Search Results (8)

Seedlessness is one of the highest valued agronomic traits in grapes. Embryo rescue in combination with marker-assisted selection have been widely applied in seedless grape breeding due to the advantages of increasing the ratio of seedless progenies and shortening the breeding cycle. However, the large number of deformed seedlings produced during embryo rescue and the lack of fast, efficient, and low-cost markers severely inhibit the process of seedless grape breeding. In this study, a total of eighty-three grape cultivars (51 seedless and 32 seeded) with diverse genetic backgrounds and two populations derived from embryo rescue, including 113 F1 hybrid individuals (60 seedless and 53 seeded), were utilized. We screened suitable media for converting malformed seedlings into normal seedlings, analyzed the association between the SNP in VviAGL11 and seeded/seedless phenotype, and developed a KASP marker related to stenospermocarpic seedlessness. Our results indicated that the transformation rate of 37.8% was obtained with MS medium supplemented with 2.0 mg·L⁻¹ of 6-BA and 0.5 mg·L⁻¹ of IBA. The presence of an A nucleotide allele at position chr18:26889437 was further confirmed to be fully associated with the stenospermocarpic seedlessness phenotype. The developed KASP marker, based on the verified SNP locus in VviAGL11, successfully distinguished the seedless and seeded genotypes with high precision and throughput. The results will contribute to enhancing the efficiency of embryo rescue and facilitate parent selection and early selection of seedless offspring with molecular markers, thereby accelerating the breeding process in seedless table grapes. Full article

Parthenocarpy and stenospermocarpy are the two mechanisms underlying the seedless fruit set program. Seedless fruit occurs naturally and can be produced using hormone application, crossbreeding, or ploidy breeding. However, the two types of breeding are time-consuming and sometimes ineffective due to interspecies hybridization barriers or the absence of appropriate parental genotypes to use in the breeding process. The genetic engineering approach provides a better prospect, which can be explored based on an understanding of the genetic causes underlying the seedlessness trait. For instance, CRISPR/Cas is a comprehensive and precise technology. The prerequisite for using the strategy to induce seedlessness is identifying the crucial master gene or transcription factor liable for seed formation/development. In this review, we primarily explored the seedlessness mechanisms and identified the potential candidate genes underlying seed development. We also discussed the CRISPR/Cas-mediated genome editing approaches and their improvements. Full article

The European grapevine (Vitis vinifera L.) is one of the world’s most widely cultivated and economically important fruit crops. Seedless fruits are particularly desired for table grapes, with seedlessness resulting from stenospermocarpy being an important goal for cultivar improvement. The establishment of an RNA in situ hybridisation (ISH) system for grape berries and ovules is, therefore, important for understanding the molecular mechanisms of ovule abortion in stenospermocarpic seedless cultivars. We improved RNA in situ hybridisation procedures for developing berries and ovules by targeting two transcription factor genes, VvHB63 and VvTAU, using two seeded varieties, ‘Red Globe’ and ‘Pinot Noir’, and two seedless cultivars, ‘Flame Seedless’ and ‘Thompson Seedless’. Optimisation focused on the time of proteinase K treatment, probe length, probe concentration, hybridisation temperature and post-hybridisation washing conditions. The objectives were to maximise hybridisation signals and minimise background interference, while still preserving tissue integrity. For the target genes and samples tested, the best results were obtained with a pre-hybridisation proteinase K treatment of 30 min, probe length of 150 bp and concentration of 100 ng/mL, hybridisation temperature of 50 °C, three washes with 0.2× saline sodium citrate (SSC) solution and blocking with 1% blocking reagent for 45 min during the subsequent hybridisation. The improved ISH system was used to study the spatiotemporal expression patterns of genes related to ovule development at a microscopic level. Full article

The development of new seedless cultivar represents one of the most important goals in table grape breeding programmes worldwide. The most common technique to obtain new seedless cultivars is embryo rescue, an approach that allows the isolation of immature embryos and their cultivation in vitro. In this study, a total of 23 crosses (developed employing one seeded and one seedless parent) were performed during two seasons (2017 and 2018) for a total of 1140 seedlings. For each cross, the principal parameters related to the efficiency of the pollination were measured (harvested bunches, collected berries, recovered embryos/seeds and plants obtained). Based on these traits, statistical analyses were performed to calculate the female and male parental efficiency and to compare the two techniques of propagation employed: embryo rescue (7.8% of plants obtained) and gamic propagation (8.4%). Finally, the segregation of the SSR marker p3_VvAGL11 was evaluated on the progeny of four crosses in which the same cultivar was used alternately as female or male parent (SugxIta/ItaxSug and CrixIta/ItaxCri). The parameters measured showed a positive correlation between berries, seeds and plants obtained, exclusively in combination with a seeded female parent. The crossing combinations investigated indicate that some genotypes outperformed others when used as female parent in terms of embryos/seeds produced. Therefore, the efficiency in terms of seedlings obtained for the seedless female parents is actually balanced with those obtained for the seeded ones. The proposed research aims to provide useful information to guide the choice of genotypes used in the genetic improvement programs of seedless grapes, to increase their efficiency. Full article

Conventional crossing of stenospermocarpic grapes for the obtainment of seedless cultivars presents some technical constraints causing embryo abortion in the early berry developmental stages. Embryo rescue technique partially overcomes these limitations, but the obtainment of viable plantlets relies on the optimization of several genetic and methodological issues. This work aimed to regenerate viable plants from immature ovules of stenospermocarpic table grape hybrids by applying a three-step in vitro culture protocol consisting of embryo development, embryo germination-rooting, and plantlet formation. The influence of parental genotypes (six “seedless × seedless” crosses), ovule sampling time (30, 40, 50 days after pollination (DAP)), and extent of embryo germination induction (4, 6, 8 weeks) was assessed on ovule fertilization, embryo development and germination, rooting, and plantlet formation to establish the best rescue time for each combination hybrid. Our optimized protocol included immature ovule isolation for 40 DAP and embryo germination induction for 8 weeks. As for genotypes, the most efficient embryo germination was recovered from hybrids of Thompson, Superior, and Regal cultivars, whereas the highest percentage of viable plants was derived from 50-DAP ovules of Luisa × Thompson progeny. Such an optimized protocol could be useful to maximize the efficiency of future breeding programs for grape seedlessness. Full article

Mexico is the leading exporter of mangos worldwide, and ‘Ataulfo’ is one of the most popular cultivars. However, their production has dramatically dropped in recent years due to the high incidence of nubbins. One of the possible causes is the presence of a delayed self-incompatibility found in this cultivar; thus, proximity to compatible cultivars may help to reduce this incidence. Nevertheless, there is a lack of studies that have rigorously tested this hypothesis in this cultivar. For two consecutive years, the present study evaluated the incidence of nubbins, as well as the quality and quantity of commercial fruits of ‘Ataulfo’ trees located at 10, 30, and 50 m away from ‘Haden’ cultivar. Additionally, the yield and economic income of different planting designs were estimated. During both sampling periods, our results clearly indicated that at 10 m away from ‘Haden’ individuals, ‘Ataulfo’ trees presented a lower incidence of nubbins and higher production of commercial fruits, and higher yield and total income per hectare than at 30 or 50 m away from them. These results indicate that planting designs of ‘Ataulfo’ trees located 10 m away from ‘Haden’ will help to satisfy the increasing demand for mangos of this cultivar in the international market. Full article

Reduced expression of MADS-box gene AGAMOUS-LIKE11 (VviAGL11) is responsible for stenospermocarpic seedlessness in bunch grapes. This study is aimed to characterize the VviAGL11 orthologous gene (VroAGL11) in native muscadine grapes (Vitis rotundifolia) at the molecular level and analyze its divergence from other plants. The VroAGL11 transcripts were found in all muscadine cultivars tested and highly expressed in berries while barely detectable in leaves. RT-PCR and sequencing of predicted ORFs from diverse grape species showed that AGL11 transcripts were conservatively spliced. The encoded VroAGL11 protein contains highly conserved MADS-MEF2-like domain, MADS domain, K box, putative phosphorylation site and two sumoylation motifs. The muscadine VroAGL11 proteins are almost identical (99%) to that of seeded bunch cultivar, Chardonnay, except in one amino acid (A79G), but differs from mutant protein of seedless bunch grape, Sultanina, in two amino acids, R197L and T210A. Phylogenetic analysis showed that AGL11 gene of muscadine and other Vitis species formed a separate clade than that of other eudicots and monocots. Muscadine grape cultivar “Jane Bell” containing the highest percentage of seed content in berry (7.2% of berry weight) had the highest VroAGL11 expression, but almost none to nominal expression in seedless cultivars Fry Seedless (muscadine) and Reliance Seedless (bunch). These findings suggest that VroAGL11 gene controls the seed morphogenesis in muscadine grapes like in bunch grape and can be manipulated to induce stenospermocarpic seedlessness using gene editing technology. Full article

Seedless inheritance has been considered a quasi-monogenic trait based on the VvAGL11 gene. An intragenic simple sequence repeat (SSR) marker, p3_VvAGL11, is currently used to opportunely discard seeded progeny, which represents up to 50% of seedlings to be established in the field. However, the rate of false positives remains significant, and this lack of accuracy might be due to a more complex genetic architecture, some intrinsic flaws of p3_VvAGL11, or potential recombination events between p3_VvAGL11 and the causal SNP located in the coding region. The purpose of this study was to update the genetic architecture of this trait in order to better understand its implications in breeding strategies. A total of 573 F1 individuals that segregate for seedlessness were genotyped with a 20K SNP chip and characterized phenotypically during four seasons for a fine QTL mapping analysis. Based on the molecular diversity of p3_VvAGL11 alleles, we redesigned this marker, and based on the causal SNP, we developed a qPCR-HRM marker for high-throughput and a Tetra-ARMS-PCR for simple predictive analyses. Up to 10 new QTLs were identified that describe the complex nature of seedlessness, corresponding to small but stable effects. The positive predictive value, based on VvAGL11 alone (0.647), was improved up to 0.814 when adding three small-effect QTLs in a multi-QTL additive model as a proof of concept. The new SSR, 5U_VviAGL11, is more informative and robust, and easier to analyze. However, we demonstrated that the association can be lost by intragenic recombination and that the e7_VviAGL11 SNP-based marker is thus more reliable and decreases the occurrence of false positives. This study highlights the bases of prediction failure based solely on a major gene and a reduced set of candidate genes, in addition to opportunities for molecular breeding following further and larger validation studies. Full article