Embryo Rescue and Moleclar Marker-Assisted Selection of Hybrid Seedless Grape

Seedless grapes play an important role in fresh food and dry production. New varieties breeding by hybridization with seedless varieties as female parents is the most effective way to cultivate seedless varieties. However, the embryos of Seedless varieties can not develop normally, so it is dicult to obtain hybrid offspring as hybrid female parent. Moreover, grape is a perennial tree species with highly heterozygous genes, with long breeding cycle and low eciency. In this study, embryo rescue technology was used to cultivate hybrid offspring by crossing with ‘Ruby Seedless’ as female parent and ‘Hongqitezao’ as male parent, so as to solve the problem that seedless varieties can not be female parent; and molecular technology was used to carry out assisted breeding research to solve the problems of long cycle and low eciency. TP-M13-SSR technique was used to carry out authenticity breeding. SCAR marker SCF27-2000 was used to detect the seedless traits of hybrid plants, phenotypic traits was used to verify the results of molecular markers, and Seedless trait-related SSR markers VMC7F2, VrSD10 and P3_VvAGL11 was used to detect and verify the genotypes of individual plants with inconsistent detection results by the two methods. In this study, a total of 384 hybrid offspring were nally obtained, and the hybridization rate was 84.43%. A total of 163 fruit-bearing plants were identied, and the phenotypes of their seeds were identied. The coincidence rate of genotypic and phenotypic analyses was 93.88%. Additionally, 305 F 1 plants were detected using the SCF27-2000 marker, and the abortion rate was 64.92%. We speculate that the inconsistent results were caused by parthenocarpy, SCF27 marker limitation, among other factors. Overall, this study shows that embryo rescue is an effective method for breeding seedless grape cultivars, and the application of molecular markers could facilitate the early identication of hybrid traits,and improve breeding eciency. the genotypes by three suggesting related with the genes linked the three One of the nineteen F 1 plants carried a band labeled with SCF27, and its genotypes at the three loci were 192/192, 99/99 and 178/178, respectively. Plants with these genotypes should be seeded, indicates that SCF27 has limitations in detecting homozygous nucleated genotypes, consistent with the conclusions of Adamblondon et et al.


Introduction
Grape (Vitis vinifera L.) is grown all over the world, and has the second largest cultivation area and yield among all fruit crops (Kong 2002). The seedless trait is an important index of grape fruit. Seedless varieties ful ll the market for table grapes, which are usually consumed fresh or as raisins after drying. Therefore, the development of seedless cultivars is an important goal of grape breeding projects. In the past, because the embryos of seedless varieties could not develop normally, seedless varieties could be selected only through conventional hybridization, with very low e ciency (Zhao et al. 2004). With the application of tissue culture technology in hybrid breeding, seedless varieties can be used as female parents to realize high-e ciency seedless grape breeding (Ramming 1982(Ramming , 1990. A number of seedless grape varieties have been developed to date using the tissue culture technology, including 'Qincui seedless', 'Qinhong seedless' (Wang et al. 2004 With the development of molecular biology techniques, molecular markers are being increasingly used in grape breeding. The simple sequence repeat (SSR) marker TP-M13-SSR uses uorescence to detect the PCR ampli cation products. Because of its good repeatability, high accuracy and simple operation procedure, the TP-M13-SSR marker has been used for grape ngerprinting and genetic relationship analysis (Ma et al. 2018; Wang et al. 2020 ).
To date, great progress has been made in the investigation of the seedless traits of grape using molecular markers. In 1998, Lahogue and colleagues developed the SCP18 and SCC8 markers linked to the seedless trait gene, and proposed the seed development inhibitor (SDI) hypothesis, according to which the seedless trait is controlled by a single dominant gene and several recessive genes (Lahogue et al. 1998). Subsequently, many scholars con rmed the existence of SDI loci through quantitative locus (QTL) mapping (Cabezas et al. 2006;Costantini et al. 2008;Doligez et al. 2002;Mejía et al. 2007). The seedless trait is mainly controlled by the main SDI gene located on chromosome 18 (Cabezas et al. 2006;Costantini et al. 2008;Mejía et al. 2007). Wang et al. (2002) developed an 18-bp speci c probe, GSLP-569, which can be used to detect non-nuclear genes; this marker was named as GSLP1. Mejia and Hinrichsen (2003) reported a sequence characterized ampli ed region (SCAR) marker, SCF27, which was associated with the seedless trait of grape. Wang et al. (2018) considered that the genes associated with SCP18 and SCC8 markers were recessive or minor genes, which could be detected in both nucleated and seedless varieties. The seedless gene probe GSLP1 could only detect seedless varieties related to 'Thompson seedless'.
The gene marked by SCF27 is a dominant or major gene, which can be used for the identi cation of seedless traits. Cabezas et al. (2006) reported the SSR marker VMC7F2. QTL analysis showed that VMC7F2 is closely linked to a 198-bp allele at the SDI site, which can be used for the seedless character detection. The MADS-box gene VvAGL11 is homologous to known genes expressed in ovule and during seed development The objective of this study is to apply embryo rescue technology and molecular biotechnology to grape seedless hybrid breeding to solve the problems of low rate of Seedless offspring and low e ciency of seedless grape breeding.

Plant materials
Eight-year-old seedless grape variety 'Thompson seedless' was used as the control. Additionally, 8-year-old 'Ruby Seedless' and 6-year-old 'Hongqitezao' grape varieties were used as female and male parents, respectively, to generate 305 F 1 hybrids in 2017 (Table 1, Fig. 1). Primer pairs for ve SSR markers and one SCAR marker used in this study are listed in Table 2.
Hybridization When 15% of the ower buds were open, the middle and upper robust in orescences of vigorous plants were selected for pollen collection. The emasculation of in orescences began 3-4 days before anthesis. The collected pollen were transferred to a 20-ml glass bottle and dried at low temperature. After emasculation, the in orescences were rinsed with clean water. Pollination was started when mucus appeared on the stigma, and was performed once a day for 3 days.
Ovule culture Young hybrid fruits were picked approximately 55 days after pollination, and placed in a 500-ml glass bottle. The fruits were washed with tap water for 20 min, soaked in 75% ethanol for approximately 1 min, and washed once with sterile water. Then, the fruits were disinfected with 0.5% (w/v) NaClO for 20 min, and washed twice with sterile water. Subsequently, the ovules were peeled, and 25-30 peeled ovules were placed in a 100-ml plastic bottle containing solid-liquid double-layer Emershad and Ramming (ER) medium (pH 6.0) supplemented with 500 mg/L CH, 1.21 g/L cysteine, 60 g/L sucrose, 3 g/L activated carbon and 0 or 7 g/L agar.

Embryo culture
After ovule culture for 8 weeks, young embryos were peeled, and embryo germination culture was started in 100-ml transparent glass test tubes containing Woody Plant (WP) medium supplemented with 20 g/L sucrose, 3 g/L activated carbon and 7 g/L agar. One young embryo was placed in each bottle.

Subculture
After the young embryo developed into healthy seedlings, the stem segments were cut and placed in 100-ml transparent plastic bottles (2-3 stem segments per bottle) containing half-strength Murashige and Skoog (1/2 MS) medium supplemented with 0.35 mg/L IBA, 20 g/L sucrose and 7 g/L agar. The stem fragments were cultured under LED light.

Transplantation of embryo rescue-derived seedlings
After the stem segments developed into seedlings, the robust seedlings were transplanted into the coconut bran: rotten leaf soil: vermiculite (2:1:1) mix under sterile conditions. The substrate and water used for transplanting seedlings were disinfected. After transplanting, the seedlings were covered with a transparent cover to maintain moderate humidity. Finally, the seedlings were transferred to the greenhouse for cultivation.
Breeding of seedless grape Grape varieties 'Ruby Seedless' and 'Hongqitezao' were crossed in mid-May 2017. The ovules were stripped in mid-July 2017, and young embryos were stripped in late October 2017. Test-tube seedlings were subcultured and propagated in early December, and transplanted in mid-January 2018. In April, the survived seedlings were transferred to the greenhouse. Hybrid seedlings were harvested in mid-November and planted in the facility greenhouse in February 2019. Some results will be obtained in 2020, while the majority of results will be obtained in 2021.

Identi cation of authentic hybrids
Using the genomic DNA of 'Ruby Seedless' and 'Hongqitezao', two pairs of SSR-speci c primers, Vchr4a-166/173bp and Vchr18a-159/172bp (Table 2), were selected from a collection of 16 primer pairs of SSR labeled primers, and used to identify authentic 305 hybrid offspring via the TP-M13-SSR technology PCR was performed under the following conditions: initial denaturation at 94℃ for 4 min, followed by 35 cycles of denaturation at 94℃ for 40 s, annealing at 55℃ or 53℃ for 40 s and extension at 72℃ for 1 min, and a nal extension at 72℃ for 10 min. The PCR products were determined via capillary electrophoresis by Beijing Junweinuo Technology Co., Ltd.
Marker assisted selection of seedless traits The seedless trait of 305 F 1 hybrids was screened using the seedless molecular marker SCF27-2000bp (Table 2), and the individual plants with the 2,000bp PCR product were identi ed. PCR was performed under the following conditions: 94℃ for 5 min, followed by 35 cycles of 94℃ for 30 s, 55℃ or 51℃ for 30 s, and 72℃ for 1.5 min, and nally 72℃ for 10 min. Ampli cation products were separated by electrophoresis on 1.8% agarose gels, and photographed.
Identi cation of seedless traits and genotyping of seedless loci A total of 163 hybrid progenies were used to monitor seed development and nucleation. The genotypes of some individual plants were determined using SSR markers such as VMC7F2, VrSD10 and P3_VvAGL11 (Table 2). PCR reactions were carried out according to the corresponding references, and PCR products were determined via capillary electrophoresis by Beijing Junweinuo Technology Co., Ltd.

Results
Breeding of seedless grape via embryo rescue In this study, 7,400 ovules were obtained from the 'Ruby Seedless' ´ 'Hongqitezao' cross. A total of 1,296 embryos were obtained using the embryo rescue technology, resulting in 454 seedlings, of which 384 survived after transplanting. At present, 163 seedlings have been obtained. The embryo stripping and germination rates were 17.51% and 35.03%, respectively, while the seedling rate was 84.58%. The fruiting rate in the third year after planting was 42.45% (Table 3). In this study, 305 robust individual plants were selected from a collection of 384 hybrid seedlings, and the genotype of these hybrids was veri ed using primers for SSR markers Vchr4a and Vchr18a, which can amplify distinct bands from 'Ruby Seedless' and 'Hongqitezao'. As shown in Table 5  The molecular marker SCF27-2000, linked to the seedless trait-controlling gene of grape, was used to identify the three grape varieties, 'Thompson seedless' (seedless control), 'Ruby Seedless' and 'Hongqitezao' (Fig. 4). The results showed that a 2,000-bp band was ampli ed from the seedless varieties 'Thompson seedless' and 'Ruby Seedless' but not from the seeded variety 'Hongqitezao'. This shows that the SCF27-2000 marker is suitable for the detection of the seedless trait in hybrid populations of 'Ruby Seedless' × 'Hongqitezao'. The results of 163 individual plants tested using the SCF27-2000 marker are shown in Table 6. Among the 163 individual plants, 98 carried the target bands, of which 92 were abortive; thus, the rate of coincidence between genotypic and phenotypic detection methods was 93.88% (Fig. 4, Table 5). Of the 65 plants that did not carry the target band of the marker, only 52 were seeds; thus, the coincidence rate was 80.00% (Fig. 4, Table 5).
Detection of the seedless trait in 305 F 1 hybrids using the SCAR marker SCF27-2000 The seedless trait of 305 F 1 hybrids was tested using the SCAR marker SCF27-2000. The results showed that 198 plants carried the 2,000-bp target band of 2000bp, accounting for 64.92% of the total samples (Table 6).  genotypes. The results obtained using VrSD10 and P3_VvAGL11 markers were identical to those obtained using the VMC7F2 marker (Table 8). The VMC7F2 marker detected two 190/190 genotypes, three 192/192 genotypes and eight 190/192 genotypes, which was ampli ed by three pairs of primers for seedless traits on 13 individual plants that did not carry 2,000-bp fragment but showed the abortion phenotype. Ten individual plants carrying the 190 genotype exhibited abortion, indicating that the phenotype was accurate, but the genotype determined using the SCF27 marker was incorrect.
Three individual plants with the 192/192 genotype showed abortion, which may be caused by the failure of normal pollination and fertilization, resulting in parthenocarpy and consequently misjudgment on behalf of the staff. The VrSD10 marker detected two 97/97 genotypes, three 99/99 genotypes, eight 97/99 genotypes and ten 97 genotypes. P3_VvAGL11 ampli ed two 188/188 genotypes, three 178/178 genotypes, eight 178/188 genotypes and ten 188 genotypes. The results obtained using VrSD10 and P3_VvAGL11 markers were identical to those obtained using the VMC7F2 marker (Table 9).  Hybridization between two seedless grape varieties or between a seeded grape variety (as male parent) and a seedless grape variety (as female parent) is an effective way to create new seedless grape varieties. Raming et al. (2000) used this approach to achieve a seedless ratio of 45-82%. In the current study, we used the seedless × seeded cross to achieve a seedless rate of 64.92%.
Most grape varieties are monoecious. The key to hybridization is to remove stamens. A proper time should be selected for emasculation. Stigma may be damaged if emasculation is conducted too early, and self-pollination may occur if the ower is emasculated too late. In this study, some problems were detected in the emasculation of 'Ruby Seedless' ×'Hongqitezao' combination, as approximately 14.10% of the offspring were self-pollinated. Sealing treatment after pollination is also important to ensure high hybridization e ciency. VMC7F2 (Pellerone et al., 2001) is an SSR marker located on grape chromosome 18, and is used for screening the seedless trait of grape cultivars.
Previous studies con rmed that VMC7F2 has a high application value, with high accuracy and stability in the process of marker-assisted selection of the seedless trait of grape ( In the current study, the genotypes of 18 F1 plants with seedless trait loci were detected by using the three markers whose molecular detection results were inconsistent with the eld identi cation results. The results showed that the genotypes detected by the three markers were almost the same, suggesting that it was related with the genes linked to the three markers.

Conclusion
The results of this study con rmed that embryo rescue is an effective method for breeding seedless grape varieties. The molecular marker technology can help realize the early identi cation of hybrid traits, reduce the number of ineffective plants, and improve the breeding e ciency. Con ict of interest The authors declare that they have no con ict of interest.

Abbreviations
Author Contributions All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Yong Wang, Yuling Li and Feng Sun. The rst draft of the manuscript was written by Yong Wang and all authors commented on previous versions of the manuscript. All authors read and approved the nal manuscript.
Data Availability The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.
Human and animal rights No human or animal material was used. The research conducted complied with all institutional and national guidelines.  Detection of three varieties by the seedless trait-speci c SCAR marker SCF27. M, Marker; 1, 'Thompson seedless'; 2, 'Ruby Seedless'; 3, 'Hongqitezao'