Stenospermocarpic Seedlessness Research Articles

Optimization of In Vitro Embryo Rescue and Development of a Kompetitive Allele-Specific PCR (KASP) Marker Related to Stenospermocarpic Seedlessness in Grape (Vitis vinifera L.).

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.

Effects of parental genotypes on embryo rescue of seedless grape hybrid

ABSTRACT The success of seedless grape embryo rescue depends on many factors. To study the effects of parental genotypes on embryo rescue, we conducted 11 hybridisation combinations using nine stenospermocarpic seedless grapes as female parents and three seedless or seeded grapes as male parents. At the optimal sampling time of each mother plant, the hybrid ovules were inoculated and cultured on a solid–liquid biphasic MM3 medium. Then, the bare embryos were removed and inoculated on a WPM medium for germination and seedling formation. The results showed that when ‘Jupiter’ (seedless) was the male parent, ‘Perlette’ had the highest rates of embryo development, germination and seedling formation, followed by ‘Kunxiang Seedless’; when ‘Su-67’ (seedless) was the male parent, ‘FZ42’ had the highest embryo development rate; when ‘Beichun’ (seeded) was the male parent, ‘Fresno Seedless’ had the best embryo rescue results, followed by ‘Qinhong No.10’. These suggest that ‘Perlette’, ‘Kunxiang Seedless’, ‘Fresno Seedless’ and ‘Qinhong No.10’ are suitable female parents for embryo rescue. When ‘FZ42’ was the female parent, ‘Su-67’ was better than ‘Jupiter’ as the male parent; when ‘Perlette’ was the female parent, ‘Jupiter’ and ‘Su-67’ as male parents had no significant difference, indicating that paternal genotype has a certain effect on embryo rescue. HIGHLIGHTS The maternal genotype has a significant effect on the embryo rescue of seedless grapes. The paternal genotype has a certain effect on embryo rescue of seedless grapes. ‘Perlette’, ‘Kunxiang Seedless’, ‘Fresno Seedless’ and ‘Qinhong No.10’ are suitable female parents. A total of 345 hybrids from 11 cross-combinations were obtained by embryo rescue.

Identification and expression analysis of autophagy-related gene family associated with embryonic development in seeded and seedless grapes (Vitis vinifera)

ABSTRACT Autophagy is an important protein degradation pathway in eukaryotes and is involved in multiple processes. To date, many autophagy-related genes had been studied in various plants, but a few studies have been reported on the role of autophagy in seed development. For centuries, seedlessness has been one of the most valuable characteristics of grapes. In this study, we identified a total of 33 ATG genes in the grape. Tissue-specific expression analysis showed that VvATGs expressed widely in V. vinifera cv. ‘Pinot Noir’, indicating autophagy has an extensive role in plant development. Furthermore, we analysed the expression profiles of VvATGs during seed development in stenospermocarpic seedless ‘Thompson Seedless’ and seeded grapes ‘Pinot Noir’, the results showed that most of VvATGs exhibited higher expression levels in stenospermocarpic seedless grapes than that of seeded ones, which implies the autophagic activity in seedless grapes is relatively brisk and VvATGs may involve in the seed abortion. And the yeast two-hybrid assay showed that VvATG7 protein interacted with VvATG10 and VvATG12 protein, respectively, indicating that they may form autophagy complex through protein interactionIn summary, the genome-wide identification, expression analysis, and protein interaction of VvATGs provide new insights regarding potential roles during plant embryonic development.

Improvement of RNA In Situ Hybridisation for Grapevine Fruits and Ovules.

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.

Optimisation of embryo rescue for cold-resistant seedless grapevine

ABSTRACT Embryo rescue is essential for the breeding of seedless grapes, but current protocols are inefficient and limit practical application. Here, we documented factors influencing embryo rescue efficiency using crosses of a cold-resistant seeded hybrid 00-1-5 (‘Muscat Hamburg’ × Vitis amurensis) as the paternal parent with four stenospermocarpic seedless grape cvs., ‘Flame Seedless’, ‘Qinhong No. 2’, ‘Qinhong No. 10’, and ‘Ruby Seedless’. The results showed that treatment of inflorescences with 30∼50 mg·L−1 IBA 14 days before flowering significantly increased the embryo development, germination, and transition to seedling for progeny of each of the four crosses. ‘Qinhong No. 2’ × 00-1-5 and ‘Qinhong No. 10’ × 00-1-5, with a large number of ovules, were cultured on solid and solid–liquid biphase MM3 media respectively, the embryo development rate on solid medium was significantly better than that on solid–liquid biphase medium. ‘Ruby Seedless’, ‘Qinhong No. 2’ and ‘Qinhong No. 10’ are more suitable for embryo rescue as maternal parent. The optimal medium for transforming deformed seedlings into normal seedlings was 2 × MS medium containing 0.2 mg·L−1 6-BA, 0.1 mg·L−1 IAA, and 1.6 mg·L−1 ZnSO4. In total, 311 embryos were rescued successfully, which were considered as potential cold-resistant seedless hybrids.

Evidence confirms the occurrence of stenospermocarpic seedless fruit development in pointed gourd (Trichosanthes dioica Roxb.)

Pointed gourd (Trichosanthes dioica) is a perennial andvegetatively propagated cucurbitaceous vegetable grownfor its tender fruits, and the occurrence of harder seedsreduces consumers’ preference. The present study wasundertaken to understand the mechanism of seedless fruitdevelopment in pointed gourd. The period of rapid fruitgrowth and development in pointed gourd took placegenerally 3-7 days after pollination due to faster cell divisionand cell enlargement. Significantly higher fruit growthrecorded in seeded clone as compare to seedless clones(49.1% for fruit length, 16.7% for fruit diameter and 55.2%for fruit weight) at harvestable fruit maturity but at the sametime no significant differences found at initial stage of fruitdevelopment. The occurrence of various mechanisms ofseedless fruit development such as vegetativeparthenocarpy, stimulative parthenocarpy andstenospermocarpy have been studied in details throughdifferent pollination treatments in both seeded and seedlessclones. In seedless clone, the growth/development of seedwas normal for first 3-4 days after pollination and becomeabnormal (aborted and degenerated embryo, shriveled anddisintegrated cotyledons, traces of aborted seeds yellowishwhitein colour and zero per cent seed germination) indicatethe existence of fertilization and subsequent abortion offertilized embryo which confirms the occurrence ofstenospermocarpic seedlessness and absence of stimulativeparthenocarpy in pointed gourd.

Molecular Characterization and Phylogenetic Analysis of MADS-Box Gene VroAGL11 Associated with Stenospermocarpic Seedlessness in Muscadine Grapes.

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.

Differential expression of the seed-specific gene ABCG20 between seedless and seeded grapes and its roles in tomato seed development

In grapevine (Vitis vinifera L.), seedlessness is classified into two distinct types: parthenocarpy and stenospermocarpy. Stenospermocarpy (seedlessness due to seed abortion) in grapevine is an agronomically valuable trait which is completely hereditary and not influenced by various environmental factors. Many hypotheses have been proposed for stenospermocarpy. However, the molecular mechanisms underlying development or abortion of the seed are still poorly understood. To identify genes involved in seed development or abortion in grape, we carried out Sequence-Related Amplified Polymorphism (SRAP)-cDNA analysis of grape seed development, comparing the seedless cultivar Youngle with the seeded cultivar Pinot noir. We identified VviABCG20, which encodes a putative ATP binding cassette G transporter, as a gene more highly expressed in ‘Pinot noir’. VviABCG20 is highly expressed in root, floral bud, seed and the expression level of VviABCG20 in all of five seeded grapevine cultivars tested was generally higher than that observed in seedless cultivars. Sequencing of VviABCG20 cDNAs from six stenospermocarpic seedless and seven seeded genotypes revealed that the seed-associated expression pattern of VviABCG20 was unrelated to sequence difference within the transcribed regions. Suppression of the tomato homolog of VviABCG20 in developing tomato fruit led to reduction in seed numbers, suggesting that LeABCG20 plays an essential role in seed development. Our experiments suggest that regulation of VviABCG20 is associated with seed development, and provide a new insight into the mechanism underlying seed abortion in stenospermocarpic grapes.

Anatomical, biochemical and transcriptome analyses of Vitis vinifera cv. 'Hongju' reveal novel information regarding the seed hardness of stenospermocarpic soft‐seed grapes

AbstractThe breeding of new stenospermocarpic seedless grape cultivars is of particular importance to the growing table grape market and grape processing industry. However, the developmental and genetic architectures of soft‐seed and seedlessness traits are unclear. In this study, we performed anatomical, histochemical and gene expression analyses to characterize the morphological and genetic basis of the seed hardness of 'Hongju', a stenospermocarpic soft‐seed grape cultivar developed by crossbreeding maternal 'Italia' and paternal 'Perlon' cultivars. We found intensively lignified medium integument layers in the seeds of hard‐seed cultivars, but these layers were degraded in the stenospermocarpic seedless cultivars beginning 35 days after flowering. The expression of genes related to the lignin and cellulose biosynthesis pathways was downregulated, whereas that of several cell‐wall loosening‐related genes was upregulated in 'Hongju' compared with 'Italia' beginning at 21 days after flowering. These results suggest that transcriptional suppression of the lignin and cellulose biosynthesis pathways coupled with reduced formation of medium integument plays an important role in the development of soft seeds in stenospermocarpic seedless grape cultivars.

Genetic variation for grapevine reproductive development

Plant reproductive development has evolved to ensure reproductive success. However, in crop species, genetic variation in reproductive development has also been targeted from domestication to current breeding programs to improve the production of fruits and seeds. In grapevine, genetic variation in reproductive developmental processes such as flower induction and initiation, inflorescence and flower development, and gamete, embryo and seed development have direct implications on most production traits, including fertility, cluster weight, cluster compactness or berry weight, but also on quality traits such as skin-to-pulp ratio or seedlessness. Lack of sexual reproduction in traditional wine-grape cultivars has allowed the accumulation of mutations that affect gamete development and viability as well as embryo and seed development. Mutations that would be lost through purifying selection in natural populations can provide interesting quality traits in grapevine cultivars. In this way, genetic variation decreasing gamete viability is behind low seed number per berry, berry size and cluster compactness phenotypes, current quality goals in clonal selection for low production and loose clusters. This trait can have negative consequences, depending on environmental conditions. Complete absence of gamete viability is behind some forms of parthenocarpic development, as in the case in the origin of ‘Corinto’ raisins. Genetic variation impairing ovule and seed development seems to be behind stenospermocarpic seedlessness, a main quality trait in table-grape breeding. All these examples point out how understanding the processes of grapevine reproductive development and particularly the existing genetic variation for these traits can help to develop more suitable and adapted cultivars for both wine and table grape production.

Gene cloning and expression analyses of WBC genes in the developing grapevine seeds

White-brown complex (WBC) transporters, also called half-size ATP binding cassette G (ABCG) transporters, are involved in many biological processes, including seed development; however, the WBC transporters in grapevines received less attention to date. To reveal the molecular characteristics of WBCs and the connection between WBCs and agronomic traits of stenospermocarpic (seedless) grapevine, we carried out a genomic census and analysis of ovule-associated expression for VvWBC genes in grapevine. We identified 30 VvWBC genes and cloned full-length complementary DNAs (cDNAs) for 20 of these. The tissue or organ-specific expression analysis showed that several VvWBCs exhibited distinct expression patterns with some showing tissue specificity. Twelve VvWBC genes were found to be expressed in the developing ovules. Moreover, the results of quantitative real-time PCR (qRT-PCR) suggested that four of twelve ovule-expressed VvWBCs have distinct expression profiles during the development of ovules between seeded and stenospermocarpic grapevines. These four genes might be involved in ovule abortion. Meanwhile, chromosome mapping, multiple sequence alignments, exon/intron structure analyses and synteny analyses were preformed on VvWBC genes. Our experiments provide a new perspective on the mechanism of stenospermocarpic seedlessness and put forward a framework for further study of WBC transporters.

Characterization of the p3-VvAGL11 Marker for Stenospermocarpic Seedlessness in Euvitis × Muscadinia Grape Hybrid Progenies

Stenospermocarpic seedlessness from Vitis vinifera L. is being introgressed into muscadine grape (Vitis rotundifolia Michx.) germplasm through the use of a cross-fertile hybrid of the two species. Recently, a sequence-tagged site (STS) molecular marker, p3_VvAGL11, has been developed which enables detection of the dominant allele controlling stenospermocarpic seedlessness in V. vinifera. This marker was evaluated in six Euvitis Planch. × Muscadinia Planch. hybrid progenies to determine its association with seedlessness in this material. The presence of the 214-bp seedlessness-associated p3_VvAGL11 allele in seedling vines resulted in a nearly 3-fold reduction in mean seed fresh weight (MSFW) and significantly reduced mean seed weight per berry (MSWB), percent berry weight composed of seed (BWCS), and mean berry weight (MBW). When the lack of lignified seed was used as the determinant of seedlessness, the p3_VvAGL11 marker was able to correctly judge seedlessness in ≈85% of the progeny. Analysis of seedlessness in the progenies was hampered by poor vigor and fruiting ability of the hybrid seedlings. The p3_VvAGL11 marker shows potential to speed the introduction of the stenospermocarpic seedlessness into Muscadinia germplasm by identifying seedless progeny at the seedling stage.

In Vitro Culture of Ovules and Embryos from Seedless Grapes (Vitis vinifera L.)

Ovules of stenospermocarpic seedless grapes were cultured under different conditions. The number of embryos was not significantly increased when ovules of Muscat Seedless were cultured on a medium supplemented with the plant growth regulators indole-3-acetic acid and gibberellic acid compared to the basal medium. No correlation was found between the ovule size of Sultanina and the presence of embryos. Comparable percentages of embryos were found in small, medium and large ovules following weekly culture from four to 10 weeks after bloom. Varying results were obtained when ovules of four open-pollinated seedless cultivars were cultured on two basal media with or without activated charcoal. In all four cultivars the highest number of embryos was found at the later culture date. With three cultivars the best results were obtained when ovules were cultured on the medium of Bouquet & Davis (1989), supplemented with activated charcoal. Isolated embryos of Muscat Seedless and Sunred Seedless were cultured under various conditions and it was found that the absence of growth regulators resulted in the highest number of plantlets with normal leaves.

Gene Cloning, Expression and Enzyme Activity of Vitis vinifera Vacuolar Processing Enzymes (VvVPEs).

Vacuolar processing enzymes (VPEs) have received considerable attention due to their caspase-1-like activity and ability to regulate programmed cell death (PCD), which plays an essential role in the development of stenospermocarpic seedless grapes ovules. To characterize VPEs and the relationship between stenospermocarpic grapes and the VPE gene family, we identified 3 Vitis vinifera VPE genes (VvβVPE, VvγVPE, and VvδVPE) from the PN40024 grape genome and cloned the full-length complementary DNAs (cDNAs) from the ‘Vitis vinifera cv. Pinot Noir’ and ‘Vitis vinifera cv. Thompson Seedless’ varietals. Each of the VPEs contained a typical catalytic dyad [His (177), Cys (219)] and substrate binding pocket [Arg (112), Arg (389), Ser (395)], except that Ser (395) in the VvγVPE protein sequence was replaced with alanine. Phylogenetic analysis of 4 Arabidopsis thaliana and 6 Vitis vinifera VPEs revealed that the 10 VPEs form 3 major branches. Furthermore, the 6 grapevine VPEs share a similar gene structure, with 9 exons and 8 introns. The 6 grapevine VPEs are located on 3 different chromosomes. We also tested the enzymatic activity of recombinant VPEs expressed in the Pichia Pastoris expression system and found that the VvVPEs exhibit cysteine peptidase activity. Tissue-specific expression analysis showed that VvδVPE is only expressed in flowers, buds and ovules, that VvγVPE is expressed in various tissues, and that VvβVPE was expressed in roots, flowers, buds and ovules. The results of quantitative real-time PCR (qRT-PCR) suggested that VvβVPE in seeded grapes increased significantly at 30 days after full-bloom (DAF), close to the timing of endosperm abortion at 32 DAF. These results suggested that VvβVPE is related to ovule abortion in seedless grapes. Our experiments provide a new perspective for understanding the mechanism of stenospermocarpic seedlessness and represent a useful reference for the further study of VPEs.

Breeding for cold-resistant, seedless grapes from Chinese wild Vitis amurensis using embryo rescue

ABSTRACTHybridisation was carried out between stenospermocarpic seedless grape cultivars (female parents) and Chinese wild Vitis amurensis or its hybrids with cold-resistance alleles (male parents). The aim of this paper was: (1) to evaluate the effects of the addition of the plant growth regulator indole-3-acetic acid (IAA) to the embryo development medium MM3 + 500 mg L−1 casein hydrolysate + 1 mmol L−1 serine; (2) to obtain a batch of seedless hybrid grape offspring by embryo rescue; and (3) to screen suitable genotypes for embryo rescue. The results indicated that adding 1.75 mg L−1 IAA promotes embryo development, germination and plantlet formation in the combinations of ‘Ruby Seedless’ × ‘00-1-5’ and ‘Beauty Seedless’ × ‘Beichun’. However, it inhibits the combinations ‘Perlette’ × ‘00-1-5’ and ‘Neptune’ × ‘00-1-5’. ‘Crimson Seedless’, ‘Ruby Seedless’ and ‘Flame Seedless’ were more suitable as female parents than ‘Neptune’, ‘Perlette’, ‘Beauty Seedless’, ‘Heshen Seedless’, ‘Jupiter’ and ‘Su-67’. The Hybrid ‘00-1-5’ performed best as the male parent for cold-resistant seedless grape embryo rescue. A total of 267 new hybrid progenies derived from 11 cross combinations were obtained by embryo rescue. GLSP1 and SCF27, two special primers of the molecular marker linked to seedless gene in grape, were employed to screen the progenies of five cross combinations for possible seedlessness. Five of 22 strains obtained from two cross combinations had the marker GLSP1-569; 43 of 89 strains obtained from three cross combinations had the marker SCF27-2000. These marked strains were preliminarily identified as being seedless.

The metacaspase gene family of Vitis vinifera L.: Characterization and differential expression during ovule abortion in stenospermocarpic seedless grapes

In both plants and animals, programmed cell death (PCD) is an indispensable process that removes redundant cells. In seedless grapes (Vitis vinifera), abnormal PCD in ovule cells and subsequent ovule abortion play key roles in stenospermocarpy. Metacaspase, a type of cysteine-dependent protease, plays an essential role in PCD. To reveal the characteristics of the metacaspase (MC) gene family and the relationship between metacaspases and the seedless trait, we identified the 6 V. vinifera metacaspases VvMC1–VvMC6, from the grape genome, using BLASTN against the 9 known Arabidopsis metacaspases. We also obtained full-length cDNAs by RT-PCR. Each of the 6 grape metacaspases contains small (p10-like) and a large (p20-like) conserved structural domains. Phylogenetic analysis of 6 grape and 9 Arabidopsis metacaspases showed that all metacaspases could be grouped into two classes: Type I and Type II. Each phylogenetic branch shares a similar exon/intron structure. Furthermore, the putative promoters of the grape metacaspases contained cis-elements that are involved in grape endosperm development. Moreover, expression analysis of metacaspases using real-time quantitative PCR demonstrated that VvMC1 and VvMC2 were able to be detected in any tissue, and VvMC3, VvMC4, VvMC5 and VvMC6 exhibited tissue-specific expression. Lastly, in cv. Thompson seedless grapes VvMC1, VvMC3, and VvMC4 were significantly up-regulated at the 35 DAF during ovule development, roughly same stage as endosperm abortion. In addition, the expression trend of VvMC2 and VvMC5 was similar between cv. Pinot Noir and cv. Thompson grape ovule development and that of VvMC6 was sustained in a relatively low level except the expression of cv. Pinot Noir significantly up-regulated in 25 DAF. Our data provided new insights into PCD by identifying the grape metacaspase gene family and provide a useful reference for further functional analysis of metacaspases in grape.

‘Faith’, ‘Gratitude’, ‘Hope’, and ‘Joy’ Seedless Table Grapes

‘Faith’, ‘Gratitude’, ‘Hope’, and ‘Joy’ seedless table grapes are new cultivars (Vitis hybrid) developed by the University of Arkansas Division of Agriculture, bringing to 12 the total number of releases from the program. These releases expand options for table grape growers for local markets in the United States. All four cultivars have nonslipskin flesh with good skin quality, fruit cracking resistance, good vine health, and winterhardiness. ‘Faith’ is blue-fruited, slight fruity to neutral in flavor, semicrisp, and ripens in late July to early August, the earliest of the four. ‘Gratitude’ berries are green with exceptional flesh crispness and neutral flavor, ripening usually in late August at this same location. ‘Hope’ is green-fruited, has a fruity flavor, is rather soft in texture, has high production potential, and ripens near 19 Aug. ‘Joy’ is blue-fruited with exceptional fruity flavor but very soft texture and ripens the first or second week of August. All ripening dates are for west–central Arkansas. All of the cultivars exhibit stenospermocarpic seedlessness. These cultivars, in addition to prior releases from the program (including ‘Mars’, ‘Jupiter’, and ‘Neptune’) (Clark, 2010), provide for a range of dates of harvest along with choices of fruit colors, shapes, textures, and flavors. These should be adapted in the midSouth and Pacific Northwest of the United States but should be further evaluated for adaptation and hardiness in the Midwest. It is envisioned that these cultivars will be best used by local-market growers, because they differ from commercial table grapes commonly found in retail markets in the United States in that these new releases tend to be smaller-fruited, softer in texture, and can vary in seed remnant size compared with most commercial cultivars. Origin

Validation Assay of p3_VvAGL11 Marker in a Wide Range of Genetic Background for Early Selection of Stenospermocarpy in Vitis vinifera L.

DNA markers technology, derived from research in molecular biology and genomics, offers great promise for plant breeding, allowing the "molecular breeding" via marker-assisted selection. Grapevine genomic resources allowed, in recent years, the characterization at molecular level of genes involved in interesting phenotypes such as stenospermocarpic seedlessness, a trait really appreciated by consumers. Recent studies in table grapes revealed that the VvAGL11 gene, member of the D-lineage MADS-box family, controls the ovule identity, and thus potentially playing an important role in stenospermocarpy. Intragenic markers of VvAGL11 have been found and tested for breeding purposes. In the present paper, we describe an in deep assay on a total of 475 genotypes derived by our own grape germplasm and seeded×seedless crosses F1 offspring, to evaluate and verify the "diagnostic" power of VvAGL11 in marker-assisted selection. We found only 8/475 that were seeded and carried the seedless-associated allele in the STS p3_VvAGL11. However, and most importantly, there were no seedless varieties without such allele. We validated the marker as a 100% effective tool for early negative selection of stenospermocarpy in Vitis vinifera L. crosses.

Marker assisted selection for seedlessness in table grape breeding

Seedlessness is one of the most appreciated traits in the table grape (Vitis vinifera L.). The development of new seedless varieties is expensive and time consuming, involving the generation and selection of thousands of hybrids each year. In seeded × seedless crosses, seedlessness commonly segregates 1:1, so molecular markers allowing for the early identification of plants that will produce seedless berries are very useful. This early selection can lead to savings in maintenance and evaluation costs, and allows additional space for larger effective progenies. The variety Sultanina has been the main source of stenospermocarpic seedlessness in table grape breeding. In a previous work, we showed a 198-bp allele at the VMC7F2 microsatellite locus as a potential marker for selection of seedless genotypes due to its close linkage to the major effect seedless QTL, SDI. In this study, we show that stenospermocarpic bred cultivars share a main haplotype around this locus not found in seeded cultivars, which facilitating the development and use of marker assisted selection (MAS) strategies for the selection of seedless plants. In this way, VMC7F2 on its own can be a very useful marker for selecting seedless individuals from segregating crosses. A MAS program based on the presence of the 198-bp allele at VMC7F2 allows the reduction of the progeny size to 54%, selecting most of the seedless individuals. In addition, our results show the existence of other possible sources of stenospermocarpic seedlessness that could provide alternative sources of genetic variation for breeding of this trait.

Molecular, genetic and transcriptional evidence for a role of VvAGL11 in stenospermocarpic seedlessness in grapevine

BackgroundStenospermocarpy is a mechanism through which certain genotypes of Vitis vinifera L. such as Sultanina produce berries with seeds reduced in size. Stenospermocarpy has not yet been characterized at the molecular level.ResultsGenetic and physical maps were integrated with the public genomic sequence of Vitis vinifera L. to improve QTL analysis for seedlessness and berry size in experimental progeny derived from a cross of two seedless genotypes. Major QTLs co-positioning for both traits on chromosome 18 defined a 92-kb confidence interval. Functional information from model species including Vitis suggested that VvAGL11, included in this confidence interval, might be the main positional candidate gene responsible for seed and berry development.Characterization of VvAGL11 at the sequence level in the experimental progeny identified several SNPs and INDELs in both regulatory and coding regions. In association analyses performed over three seasons, these SNPs and INDELs explained up to 78% and 44% of the phenotypic variation in seed and berry weight, respectively. Moreover, genetic experiments indicated that the regulatory region has a larger effect on the phenotype than the coding region. Transcriptional analysis lent additional support to the putative role of VvAGL11's regulatory region, as its expression is abolished in seedless genotypes at key stages of seed development. These results transform VvAGL11 into a functional candidate gene for further analyses based on genetic transformation.For breeding purposes, intragenic markers were tested individually for marker assisted selection, and the best markers were those closest to the transcription start site.ConclusionWe propose that VvAGL11 is the major functional candidate gene for seedlessness, and we provide experimental evidence suggesting that the seedless phenotype might be caused by variations in its promoter region. Current knowledge of the function of its orthologous genes, its expression profile in Vitis varieties and the strong association between its sequence variation and the degree of seedlessness together indicate that the D-lineage MADS-box gene VvAGL11 corresponds to the Seed Development Inhibitor locus described earlier as a major locus for seedlessness. These results provide new hypotheses for further investigations of the molecular mechanisms involved in seed and berry development.