Targeted sequencing of the \u3ci\u3ePanicum miliaceum\u3c/i\u3e gene space and genotyping of variant sites from population genetics studies, combined in a single assay, as a tool for broomcorn millet assisted breeding

Broomcorn millet (Panicum miliaceum L.) is a prehistorical cereal, today cultivated as a minor crop with low yields but with a renewed interest for its high water use effciency and gluten-free grains. To reverse the downward trend in broomcorn millet cultivation, the crop needs genetic improvement and creation of novel genetic variation to increase productivity. In order to facilitate genomics-assisted breeding, we designed a reduced representation genome-sequencing assay that investigates 1.8% of the nuclear DNA in a targeted and reproducible way, with an intensity of genomic sampling that is a direct function of local recombination rate. We used this tool and set up bioinformatics analyses tailored to the polyploid genome of P. miliaceum for maternity and paternity testing, quantification and genomic distribution of homozygous regions and estimation of parental genome contribution for individual seedlings in advanced inbred lines from a breeding program and compared their genomic composition with registered varieties. We found several clues that suggest that the genetic purifcation process to ensure genetic uniformity is incomplete in varieties of this species. Residual heterozygosity was detected in the genome of three registered varieties ranging from 4.4 to 6.25% of their haploid genome length. Other registered varieties show genome-wide homozygosity. We found, however, evidence of intravarietal genetic variation in three cases that suggest that the breeder seed or commercial seed production had fixed by self-pollination multiple inbred lines with very similar, though not identical, genotypes within each variety

Identification of stable QTLs for vegetative and reproductive traits in the microvine (Vitis vinifera L.) using the 18 K Infinium chip

UMR AGAP - équipe DAAV - Diversité, adaptation et amélioration de la vigne[b]Background[/b] [br/]The increasing temperature associated with climate change impacts grapevine phenology and development with critical effects on grape yield and composition. Plant breeding has the potential to deliver new cultivars with stable yield and quality under warmer climate conditions, but this requires the identification of stable genetic determinants. This study tested the potentialities of the microvine to boost genetics in grapevine. A mapping population of 129 microvines derived from Picovine x Ugni Blanc flb, was genotyped with the Illumina® 18 K SNP (Single Nucleotide Polymorphism) chip. Forty-three vegetative and reproductive traits were phenotyped outdoors over four cropping cycles, and a subset of 22 traits over two cropping cycles in growth rooms with two contrasted temperatures, in order to map stable QTLs (Quantitative Trait Loci). [br/][b]Results[/b] [br/]Ten stable QTLs for berry development and quality or leaf area were identified on the parental maps. A new major QTL explaining up to 44 % of total variance of berry weight was identified on chromosome 7 in Ugni Blanc flb, and co-localized with QTLs for seed number (up to 76 % total variance), major berry acids at green lag phase (up to 35 %), and other yield components (up to 25 %). In addition, a minor QTL for leaf area was found on chromosome 4 of the same parent. In contrast, only minor QTLs for berry acidity and leaf area could be found as moderately stable in Picovine. None of the transporters recently identified as mutated in low acidity apples or Cucurbits were included in the several hundreds of candidate genes underlying the above berry QTLs, which could be reduced to a few dozen candidate genes when a priori pertinent biological functions and organ specific expression were considered. [br/][b]Conclusions[/b] [br/]This study combining the use of microvine and a high throughput genotyping technology was innovative for grapevine genetics. It allowed the identification of 10 stable QTLs, including the first berry acidity QTLs reported so far in a Vitis vinifera intra-specific cross. Robustness of a set of QTLs was assessed with respect to temperature variatio

The Rpv3 locus in grapevine: DNA variation and relevance for conventional breeding

This thesis is composed of two chapters. In the first chapter, I report the sequencing and assembly of a haplotype of the Rpv3 locus that confers race-specific resistance to Plasmopara viticola. Full-length sequences of the alleles were reconstructed for all genes in the locus and cloned from the resistant haplotype into an expression vector. Agrobacterium-mediated transformation of embryogenic calli from susceptible grapevines has been initiated for the functional characterization of each allele. During my PhD, I also worked on a grapevine breeding program for stacking major resistance genes against downy and powdery mildews and selecting high-quality resistant varieties via conventional breeding. Based on these experience, a review article entitled "Molecular grape breeding techniques" written by me and my supervisor will be published as a chapter in the book "Grape breeding programs for the wine industry" edited by Andrew G. Reynolds for the Woodhead Publishing series in Food Science, Technology and Nutrition. This article is also reported in the second chapter of this thesis

Genetic, morphological and chemical characterisation of the grape variety 'Probus' (Vitis vinifera L.)

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Extent of wild–to–crop interspecific introgression in grapevine (Vitis vinifera) as a consequence of resistance breeding and implications for the crop species definition

Over the past two centuries, introgression through repeated backcrossing has introduced disease resistance from wild grape species into the domesticated lineage Vitis vinifera subsp. sativa. Introgression lines are being cultivated over increasing vineyard surface areas, as their wines now rival in quality those obtained from preexisting varieties. There is, however, a lot of debate about whether and how wine laws defining commercial product categories, which are based on the classification of V. vinifera and interspecific hybrid grapes, should be revised to accommodate novel varieties that do not fit either category. Here, we developed a method of multilocus genotype analysis using short–read resequencing to identify haplotypic blocks of wild ancestry in introgression lines and quantify the physical length of chromosome segments free–of–introgression or with monoallelic and biallelic introgression. We used this genomic data to characterize species, hybrids and introgression lines and show that newly released resistant varieties contain 76.5–94.8% of V. vinifera DNA. We found that varietal wine ratings are not always commensurate with the percentage of V. vinifera ancestry and linkage drag of wild alleles around known resistance genes persists over at least 7.1–11.5 Mb, slowing down the recovery of the recurrent parental genome. This method also allowed us to identify the donor species of resistance haplotypes, define the ancestry of wild genetic background in introgression lines with complex pedigrees, validate the ancestry of the historic varieties Concord and Norton, and unravel sample curation errors in public databases

Genotyping apple (Malus x domestica Borkh) heirloom germplasm collected and maintained by the Regional Administration of Friuli Venezia Giulia (Italy)

This paper reports the genetic diversity of apple germplasm collected in the Friuli Venezia Giulia region (Italy). The collection, maintained in three different locations, was represented by local varieties likely originated as chance seedlings admixed with varieties introduced since centuries from neighbour countries, for most of which the name and origin was lost with the time. A preliminary procedure described in the paper started from 469 molecular profiles analysed at 15 Simple Sequence Repeat (SSR) markers and allowed to identify the \u2018true-to-type\u2019 genotypes among those maintained in multiple locations. The set of the remaining 233 accessions was further reduced to 133 unique profiles by removing 100 synonymy. The flow cytometry analysis allowed to identify as much as 55 triploids (41.4%), whose state was confirmed by field observations on leaf size and the occurrence of triallelic profiles at several SSR markers. The remaining 78 diploid accessions were analysed for their genetic diversity, that is the number of alleles, observed and expected heterozygosity, polymorphism information content (PIC), the frequency of null alleles and the probability of identity for unrelated and full-sib genotypes. The paper provides a critical evaluation of the SSR markers adopted for the study, discusses the genetic diversity observed in the apple collection examined as well as the high occurrence of triploids compared with other apple collections described in the literature

Historical Introgression of the Downy Mildew Resistance Gene Rpv12 from the Asian Species Vitis amurensis into Grapevine Varieties

The Amur grape (Vitis amurensis Rupr.) thrives naturally in cool climates of Northeast Asia. Resistance against the introduced pathogen Plasmopara viticola is common among wild ecotypes that were propagated from Manchuria into Chinese vineyards or collected by Soviet botanists in Siberia, and used for the introgression of resistance into wine grapes (Vitis vinifera L.). A QTL analysis revealed a dominant gene Rpv12 that explained 79% of the phenotypic variance for downy mildew resistance and was inherited independently of other resistance genes. A Mendelian component of resistance\u2013a hypersensitive response in leaves challenged with P. viticola\u2013was mapped in an interval of 0.2 cM containing an array of coiled-coil NB-LRR genes on chromosome 14. We sequenced 10-kb genic regions in the Rpv12+ haplotype and identified polymorphisms in 12 varieties of V. vinifera using next-generation sequencing. The combination of two SNPs in single-copy genes flanking the NB-LRR cluster distinguished the resistant haplotype from all others found in 200 accessions of V. vinifera, V. amurensis, and V. amurensis x V. vinifera crosses. The Rpv12+ haplotype is shared by 15 varieties, the most ancestral of which are the century-old \u2018Zarja severa\u2019 and \u2018Michurinets\u2019. Before this knowledge, the chromosome segment around Rpv12+ became introgressed, shortened, and pyramided with another downy mildew resistance gene from North American grapevines (Rpv3) only by phenotypic selection. Rpv12+ has an additive effect with Rpv3+ to protect vines against natural infections, and confers foliar resistance to strains that are virulent on Rpv3+ plant

Gene duplication and transposition of mobile elements drive evolution of the Rpv3 resistance locus in grapevine

A wild grape haplotype (Rpv3-1) confers resistance to Plasmopara viticola. We mapped the causal factor for resistance to an interval containing a TIR-NB-LRR (TNL) gene pair that originated 1.6-2.6 million years ago by a tandem segmental duplication. Transient coexpression of the TNL pair in Vitis vinifera leaves activated pathogen-induced necrosis and reduced sporulation compared to control leaves. Even though transcripts of the TNL pair from the wild haplotype appear to be partially subject to nonsense-mediated mRNA decay, mature mRNA levels in a homozygous resistant genotype were individually higher than the mRNA trace levels observed for the orthologous single-copy TNL in sensitive genotypes. Allelic expression imbalance in a resistant heterozygote confirmed that cis-acting regulatory variation promotes expression in the wild haplotype. The movement of transposable elements had a major impact on the generation of haplotype diversity, altering the DNA context around similar TNL coding sequences and the CG content in their proximal 5' intergenic regions. The wild and domesticated haplotypes also diverged in conserved single-copy intergenic DNA, but the highest divergence was observed in intraspecific and not in interspecific comparisons. In this case, introgression breeding did not transgress the genetic boundaries of the domesticated species, because haplotypes present in modern varieties sometimes predate speciation events between wild and cultivated species