Breeding new seedless table grapevines for a more sustainable viticulture in Mediterranean climate.

The growing demand for sustainable and environmentally friendly viticulture is leading to a multiplication of breeding programs aimed at obtaining vines that are resistant to powdery mildew (PM) and downy mildew (DM), the two most damaging vine diseases. In Puglia, the most important Italian region for the production of table grapes, an extensive crossing program was launched in 2015 with 113 crosses, including elite table varieties, seedless varieties, and resistant varieties. The main seedling production parameters were measured for each cross. In particular, berries harvested as well as the number of seeds and seedlings obtained were considered. Approximately 103,119 seedlings were obtained and subjected to marker-assisted selection for seedlessness using the marker VvAGL11 and for resistance to PM and DM with appropriate markers. Approximately one third (32,638) of the progenies were selected as putative seedless and seventeen thousand five hundred-nine (17,509) were transferred to the field for phenotypic evaluation, including 527 seedless individuals putatively resistant, of which 208 confirmed to be resistant to DM, 22 resistant to PM, and 20 individuals that combined resistance and seedlessness traits. The work discusses the effects of parental combinations and other variables in obtaining surviving progeny and pyramiding genes in table grapes and provides useful information for selecting genotypes and increasing the efficiency of breeding programs for seedless disease-resistant grapes.

Fine Mapping and Candidate Gene Analysis of Pm36, a Wild Emmer-Derived Powdery Mildew Resistance Locus in Durum Wheat.

Powdery mildew (PM) is an economically important foliar disease of cultivated cereals worldwide. The cultivation of disease-resistant varieties is considered the most efficient, sustainable and economical strategy for disease management. The objectives of the current study were to fine map the chromosomal region harboring the wild emmer PM resistance locus Pm36 and to identify candidate genes by exploiting the improved tetraploid wheat genomic resources. A set of backcross inbred lines (BILs) of durum wheat were genotyped with the SNP 25K chip array and comparison of the PM-resistant and susceptible lines defined a 1.5 cM region (physical interval of 1.08 Mb) harboring Pm36. The genetic map constructed with F2:3 progenies derived by crossing the PM resistant line 5BIL-42 and the durum parent Latino, restricted to 0.3 cM the genetic distance between Pm36 and the SNP marker IWB22904 (physical distance 0.515 Mb). The distribution of the marker interval including Pm36 in a tetraploid wheat collection indicated that the positive allele was largely present in the domesticated and wild emmer Triticum turgidum spp. dicoccum and ssp. dicoccoides. Ten high-confidence protein coding genes were identified in the Pm36 region of the emmer, durum and bread wheat reference genomes, while three added genes showed no homologous in the emmer genome. The tightly linked markers can be used for marker-assisted selection in wheat breeding programs, and as starting point for the Pm36 map-based cloning.

How to Choose a Good Marker to Analyze the Olive Germplasm (Olea europaea L.) and Derived Products.

The olive tree (Olea europaea L.) is one of the most cultivated crops in the Mediterranean basin. Its economic importance is mainly due to the intense production of table olives and oil. Cultivated varieties are characterized by high morphological and genetic variability and present a large number of synonyms and homonyms. This necessitates the introduction of a rapid and accurate system for varietal identification. In the past, the recognition of olive cultivars was based solely on analysis of the morphological traits, however, these are highly influenced by environmental conditions. Therefore, over the years, several methods based on DNA analysis were developed, allowing a more accurate and reliable varietal identification. This review aims to investigate the evolving history of olive tree characterization approaches, starting from the earlier morphological methods to the latest technologies based on molecular markers, focusing on the main applications of each approach. Furthermore, we discuss the impact of the advent of next generation sequencing and the recent sequencing of the olive genome on the strategies used for the development of new molecular markers.

Mapping Powdery Mildew (Blumeria graminis f. sp. tritici) Resistance in Wild and Cultivated Tetraploid Wheats.

Wheat is the most widely grown crop and represents the staple food for one third of the world's population. Wheat is attacked by a large variety of pathogens and the use of resistant cultivars is an effective and environmentally safe strategy for controlling diseases and eliminating the use of fungicides. In this study, a collection of wild and cultivated tetraploid wheats (Triticum turgidum) were evaluated for seedling resistance (SR) and adult plant resistance (APR) to powdery mildew (Blumeria graminis) and genotyped with a 90K single nucleotide polymorphism (SNP) array to identify new sources of resistance genes. The genome-wide association mapping detected 18 quantitative trait loci (QTL) for APR and 8 QTL for SR, four of which were identical or at least closely linked to four QTL for APR. Thirteen candidate genes, containing nucleotide binding sites and leucine-rich repeats, were localized in the confidence intervals of the QTL-tagging SNPs. The marker IWB6155, associated to QPm.mgb-1AS, was located within the gene TRITD1Av1G004560 coding for a disease resistance protein. While most of the identified QTL were described previously, five QTL for APR (QPm.mgb-1AS, QPm.mgb-2BS, QPm.mgb-3BL.1, QPm.mgb-4BL, QPm.mgb-7BS.1) and three QTL for SR (QPm.mgb-3BL.3, QPm.mgb-5AL.2, QPm.mgb-7BS.2) were mapped on chromosome regions where no resistance gene was reported before. The novel QTL/genes can contribute to enriching the resistance sources available to breeders.

A Robust DNA Isolation Protocol from Filtered Commercial Olive Oil for PCR-Based Fingerprinting.

Extra virgin olive oil (EVOO) has elevated commercial value due to its health appeal, desirable characteristics and quantitatively limited production, and thus it has become an object of intentional adulteration. As EVOOs on the market might consist of a blend of olive varieties or sometimes even of a mixture of oils from different botanical species, an array of DNA-fingerprinting methods have been developed to check the varietal composition of the blend. Starting from a comparison between publicly available DNA extraction protocols, we set up a timely, low-cost, reproducible and effective DNA isolation protocol, which allows an adequate amount of DNA to be recovered even from commercial filtered EVOOs. Then, in order to verify the effectiveness of the DNA extraction protocol herein proposed, we applied PCR-based fingerprinting methods starting from the DNA extracted from three EVOO samples of unknown composition. In particular, genomic regions harboring nine simple sequence repeats (SSRs) and eight genotyping-by-sequencing-derived single nucleotide polymorphism (SNP) markers were amplified for authentication and traceability of the three EVOO samples. The whole investigation strategy herein described might favor producers in terms of higher revenues and consumers in terms of price transparency and food safety.

Genetic Structure Analysis of a Collection of Tunisian Durum Wheat Germplasm.

The Tunisian durum wheat germplasm includes modern cultivars and traditional varieties that are still cultivated in areas where elite cultivars or intensive cultivation systems are not suitable. Within the frame of a collection program of the National Gene Bank of Tunisia (NGBT), durum wheat germplasm was collected from different Tunisian agro-ecological zones. The collected samples were studied using simple sequence repeats (SSRs) markers to explore the genetic diversity and evaluate the genetic structure in Tunisian germplasm. The results demonstrated significant diversity in the Tunisian durum wheat germplasm, with clear differentiation between traditional varieties and modern cultivars. The population structure analysis allowed the identification of five subpopulations, two of which appear to be more strongly represented in germplasm collected in central and southern Tunisia, where environmental conditions at critical development phases of the plant are harsher. Moreover these subpopulations are underrepresented in modern varieties, suggesting that traits of adaptation useful for breeding more resilient varieties might be present in central and southern germplasm. Moreover, our results will support, the activity of in situ on farm conservation of Tunisian durum wheat germplasm started by the National Gene Bank of Tunisia along with the ex situ approach.

Development of a new wheat microarray from a durum wheat totipotent cDNA library used for a powdery mildew resistance study.

Totipotent cDNA libraries representative of all the potentially expressed sequences in a genome would be of great benefit to gene expression studies. Here, we report on an innovative method for creating such a library for durum wheat (Triticum turgidum L. var. durum) and its application for gene discovery. The use of suitable quantities of 5-azacytidine during the germination phase induced the demethylation of total DNA, and the resulting seedlings potentially express all of the genes present in the genome. A new wheat microarray consisting of 4925 unigenes was developed from the totipotent cDNA library and used to screen for genes that may contribute to differences in the disease resistance of two near-isogenic lines, the durum wheat cultivar Latino and the line 5BIL-42, which are respectively susceptible and resistant to powdery mildew. Fluorescently labeled cDNA was prepared from the RNA of seedlings of the two near-isogenic wheat lines after infection with a single powdery mildew isolate under controlled conditions in the greenhouse. Hybridization to the microarray identified six genes that were differently expressed in the two lines. Four of the sequences could be assigned putative functions based on their similarity to known genes in public databases. Physical mapping of the six genes localized them to two regions of the genome: the centromeric region of chromosome 5B, where the Pm36 resistance gene was previously localized, and chromosome 6B.

Molecular identification of a new powdery mildew resistance gene on chromosome 2BS from Triticum turgidum ssp. dicoccum.

Powdery mildew caused by the fungal pathogen Blumeria graminis f. sp. tritici (Bgt), is a destructive foliar disease on wheat in many regions of the world. Triticum turgidum ssp. dicoccum (2n=4x=28) shows particular promises as a donor source of useful genetic variation for several traits, including disease resistances that could be introgressed to cultivated wheats. Accession MG5323, resistant to powdery mildew, was crossed to the susceptible durum cultivar Latino and a set of 122 recombinant inbred lines (RILs) was produced. F1 and F2 progenies and the RIL population were tested with one isolate of Blumeria graminis and data obtained indicated that a single dominant gene, temporarily designated Ml5323, controlled resistance at the seedling stage. Molecular markers were used to characterize and map the powdery mildew resistance gene. Twelve microsatellite markers were linked to the resistance gene, and among them, EST-SSR CA695634 was tightly linked to the resistance gene, which was assigned to chromosome arm 2BS and physically mapped to the gene rich region of fragment length (FL) 0.84-1.00. An allelism test showed that the Ml5323 gene and the resistant gene Pm26 of ssp. dicoccoides localized in the same bin, are not allelic and tightly linked.