苹果S基因型数据库的建立与使用

苹果一般自花授粉不能结实,需配置一定比例的授粉品种,且依靠昆虫或人工授粉才能获得经济产量。苹果属于配子体型自交不亲和性类型,受17号染色体末端S基因座的复等位基因控制,最早人们将‘国光’品种2个同源染色体上控制自交不亲和性的等位基因分别命名为S_1和S_2之后(S基因型为S_1S_2),截至目前已从苹果属植物中鉴定出S_1、S_2、S_3、S_4、S_5、S_6、S_7、S_8、S_9等31种S等位基因,确定了‘鸡冠’‘富士’‘嘎拉’‘成保光’等1 000多个品种的S基因型,分别为S_3S_5、S_1S_9、S_2S_5和S_1S_2等。当‘国光’与‘鸡冠’相互授粉时,由于2个S基因型不同,相互授粉可结实,表现为亲和;当‘国光’与‘富士’相互授粉时,由于有一个S等位基因相同,有一半花粉可授粉受精结实,表现为半亲和;当‘国光’与‘成保光’相互授粉时,由于S基因型完全相同,相互授粉不结实,表现为不亲和。由于S基因型直接决定了相互授粉的亲和程度,可为栽培生产授粉树的配置和杂交育种亲本选配提供参考和帮助,因此,本研究室与日本名古屋大学合作,在对中国国家苹果资源圃和日本国家苹果资源圃S基因型大量鉴定的基础上,建立了苹果S基因型数据库,配有中、日、英等文字的操作页面,该网站可根据用户的需要,给出相应品种的对应授粉品种,用户再根据花期相遇、花粉育性等从中挑选出最适合的授粉组合。     

20个苹果品种的S基因型鉴定

以‘富士’、‘华瑞’、‘华硕’和‘华星’等20个苹果品种为材料,利用S等位基因高度保守氨基酸序列FTQQYQ和anti-1/MIWPNV设计的S基因通用引物,以及S等位基因多态性序列设计的19对特异引物,PCR扩增、测序以鉴定20个品种的S基因型;并用‘华瑞’和‘华硕’分别与‘美八’、‘锦秀红’、‘华冠’和‘富士’进行授粉试验验证S基因型的准确性。PCR结果表明:通用引物扩增S等位基因时,仅‘富士’、‘华瑞’、‘华硕’、‘华星’、‘美八’和‘红脆宝’6个品种有效地扩增出2条特异的S等位基因条带,其S基因型有S1S_9、S_9S_(24)、S_5S_9和S_5S_(24)等4种;19对特异引物扩增S等位基因时,‘华帅’等14个品种扩增得到2条特异性条带,S基因型有S_(10)S_(19)、s_2s_3、s_2S_5、s_3S_(10)、s_2S_9、S_5S_(24)、S_9S_(10)、s_3S_(10)和S_5S_9等9种。因此,20个苹果品种的S基因型分别为:‘富士’S1S_9,‘华瑞’和‘华硕’S_9S_(24),‘华星’、‘美八’和‘红珍珠’S_5S_9,‘红脆宝’、‘华玉’和‘99-1-29’S_5S_(24),‘华帅’S_(10)S_(19),‘金玉’s_2s_3,‘早红’、‘华美’和‘嘎拉’s_2S_5,‘Seokwang’s_3S_(10),‘锦秀红’、‘蜜玉’和‘华冠’s_2S_9,‘绿佳’S_9S_(10),‘信浓红’s_3S_(10)。2015和2016年‘华瑞’与‘华硕’的正反交组合坐果率较低(低于15.52%);而‘华瑞’和‘华硕’分别与‘美八’、‘锦秀红’和‘华冠’、‘富士’品种的正反交组合坐果率较高(高于46.30%)。因此,本试验中相同S基因型的授粉组合其坐果率较低,不同S基因型的授粉组合其坐果率较高,授粉试验支持S基因型鉴定结果。     

4个枇杷品种S基因型鉴定及新基因S_(31)-RNase序列分析

【目的】鉴定‘黄密’、‘贵妃’、‘早红’和‘软条白沙’4个枇杷品种的S基因型,为其生产栽培合理选择授粉树及杂交育种亲本选择提供科学依据。【方法】以苹果S基因高度保守区设计兼并引物对4个品种的基因组DNA进行PCR扩增,片段回收、克隆及测序,分别采用Blast软件和Bioedit软件进行同源性检索和结构分析。【结果】从参试的4个品种中共分离了4个S等位基因,分别为S2、S5、S6和S31,其中S31-RNase为新分离的枇杷S-RNase基因,Gen Bank登录号为:KC131133。所克隆获得的4个枇杷S-RNase基因均克隆到4个保守区(C2、C3、RC4和C5)和1个高变区(HV),具有与苹果S基因相同的氨基酸序列结构。【结论】确定了参试4个枇杷品种S基因型分别为:‘贵妃’S2-S6、‘黄密’S2-S5、‘早红’S5-S6、‘软条白沙’S6-S31。     

桃若干重要特异性状的遗传趋向分析

为了明确桃树菊花花形、窄叶形、白化与黄化叶色及盘龙形与垂枝形树形等6个性状的遗传特性,以‘粉菊花桃’ב红根甘肃桃1号’、‘红花重瓣垂枝桃’(简称‘红垂枝’)ב粉菊花桃’、‘S9’ב粉菊花桃’、‘粉菊花桃’ב瑞光2号’、‘红珊瑚’×(‘粉菊花桃’ב瑞光2号’)为组合配制F_1和F_2代,研究菊花花形、叶片白化及垂枝树形的遗传特性;以‘98-4-32’ב金蜜狭叶桃’、‘99-7-14’ב金蜜狭叶桃’及‘99-7-15’ב金蜜狭叶桃’为组合配制F_1和F_2代,研究窄叶形遗传趋向;以‘北京2-7’ב白花山碧桃’为组合配制F_1和F_2代,研究叶片黄化遗传特点;以‘红垂枝’ב帚形山桃’及‘96-7-56’ב帚形山桃’为组合配制F_1和F_2代,研究盘龙形树形的遗传。结果表明:在蔷薇花形与菊花花形组合中,"蔷薇形/菊花形"表现出2对等位基因(Ch/ch及Ch_2/ch_2)控制的遗传特性,且蔷薇形对菊花形为显性;在铃形花形与菊花花形组合中,F_1代蔷薇形表现为二者的中间类型。在‘98-4-32’ב金蜜狭叶桃’及‘99-7-14’ב金蜜狭叶桃’组合中,"宽叶/窄叶"表现出2对等位基因(Nl/nl及Nl_2/nl_2)控制的遗传特性,且宽叶对窄叶为显性;在‘99-7-15’ב金蜜狭叶桃’组合中,"宽叶/窄叶"表现出1对等位基因控制的遗传特性。叶片"正常/白化"表现出1对等位基因(Wl/wl)控制的遗传特性,且正常对白化为显性,白化基因可能来自‘粉菊花桃’;叶片"正常/黄化"表现出2对等位基因(Yl/yl及Yl_2/yl_2)控制的遗传特性,其中1对为显性时对另一对具有抑制作用,黄化基因可能来自‘白花山碧桃’。直立树形同时表现为盘龙形与垂枝形及盘龙形与开张形的中间类型;"开张形/垂枝形"表现出1对等位基因(We/we)控制的遗传特性,且开张对垂枝为显性。结论:菊花花形由2对隐性基因(chchch_2ch_2)控制;窄叶形可能由2对隐性基因(nlnlnl_2nl_2)控制;叶片白化由1对隐性基因(wlwl)控制;叶片黄化可能由2对等位基因(Yl_yl_2yl_2或Yl_2_ylyl)控制;仅盘龙形与直立形由1对等位基因(Br_2/br_2)控制,其中盘龙形基因型为br_2br_2;垂枝形由1对隐性基因(wewe)控制。     

'寒富'苹果自花结实性分析及S基因型鉴定

 本研究以苹果‘寒富’品种及其亲本为试材,研究其自交亲和性并鉴定其S基因型。田间授粉试验结果显示,‘寒富’品种表现为自花结实,其母本‘东光’品种亦自花结实;而父本‘富士’品种自花授粉结实率极低,初步推断‘寒富’品种的自花结实性可能遗传自其母本‘东光’。利用PCR技术鉴定'寒富'品种S基因型为S1S9。由于‘东光’品种存在同名异物问题,本研究除日本‘东光’品种外,对辽宁省果树研究所、吉林省果树研究所、沈阳农业大学等单位保存的中国'东光'品种的S基因型进行了鉴定,发现除辽宁省果树研究所保存的'东光'品种S基因型为S2S19,与‘寒富’品种无亲缘关系外,其它两个单位保存的'东光'品种中均存在S9基因型,可能为'寒富'杂交育种时的原始母本。     

组培条件下苹果实时定量PCR内参基因的筛选

【目的】筛选在组织培养条件下苹果苗中稳定表达的内参基因。【方法】应用实时荧光定量PCR技术,分析了18S r RNA,GAPDH,TUB,UBQ,ACT,EF-1α六个常用植物内参基因在苹果组培苗不同基因型、不同组织、不同继代时间的m RNA表达差异情况。供试的4个苹果基因型为‘长枝富士’‘短枝富士’‘红珍珠海棠’、砧木‘A8-11’;继代培养的不同组织为茎、叶、整株,生根培养的不同组织为根、茎、叶、整株;不同继代期间为10、20、30、40、50、60、70、80 d。【结果】经Ge Norm软件对6个内参基因的表达稳定性进行评价,ACT和EF-1α在苹果组培苗的不同组织和不同继代时间的基因表达分析中较稳定,UBQ和EF-1α在苹果组培苗不同品种中表达均稳定。【结论】组培条件下,苹果基因表达相关研究的理想内参基因为ACT、UBQ和EF-1α。     

苹果花粉中与花柱S-RNase互作的γ-硫堇筛选及鉴定

以‘国光’苹果（S1S2）花粉为材料,成功构建了酵母pGADT7-cDNA文库,转化效率约为1.2×106.μg-1,插入片段大小在300~2000bp之间。通过酵母双杂交方法,用苹果S2-RNase的C2HVC3区筛选文库获得一个长505bp的cDNA片段,经分析发现该cDNA序列上有一个261bp的开放阅读框（ORF）,该ORF编码一个具N端信号肽的多肽,该成熟多肽由64个氨基酸组成,富含带正电荷的赖氨酸和精氨酸,其C端有8个半胱氨酸和一个γ-硫堇功能域,结构预测显示其具有一个α螺旋,3个β折叠,4个二硫键,由此,推断其为苹果γ-硫堇,命名为MdD1。RT-PCR分析发现：MdD1基因在‘国光’苹果叶片、萼片、花瓣、子房、花药和花柱等组织中都有表达,但花药中表达量最高。酵母双杂交结果表明MdD1除了与苹果S2-RNase的C2HVC3区互作外,还与S1-RNase的C2HVC3区,S1、S2-RNase的成熟多肽区存在互作。初步认为：苹果γ-硫堇可能通过与S-RNase非特异性互作,作为花粉非S因子参与自交不亲和反应。     

宁夏主要果树花器官及幼果霜冻临界温度比较研究

以宁夏主要果树苹果、梨、杏、李子不同花器官及幼果为试材,利用能够准确模拟霜冻降温过程的模拟霜箱,研究其花期及幼果期过冷却点和结冰点,比较不同果树及同一果树不同器官抗寒性及受霜冻的临界温度,为果园霜冻监测、预报预警及霜冻防御提供参考。结果表明:苹果、李子幼果的抗寒性明显差于花器官。苹果开花期各器官的抗寒性从强到弱为子房花蕾;李子花器官(花蕾、子房)之间抗寒性差异不大,明显强于叶片和幼果;梨和杏各主要器官抗寒性从强到弱为花蕾幼果子房。宁夏苹果、梨、杏和李子蕾期和花期若遇低于-5.2℃的低温将可能全部受冻,幼果遇低于-4.4℃的低温,则幼果将全部受冻。3个品种的李子花器官抗寒性从强到弱为‘红美丽’‘尤萨’‘龙园秋李’;幼果抗寒性从强到弱为‘龙园秋李’‘红美丽’‘尤萨’。2个杏品种在花期‘金太阳’比‘李梅杏’的耐冻性强,幼果期‘金太阳’的耐冻性弱于‘李梅杏’。     

60个苹果新品种S基因型鉴定

绝大多数苹果品种具有自交不亲和的特性，生产中需要搭配授粉树完成异花授粉。在苹果中已经分离鉴定了50多个S-RNase基因，对于一些新选育品种的S基因型至今未得到鉴定。利用S-RNase基因特异性PCR分析的方法鉴定了60份苹果新种质资源的S基因型，在60份苹果种质资源中，有56份品种的S基因型是首次鉴定到，并首次鉴定到了一个红肉苹果品种‘红色之爱’的S基因型，其S基因型是S19S？。新品种S基因型的鉴定能够为苹果育种亲本的选择及授粉树的合理搭配提供依据。     

‘早红香’苹果自交后代胚培养及其S基因型鉴定

为获得苹果自交后代纯合基因型个体、加快育种进程,对‘早红香’苹果田间自花授粉得到的种子进行胚培养。结果表明：长出真叶前留2片子叶,真叶长出后去掉1片子叶的胚成苗较好,1片子叶的次之,不留子叶的较差;28株‘早红香’苹果自交后代中含有纯合基因型的植株为15株,其S基因型S1S9、S1S1、S9S9的分离比例为13：8：7—2：1：1,符合孟得尔分离定律,可以得到不同S基因型的子代纯合基因型个体。     

8个中国梨品种S基因型的分子鉴定

为鉴定8个中国梨品种的S基因型,使用梨自交不亲和基因(S-RNase)特异引物"FTQQYQ"和"anti-ⅡWP-NV",对8个梨品种的基因组DNA进行特异扩增,并对扩增片段进行回收、克隆、测序。使用生物信息学软件对各序列分析和经同源性搜索分析后,确定了各品种的S基因型。结果分别是:兰州花长把为S19S22,青面为S1S18,黄面为S1S12,早蜜为S19S29,大面黄为S1S19,无子黄为S28S16,大青皮为S34S19及金锤子为S16S19。     

Determining the S-genotypes of several sweet cherry cultivars based on PCR-RFLP analysis

Summary

Based on the cDNA sequences encoding sweet cherry self-incompatibility associated ribonucleases (S-RNases), a PCR-based S-allele typing system for sweet cherry cultivars has been recently developed. Using this technique, we determined S-genotypes of the three newly released Japanese cvs Kouka-Nishiki, Beni-Sayaka and Beni-Shuho and one British cv Merton Glory that was classified as a Universal Donor, which is able to be used as a pollen donor for all cultivars in pollen incompatibility groups I to XIII. Furthermore, we also determined the partial sequences of the S-RNase genes of ‘Rainier’ (S¹S⁴)‘ and ‘Sato-Nishiki (S³S⁶)’,which leads to the development of a more reliable S-allele identification method of PCR-RFLP for sweet cherry cultivars. Total DNA isolated from leaves of the four cultivars along with those from ten cultivars with known S-genotypes were PCR amplified with two sets of primers that were designed from DNA sequences encoding the signal peptide (Pru-T2) and two conserved domains (Pru-C2 and Pru-C4R) of sweet cherry S-RNases. By comparing the size of PCR products on agarose gel, the 5-genotypes of ‘Kouka-Nishiki’, ‘Beni-Sayaka’, ‘Beni-Shuho’ and ‘Merton Glory’ were suggested to be S¹S³, S¹S⁶, S⁴S⁶, and S⁴S⁶, respectively. Two of these three S-genotypes (S¹S⁶ and S⁴S⁶) were found for the first time. DNA sequencing of PCR products from S-alleles of ‘Rainier’ and ‘Sato-Nishiki’ revealed that Ban II, Nru I, Apa LI and Ava I sites, respectively, were unique in the S¹-, S³-, S⁴- and S⁶- sequences flanked by Pru-T2 and Pru-C4R primers. RFLP analysis of the PCR products using these enzymes confirmed that S¹-, S³-, S⁴- and S⁶-alleles of the four cultivars contained the respective restriction enzyme recognition sites.     

Highbush Blueberry Cultivar Trial in Ontario,Canada

ABSTRACT

A replicated trial of eleven cultivars was planted at Simcoe, Ontario in 1991. Plants were first harvested in 1994. After eight harvest seasons, ‘Bluegold’ was the highest yielding cultivar, averaging 6.8 t/ha, and ‘Toro’ had the largest berries, averaging 1.81 g per berry. ‘Bluetta’ was the earliest cultivar with on average 50% of its yield picked by 16 July and ‘Elliott’ the latest, with on average 50% of its yield picked by 27 August. Overall, ‘Duke’ proved to have the best combination of characteristics of the early cultivars, and ‘Nelson’ was the most impressive late cultivar.     

Analysis of S-alleles by PCR for determination of compatibility in the ‘Red Delicious’ apple orchard

Summary

In a previous study, the apple ‘Jonathan’ was shown to be semi-compatible with the ‘Topred’ sport of ‘Red Delicious’, the main cultivar in Israel. This incompatibility led to reduced pollination potency, explaining the low yield obtained from ‘Topred’ rows adjacent to ‘Jonathan’. Hence, the importance of identifying the level of compatibility for all ‘Topred’ pollinators became apparent. By analysing S-allele by PCR with primers for the various S-alleles of the cultivars in the ‘Topred’ orchard, including S10 and S28, which were cloned in this study, it was evident that apart from ‘Jonathan’, the common pollinators for ‘Topred’ are fully compatible with it. The ability to identify all S-alleles in the ‘Topred’ orchard was also utilized to resolve the observation that, although ‘Topred’ is considered self-incompatible, self-pollinated, net-caged, ‘Topred’ flowers set some fruit. Applying PCR-analysis of S-alleles to the progeny of the suspected self-pollinated ‘Topred’ fruit revealed that the fruit-set was the outcome of cross-pollination, perhaps the consequence of fertilization by wind carried pollen. Hence, the notion that ‘Topred’ is totally self-incompatible was confirmed and the efficiency of progeny PCR S-allele analysis for the determination of the pollen donor was exhibited.     

Resistance to white rot infections in bulb onion seed lots

Five seed lots and the self- and open-pollinated progenies of the cultivar ‘Ailsa Craig’ were tested for disease reaction to Sclerotium cepivorum, the causal fungus of onion white rot. Field tests were conducted on S. cepjuorum-infested soil in Burnaby, British Columbia, from 1976 to 1980. No significant differences were observed between ‘Ailsa Craig’ and ‘Autumn Spice’ with respect to disease resistance during the 1979 and 1980 tests, whereas resistance of ‘Ailsa Craig’ had been noted in previous years. Tests indicated that resistance varied with seed lot, thus providing a possible explanation for the 1979 and 1980 results. ‘Ailsa Craig’ selfed progeny selection had significantly fewer infections than its parental seed lot and the local recommended cultivar ‘Autumn Spice’. Resistance to infection by S. cepivorum should be assigned to seed lots or breeding lines and not to presently named cultivars. These results suggest that differences in infection by S. cepivorum occur, and are available for the development of white rot resistant cultivars.     

槜李等15个李品种S基因型鉴定及其多态性分析

利用李属S-RNase基因特异性引物,对15个供试李品种进行PCR扩增,共获得30个目的条带。对这些目的条带进行测序鉴定出15个李品种的S基因型。通过与NCBI中利用BLASTn与GenBank+EMBL+DDBJ+PDB等数据库中的序列比对,结果表明,其中9个为新S-RNases基因,对9个新S-RNases核苷酸序列进行分析发现,位于高变区内的内含子大小为141～1758bp,其同源性为33.9%(S-18～S-19)～81.6%(S-20～S-21),表现出丰富的长度和序列多态性;编码区的核苷酸序列比对结果,其同源性为73.3%(S-16～S-19)～91.7%(S-17～S-22);其推导氨基酸序列相似性为67.3%(S-16～S-19)～89.1%(S-17～S-22);包含李属S-RNase一级结构所共有的C2、C3保守区和高变区(RHV)。系统进化分析表明,9个新S-RNases与李属其它树种S-RNases聚类在一起,归属为李亚科(Prunoideae)。     

甜樱桃SFB4与SFB4’基因的鉴别

 本研究以最近发现的李属花粉决定子候选基因“S 单元型特异F-box蛋白基因(SFB ) ”为基础, 根据甜樱桃SFB 4基因设计引物, 从自交不亲和‘雷尼’和‘佳红’以及自交亲和的‘斯坦拉’总DNA中分别扩增出SFB 4和SFB 4’基因的部分序列。经测序结果发现: SFB 4’基因比SFB 4基因缺失了4个碱基TAAA。根据这个缺失差异, 设计了一对引物BFP200和BFP201, 这对引物只能扩增SFB 4’基因,而不能扩增SFB 4基因, 从而利用SFB 基因区分开了甜樱桃自交不亲和的S4和自交亲和的S4’单元型。     

S-allele genotypes of apple pollenizers,cultivars and lineages including those resistant to scab

Summary

We examined S-allele genotypes of ten apple cultivars and species to determine their possible usefulness as pollenizers for all apple cultivars. ‘Dolgo’ did not contain any known S-RNases encoded at the S-locus, suggesting its possible usefulness as a pollenizer for almost all apple cultivars. We also identified and confirmed the S-allele genotypes of 18 apple cultivars by polymerase chain reaction (PCR)-digestion analysis. The S-genotype of ‘Kiou’ (S₁S₇), ‘Korei’ (S₃S₂₈), ‘Korin’ (S₁S₉), ‘Kotoku’ (S₁S₂₈), ‘Kyokkou (S₇S₂₅), ‘Lobo’ (S₁S₇), ’Mahe 7’ (S₂S₇), ‘Mellow’ (S₂S₃), ‘Takahara’ (S₃S₉) and ‘Warabi’ (S₉S₂₈) were confirmed by pollination results. These cultivars seemed not to have originated from the expected seed or pollen parents or, in the case of ‘Lobo’, might have been mislabelled. Finally, we identified the S-allele genotypes of ‘Prima’ (S₂S₁₀), ‘Querina’ (S₃S₉) and ‘Yoko’ × ‘Prima’ (S₃S₁₀), which are resistant to scab.     

Across- and along-row pollen dispersal in high-density apple orchards: Insights from allozyme markers

Summary

The organization of most high-density orchards of apple (Malus x domestica Borkh.) into single-cultivar rows is often viewed as an impediment to effective pollen dispersal and pollination; however, few direct estimates of pollen dispersal are available to assess the extent of this problem. We estimated the magnitude of pollen dispersal across rows (pollinator cultivars: Idared, Vista Bella, Granny Smith), and along rows, (pollinator cultivars Fuji, Paulared, Golden Russet), in two high-density orchards in Ontario, Canada. Fruit were sampled from trees at regular intervals along transects that extended beyond (either across or along row) each pollinator cultivar and the percentage of seeds that were sired by the pollen donor was estimated using allozyme markers. Seeds sired by the pollinator cultivar were found from seven (‘Granny Smith’) to 18 rows (‘Idared’) from the nearest pollinator tree and from seven (‘Fuji’, ‘Paulared’) to five trees (‘Granny Smith’) down the same row. Measured in metres, the maximum pollen dispersal distance was greater across rows (62.4.m) than along rows (13.7.m). However, the average dispersal distance across rows, expressed in metres or trees, (17.4.±m.3.6, 3.6.±0.7 trees) did not differ from that along rows (5.8.m.±4.1, 2.7.±0.8 trees). These results are at odds with previous studies that indicate that honeybees move farther along rows than across, and have important implications for the design of commercial apple orchards.