structure in a worldwide walnut (Juglans regia L.) germplasm using SSR markers.

Persian or English walnut (Juglans regia L.), the walnut species cultivated for nut production, is one of the oldest food sources known and is grown worldwide in temperate areas. France is the 7th leading producer as of 2016 with 39 kt. Deciphering walnut genetic diversity and structure is important for efficient management and use of genetic resources. In this work, 253 worldwide accessions from the INRA walnut germplasm collection, containing English walnut and several related species, were genotyped using 13 SSR (Single Sequence Repeat) markers selected from the literature to assess diversity and structure. Genetic diversity parameters showed a deficiency of heterozygotes and, for several SSRs, allele-specificities among the accessions tested. Principal Coordinate Analysis (PCoA) showed the 253 accessions clustered in largely in agreement with the existing botanical classification of the genus. Among the 217 J. regia accessions, two main clusters, accessions from Eastern Europe and Asia, and accessions from Western Europe and America, were identified using STRUCTURE software. This was confirmed by Principal Coordinate Analysis and supported by Neighbor-Joining tree construction using DARwin software. Moreover, a substructure was found within the two clusters, mainly according to geographical origin. A core collection containing 50 accessions was selected using the maximum length sub-tree method and prior knowledge about their phenotype. The present study constitutes a preliminary population genetics overview of INRA walnut genetic resources collection using SSR markers. The resulting estimations of genetic diversity and structure are useful for germplasm management and for future walnut breeding programs.

A single Banana streak virus integration event in the banana genome as the origin of infectious endogenous pararetrovirus.

Sequencing of plant nuclear genomes reveals the widespread presence of integrated viral sequences known as endogenous pararetroviruses (EPRVs). Banana is one of the three plant species known to harbor infectious EPRVs. Musa balbisiana carries integrated copies of Banana streak virus (BSV), which are infectious by releasing virions in interspecific hybrids. Here, we analyze the organization of the EPRV of BSV Goldfinger (BSGfV) present in the wild diploid M. balbisiana cv. Pisang Klutuk Wulung (PKW) revealed by the study of Musa bacterial artificial chromosome resources and interspecific genetic cross. cv. PKW contains two similar EPRVs of BSGfV. Genotyping of these integrants and studies of their segregation pattern show an allelic insertion. Despite the fact that integrated BSGfV has undergone extensive rearrangement, both EPRVs contain the full-length viral genome. The high degree of sequence conservation between the integrated and episomal form of the virus indicates a recent integration event; however, only one allele is infectious. Analysis of BSGfV EPRV segregation among an F1 population from an interspecific genetic cross revealed that these EPRV sequences correspond to two alleles originating from a single integration event. We describe here for the first time the full genomic and genetic organization of the two EPRVs of BSGfV present in cv. PKW in response to the challenge facing both scientists and breeders to identify and generate genetic resources free from BSV. We discuss the consequences of this unique host-pathogen interaction in terms of genetic and genomic plant defenses versus strategies of infectious BSGfV EPRVs.

Creation of a BAC resource to study the structure and evolution of the banana (Musa balbisiana) genome.

The first bacterial artificial chromosome (BAC) library of the banana species Musa balbisiana 'Pisang Klutuk Wulung' (PKW BAC library) was constructed and characterized. One improved and one novel protocol for nuclei isolation were employed to overcome problems caused by high levels of polyphenols and polysaccharides present in leaf tissues. The use of flow cytometry to purify cell nuclei eliminated contamination with secondary metabolites and plastid DNA. Furthermore, the usefulness of the inducible pCC1BAC vector to obtain a higher amount of BAC DNA was demonstrated. The PKW BAC library represents nine haploid genome equivalents of M. balbisiana and its mean insert size is 135 kb. It consists of two sublibraries, of which the first one (SN sublibrary with 24,960 clones) was prepared according to an improved standard nuclei isolation protocol, whereas the second (FN sublibrary with 11,904 clones) was obtained from flow-sorted nuclei. Screening with 12 RFLP probes, which were genetically anchored to 8 genetic linkage groups of the banana species Musa acuminata, revealed an average of 11 BAC clones per probe, thus confirming the genome coverage estimated based on the insert size, as well as a high level of conservation between the two species of Musa. Localization of selected BAC clones to mitotic chromosomes using FISH indicated that the BAC library represented a useful resource for cytogenetic mapping. As the first step in map-based cloning of a genetic factor that is involved in the activation of integrated pararetroviral sequences of Banana streak virus (BSV), the BSV expressed locus (BEL) was physically delimited. The PKW BAC library represents a publicly available tool, and is currently used to reveal the integration and activation mechanisms of BSV sequences and to study banana genome structure and evolution.