Development and application of KASP marker for high throughput detection of the seedless trait in grapevine

Molecular marker-assisted selection(MAS) can significantly accelerate and improve the efficiency of the breeding process in seedless grape cultivars. In this study, we developed the KASP_VviAGL11 and VviAGL11_410 markers based on a single nucleotide polymorphism(SNP) site(Chr18: 26889437(A/C)) of the VviAGL11 gene, and compared them with previously reported SSR markers p3_VvAGL11 and 5U_VviAGL11 by testing 101 cultivars and 81 F1 hybrid progenies. The results showed that both of the proposed markers obtained 100% accuracy rates in detecting allele A, which was closely associated with the seedless trait in grapes, while p3_VvAGL11 and 5U_VviAGL11 had lower accuracy rates due to their tendency to produce false positives. After careful evaluation of the technical advantages and disadvantages associated with these markers, we concluded that KASP_VviAGL11 was superior in terms of simplicity,cost-effectiveness, efficiency, and accuracy. Thus, we optimized the process of molecular MAS for seedless grapes,focusing on the KASP_VviAGL11 marker as a central component, to provide key technical support for the development of new seedless grape cultivars.

Screening Test of Small Seedless Watermelon Varieties in Beijing Area

[Objectives]This study was conducted to select small seedless watermelon varieties with excellent quality and high yield suitable for planting in Beijing.[Methods]Under the conditions of facility cultivation in Beijing,a screening test was conducted on seven small seedless watermelon varieties for growth process,field traits,yield traits,fruit quality,and disease resistance.[Results]Varieties Mitong,Motong and Tianbao Xiaowuzi had a short growth period and relatively stable growth,and bore fruit easily,and their female flowers were closely spaced.Their yields exceeded 60000 kg/hm~2,ranking in the top three among all varieties.The fruit had a thin skin,a bright red flesh color,and a high mass fraction of soluble solids in the center.The flesh was tight or crispy,and tasted good.They also showed good disease resistance.Overall,they showed excellent comprehensive agronomic traits,and are thus suitable for facility cultivation in Beijing and its surrounding areas.[Conclusions]This study provides a theoretical basis for the upgrading of varieties and the increase in production and farmers'income in the suburbs of Beijing.

Effects of Different Concentrations of Paclobutrazol on Seedling Quality of Small Seedless Watermelon in Autumn

[Objectives]The paper was to explore the effects of different concentrations of paclobutrazol on seedling quality of small seedless watermelon in autumn.[Methods]The leaves of watermelon seedlings were sprayed with 30,50,80 and 120 mg/L paclobutrazol at cotyledon expansion stage,and those sprayed with tap water were used as control.[Results]All the treatments of paclobutrazol effectively inhibited the excessive growth of watermelon at seedling stage,significantly enhanced the leaf quality of watermelon seedlings and promoted root growth.The seedlings treated by 50 mg/L paclobutrazol had the best performance compared with the control:a decrease of hypocotyl by 37.9%,an increase of plant dry weight by 46.55%,significantly increased seedling strength index,deep green leaf color,thick leaves,an increase of specific leaf weight by 28.13%,and significantly heavier root weight and longer length.[Conclusions]The results will provide a reference for strong seedling cultivation of small seedless watermelon.

An Effective Method for Extracting Total RNA from Young Embryo of Seedless Litchi

A total RNA extraction method for young embryo of seedless litchi was introduced. CTAB, Phenol （saturated with water）, chloroform, Guanidine isothioeyanate were used as main extraction reagents. Polyphenolie compounds were removed effectively by added PVP into the extraction buffer solution. RNA was purified intensively by phenol, chloroform extraction, and ethanol deposition after deposited by LiCl. Both the results of formaldehyde denatured agarose gel eleetrophoresis and ultraviolet spectrophotometer analysis showed high integrity and purity of RNA. So the quality of extracted RNA could meet the demand of most molecular biology experiments that require higher quality RNA.

Flame Seedless葡萄胚珠、胚乳及胚发育与败育的研究

对Flame Seedless葡萄胚珠、胚乳及胚的发育和败育过程进行了系统的细胞学观察,并以有核葡萄品种北醇为对照对Flame Seedless的胚珠重量及纵横径进行了测定.结果表明:(1)Flame Seedless内、外珠被及珠孔发育异常,20%～30%的子房未出现受精现象,受精的胚珠重量和纵横径随着胚珠的发育,先呈增加趋势,在某一时期达到最高值,随后下降,最终退化为痕迹.(2)大约有30%的胚乳核能够进行正常分裂,形成胚乳组织,胚乳在花后30 d开始退化,其余胚乳核分裂异常.合子在盛花后第19天进行第一次分裂,经过二细胞原胚→多细胞原胚→球形胚,此后胚开始退化;也有的合子始终不发生分裂,最终退化.(3)由于胚乳分裂异常和提早退化,胚缺乏营养致使其发育中止和败育.(4)Flame Seedless胚挽救的最佳接种时期在花后第37天.

Influence of Gamma Irradiation on Seedless Citrus Production: Pollen Germination and Fruit Quality

Seedlessness is a desirable characteristic in citrus fruits sold for fresh consumption. Gamma irradiation is widely used to obtain seedless citrus fruits. Here, different clones of the self-incompatible parthenocarpic ‘Moncada’ mandarin, obtained by gamma irradiation, were studied to assess seedlessness, pollen germination, fruit characteristics and qua- lity attributes. Findings indicate that irradiation altered aspects other than seedlessness, such as pollen germination, and some of the clones presented different weight, size, acidity and maturity index. Fruit quality and nutritional bio- components were affected differently;some clones presented no changes compared to the control ‘Moncada’ man-darin, while other clones showed significant differences. In general, all clones examined presented low seed numbers and re- duced pollen viability. Some of these clones, which ripen late in the season and whose fruit quality is maintained or improved, are in the process of registration.

Rapid Seedless Synthesis of Gold Nanoplates with Microscaled Edge Length in a High Yield and Their Application in SERS

We report a facile and reproducible approach toward rapid seedless synthesis of single crystalline gold nanoplates with edge length on the order of microns.The reaction is carried out by reducing gold ions with ascorbic acid in the presence of cetyltrimethylammonium bromide(CTAB).Reaction temperature and molar ratio of CTAB/Au are critical for the formation of gold nanoplates in a high yield,which are,respectively,optimized to be 85 °C and 6.The highest yield that can be achieved is 60 % at the optimized condition.The synthesis to achieve the microscaled gold nanoplates can be finished in less than 1 h under proper reaction conditions.Therefore,the reported synthesis approach is a time-and costeffective one.The gold nanoplates were further employed as the surface-enhanced Raman scattering substrates and investigated individually.Interestingly,only those adsorbed with gold nanoparticles exhibit pronounced Raman signals of probe molecules,where a maximum enhancement factor of 1.7 9 10~7 was obtained.The obtained Raman enhancement can be ascribed to the plasmon coupling between the gold nanoplate and the nanoparticle adsorbed onto it.

QTL-seq analysis of the seed size trait in grape provides new molecular insights on seedlessness

Seedlessness in grape(Vitis vinifera)is an important commercial trait for both the fresh and drying markets.However,despite numerous studies,the mechanisms and key genes regulating grape seedlessness are mostly unknown.In this study,we sequenced the genomes of the V.vinifera seeded cultivar‘Red Globe’,the seedless cultivar‘Centennial Seedless’,and the derived hybrids.Nonsynonymous single nucleotide polymorphisms(SNPs)were identified by genome sequencing and analyzed using published transcriptome data.Nonsynonymous SNPs occurred in genes related to seed development,which were identified as protein kinases,transcription factors,and cytochrome P450 s and showed differential expression during ovule development in both seeded and seedless grapes.These nonsynonymous SNP-associated genes were mainly involved in biological processes such as hormone balance,seed coat and endosperm development,reproductive organ development,oxidation and reduction,senescence and cell death.A potential quantitative trait locus(QTL)region associated with seed size was characterized based on the SNP-index,and expression analysis of candidate genes in the QTL region during ovule development in multiple seeded and seedless grape cultivars were conducted.Three SNPs were further subjected to SNa Pshot analysis and one SNP in G8 showed 67.5%efficiency in the grape progeny validation.Overall,the data obtained in this study shed light on the differences in seed development between seeded and seedless progeny at the genomic level,which provides valuable resources for future functional studies and grape breeding.

Immature Embryo Rescue and in Vitro Development Evaluation of Intraspecific Hybrids from Brazilian Seedless Grapevine“Superior×Thompson”Clones

The fruit production for export is an economically significant activity in the Valley of S?o Francisco River, especially in the irrigated lands of Petrolina-PE/Juazeiro-BA, Brazil. The development of new genetic material most suitable to the tropical climate and the demands of the consumer market have led to the selection of new seedless grapes cultivars. In this case, the use of the embryo rescue technique has produced satisfactory results for obtaining such materials, especially in the semiarid region. This study aimed to evaluate the in vitro development of intraspecific hybrids of grapevine (Vitis vinifera L.), derived from the rescue of immature embryos resultant from the crossing of “Superior Seedless” and “Thompson Seedless” Brazilian clones. To establish and develop the cultivation, the culture media was supplemented with 30 g/L sucrose, 0.1 g/L myo-inositol, 0.002 g/L glycine, 0.1 mg/L indoleacetic acid (IAA), 6.5 g/L of agar, adjusted pH to 5.7. The experiment was evaluated after 90 days. The variables measured were: number of nodes, number of leaves, plant height (cm), number of roots and length (cm) of the root system and internodes. The period of 60 days of in vitro culture of ovules resulted in the highest values of embryos (about 50%), as well as better characterized developmental stages with higher germination (47.3%). The three types of hybrid grapes evaluated in micropropagation showed very similar values of the measured parameters, even having originated from embryos of different developmental stages.

Cloning and Sequence Analysis of a Cysteine Proteinase Inhibitor Gene from Seedless Litchi

[Objective] This study aimed to clone and analyze the cysteine proteinase inhibitor gene from seedless litchi. [Method] According to the EST se- quence of cysteine proteinase inhibitor in constructed SSH snhtraetive library of seedless litchi abortion, nucleotide sequence of the cysteine proteinase inhibitor gene was obtained by using RACE technology and analyzed by using bioinformatics software. [ Result ] A cysteine protease inhibitor gene was obtained with the sequence of 635 bp containing a 321 bp open reading frame. It was predicted that the erlcoded protein contained 106 amino acids with conserved domain of cysteine proteinase inhibitor and had relatively high homology with the cysteine proteinase inhibitor gene of several species, [ Conclusion] This study laid the foundation for further ex- ploring the physiological functions of this cysteine proteinase inhibitor gene in plants.

Effect of Different Shading Practices Used at Pre-harvest Stage on Quality and Storage Life of Sultana Seedless Grapes

Covering the grapevine rows to delay maturity and thus the harvest date becomes a widespread practice at Sultana seedless vineyards. The research work was carried out with different covers to examine their effects on grape quality and storage life in 2009 and 2010. Grapevines were covered with three different densities （shading ratios： 35%, 55%, 75%） at the veraison period. Unshaded （0%） control grapevines and grapevines shaded under different ratios were covered with polypropylene cross-stitch just before harvest maturity. Grapes under cover were harvested nearly one month after than those maturing under open conditions. Grape clusters were packed in PE bags and pre-cooled after harvest and then SO2-generating pads has been put on top, bags were sealed. In the second year at the 120th day of the storage, decay development has been observed in all applications. Total soluble solids content, maturity index and a＊ colour value were found lower in 55% and 75% shaded grapes compared to unshaded grapevines. Still, berry removal force and hue angle value in 55% and 75% of shaded grapes were higher than unshaded grapes. Effects of shading ratios on these parameters decrease or disappear at the end of storage.

Influence of Bioregulator Gibberellic Acid on Some Technological Characteristics of Cluster and Berry from Some Seedless Grape Varieties

Solutions of gibberellic acid prepared at three concentration levels including 5, 10 and 20 mg/L, were applied at two seedless grape varieties, Thompson and Belgrade, by spraying, during the three different periods of the vine growing： before blooming, after blooming and before veraison, in order to study their influence on some cultural technological characteristics. Dimension and shape of the cluster and berry, mechanical characteristics of the berries and chemical content of the must （sugar and total acids） has been also investigated. It was noticed that the concentration of gibberellic acid had influence on the technological characteristics of the berries in all grape growing periods tested. The addition of gibberellic acid at concentration of 20 mg/L increased the weight of the cluster and berry, and increased the transportability of the berries belonging to the two seedless varieties.

A Good Seedless Ponkan Cultivar—Qianyang Seedless Ponkan

Qianyang Seedless Ponkan selected from common ponkan is a sport deviation cultivar without any seeds in its fruit. Matures in mid November, it has bigger avg.124.5g fruit, and the yield is 30 t ha-1 after 5 years' growing. There are no obvious in-year or off-year phenomena. Its fruits can store well without any treatments at room temperature for three or four months.

葡萄无核晚熟新品种中葡萄18号的选育

中葡萄18号是以无核紫、玫瑰香杂交育种结合无核胚挽救技术选育出的无核晚熟新品种。该品种自然坐果平均单穗质量600 g,平均单粒质量7.3 g,果粒长椭圆形,紫色或紫红色,可溶性固形物含量(w,后同)18.0%,总糖含量16.2%,总酸含量3.06 g·kg^(-1),糖酸比达到54∶1,单宁含量686 mg·kg^(-1),风味甜香,品质上等。在河南郑州露地栽培成熟期为9月初。该品种树势中庸,适宜避雨栽培。

大果少核椪柑新品种华柑3号的选育

华柑3号是通过鄂柑1号椪柑珠心胚实生苗倍性变异而培育的四倍体大果少核椪柑新品种。果实扁圆形,果实大,平均横径74.08 mm,纵径62.63 mm,平均单果质量179.94 g,平均果皮厚度3.45 mm。果肉橙黄色,肉质脆嫩,酸甜爽口,风味浓,可溶性固形物含量(w,后同)为14.49%,可滴定酸含量为1.16%,可食率为71.6%,化渣性好;种子少,平均2.7粒·果^(-1);树势中等偏强,树姿较直立,结果母枝以春梢和秋梢为主;在湖北武汉地区,12月中下旬果实成熟,可挂树至翌年1月;丰产性中等,按3.0 m×4.0 m密度定植的5年生树,每666.7 m^(2)产量可达1500 kg;适宜在年均温度16℃左右,冬季无霜冻、绝对最低温度在-5℃以上的区域种植。

浙江地方特色品种温岭高橙无核潜力新种质创制及分子鉴定

【目的】温岭高橙是浙江台州地方特色品种,栽培历史悠久,且具有一定的保健和药用价值,但果实有核限制了其在台州以外地区的发展,因此利用细胞工程高效育种技术对温岭高橙进行无核化改良。【方法】通过2条育种途径,一是以二倍体温岭高橙为母本与异源四倍体为父本倍性杂交,授粉后90 d利用幼胚离体挽救培养和流式细胞仪倍性鉴定创制三倍体无核新种质;二是以雄性不育胞质杂种华柚2号为母本与温岭高橙有性杂交,通过成熟种子催芽播种,创制二倍体无核新种质。利用SSR分子标记对杂交后代进行分子鉴定。【结果】以温岭高橙为母本、异源四倍体NS和BDZNS为父本配置2个倍性杂交组合,授粉145朵花,坐果74个;胚抢救幼嫩种子1520粒,离体培养获得再生植株553株;用流式细胞仪对再生植株进行倍性鉴定,获得三倍体45株、四倍体8株;以华柚2号为母本与温岭高橙有性杂交,授粉69朵花,坐果20个,获得成熟种子312粒;通过催芽播种获得实生后代203株,倍性鉴定均为二倍体;用SSR分子标记对2个倍性杂交组合的21株三倍体和8株四倍体后代及华柚2号×温岭高橙随机选取的29株二倍体后代进行分子鉴定,显示所鉴定的三倍体及二倍体后代均含有父本特异条带,为其杂交双亲的有性后代,而倍性杂交获得的四倍体后代与母本条带完全一致,推测其为母本珠心细胞自然加倍形成的同源四倍体(双二倍体)。【结论】研究结果为温岭高橙无核新品种培育奠定了宝贵的材料基础。

鲜食葡萄无核化研究进展

优质、大粒、无核鲜食葡萄深受消费者青睐,利用植物生长调节剂进行葡萄无核化生产已成为获取优质、大粒、无核鲜食葡萄的重要途径,并在生产上得到广泛应用。本文综述了葡萄无核化机理、常用植物生长调节剂种类及其使用浓度和处理方法、无核化栽培技术及需注意的问题等,以期为葡萄无核化生产提供理论依据和参考。

无籽西瓜新品种墨绿无籽5号的选育

墨绿无籽5号是以自交系GR4x-2为母本,以2x-55为父本配制而成的大果型无籽西瓜一代杂种。中晚熟,早春大棚栽培全生育期112 d(天)左右,果实发育期40 d(天)左右。果实正圆形,果皮墨绿色覆深绿色网条纹,果皮厚度1.2 cm,硬度27 kg·cm^(-2)左右,耐裂、耐贮运;瓜瓤红色,中心可溶性固形物含量约12.1%,边部可溶性固形物含量约9.8%。

葡萄褐纹病菌侵染过程的细胞学特性研究

为明确褐纹病菌侵染葡萄叶片的过程,采用光学显微镜、扫描电镜和透射电镜等技术手段,系统研究了葡萄褐纹病菌在‘无核白’和‘克瑞森无核’葡萄叶片上的侵染过程。结果表明:葡萄褐纹菌分生孢子在‘无核白’‘克瑞森无核’葡萄叶片接种1 d后均可萌发;后期发育过程,‘克瑞森无核’葡萄叶片上菌丝细长,生长停滞;‘无核白’葡萄叶片表面初生菌丝密布,持续在寄主体表生长扩展;接种3 d,‘无核白’葡萄叶片的表面形成了密集的菌丝网,同时观察到菌丝从气孔或直接入侵方式进入叶片表皮组织内部,随后,菌丝以胞间和胞内生长的方式扩展;接种6 d,菌丝达到上表皮,随菌丝在叶片组织内不断地生长扩展,使得寄主细胞坏死、解体。可见:‘无核白’是亲和葡萄褐纹病菌品种,而‘克瑞森无核’葡萄非亲和褐纹病菌。

葡萄无核性状分子标记通用性验证

【目的】利用葡萄自然群体对已有9个无核分子标记进行评价,验证其无核检测通用性效果,为加快无核葡萄新品种选育进程提供技术支持。【方法】以88个无核种质和120个有核种质组成的自然群体为试材,对国内外已发表的9个常用葡萄无核分子标记进行通用性验证。【结果】SCAR类型标记GSLP1-569、SCC8-1080和SCF27-2000的鉴定准确率分别为57.12%、72.20%和75.38%,无核检测率分别为90.51%、79.18%和67.82%。SSR类型标记p1-VvAGL11、p2-VvAGL11、p3-VvAGL11、5U_VviAGL11、VMC7F2和VVSD10的鉴定准确率分别是88.47%、67.43%、71.94%、68.47%、67.99%和61.60%,无核检测率分别是87.74%、77.22%、90.72%、90.80%、79.10%和63.03%;卡方分析表明与无核表型极显著相关的等位点分别是250 bp、171 bp、195 bp、315 bp、197 bp和105 bp。【结论】SCAR标记SCF27-2000和SSR标记p1-VvAGL11准确率高,综合表现最优,适用于无核葡萄新品种的分子辅助选择。