甜樱桃(Prunus avium L.)品种S基因型鉴定

根据蔷薇科S-RNase基因(S基因)高度保守区C2和RC4区设计一对特异引物PruC2和PruC4R,对甜樱桃品种的基因组DNA进行S基因特异PCR扩增。克隆S基因的扩增片段,核酸序列在GenBank上搜索,确定了4种S基因的核酸序列和大小。结果表明,在琼脂糖凝胶上位置相同的扩增带其核酸序列相同,是同一种S基因。4种S基因扩增片段的大小分别是：S1为677bp,S3为762bp,S4为945bp,S6为456bp。参试的自交不亲和品种的S基因型分别是：红灯、红艳、早红宝石和先锋相同,为S1S3;抉择、红丰和那翁相同,为S3S4;大紫为S1S6;长把红为S1S4;养老为S2S6;自交亲和品种外引7号和斯太拉为S3S4。     

萝卜自交不亲和基因SLG6的CAPS标记的开发

萝卜是我国的主要蔬菜之一,其杂种优势十分明显,培育自交不亲和系是萝卜杂种优势育种的主要途径之一.本研究根据萝卜自交不亲和基因SLG6序列设计特异引物,以8个自交系为材料,其中自交不亲和系和自交亲和系各4个,扩增SLG6基因第232～711bp之间的单拷贝片段,8个材料均获得了一条480bp的特异片段.用限制性内切酶TaqⅠ对该片段进行酶切,自交不亲和系均产生约125bp和244bp的片段,其中,244bp的片段为自交不亲和系所特有,可作为SLG6基因的CAPS标记用于萝卜自交不亲和基因SLG6的检测;而自交亲和系则具有与自交不亲和系相同的125bp的片段和不同的多态性片段.     

梨自交不亲和基因cDNA芯片杂交条件优化及应用北大核心

[目的]优化梨自交不亲和基因(S-RNase或S基因)c DNA芯片杂交条件,利用芯片检测梨品种S基因型。[方法]提取梨品种雌蕊RNA,Cy3标记引物RT-PCR获得S基因荧光标记特异c DNA序列。设置不同杂交条件,用已知S基因型品种荧光标记的PCR产物在不同条件下分别与芯片杂交,杂交信号分析芯片杂交效果。用芯片优化杂交体系鉴定梨品种未知S基因型,DNA测序验证芯片鉴定结果。[结果]芯片杂交最佳条件:杂交温度42℃,杂交时间8~9 h,PCR纯化产物终浓度为200 ng·μl-1。优化杂交条件下芯片鉴定晚咸丰、秀水、丽江马占梨1、湘菊、木通梨、甘甜、弥渡小红梨、丽江大中古、金晶和弥渡火把等梨品种S基因型分别为:Pp S15Pp S52、Pp S4Pp S5、Pb S22Pp S37、Pp S1Pp S2、Pp S1Pp S3、Pp S13Pp S15、Pp S12Pb S42、Pb S21Pb S22、Pp S3Pp S60和Pp S5Pp S5。DNA测序验证各品种所含S基因与芯片鉴定结果一致。[结论]梨自交不亲和基因c DNA芯片优化杂交条件后可准确鉴定梨品种所含已鉴定的S基因资源。     

梨自交不亲和基因cDNA芯片杂交条件优化及应用

[目的]优化梨自交不亲和基因(S-RNase或S基因)c DNA芯片杂交条件,利用芯片检测梨品种S基因型。[方法]提取梨品种雌蕊RNA,Cy3标记引物RT-PCR获得S基因荧光标记特异c DNA序列。设置不同杂交条件,用已知S基因型品种荧光标记的PCR产物在不同条件下分别与芯片杂交,杂交信号分析芯片杂交效果。用芯片优化杂交体系鉴定梨品种未知S基因型,DNA测序验证芯片鉴定结果。[结果]芯片杂交最佳条件:杂交温度42℃,杂交时间8~9 h,PCR纯化产物终浓度为200 ng·μl-1。优化杂交条件下芯片鉴定晚咸丰、秀水、丽江马占梨1、湘菊、木通梨、甘甜、弥渡小红梨、丽江大中古、金晶和弥渡火把等梨品种S基因型分别为:Pp S15Pp S52、Pp S4Pp S5、Pb S22Pp S37、Pp S1Pp S2、Pp S1Pp S3、Pp S13Pp S15、Pp S12Pb S42、Pb S21Pb S22、Pp S3Pp S60和Pp S5Pp S5。DNA测序验证各品种所含S基因与芯片鉴定结果一致。[结论]梨自交不亲和基因c DNA芯片优化杂交条件后可准确鉴定梨品种所含已鉴定的S基因资源。     

编码嗜麦芽黄单胞菌terminase-like蛋白基因的克隆与分析

根据Grover报道的XanthomonasmaltophiliaCG类受体的 342bp核酸序列设计的一特异引物P2和随机引物进行PCR扩增 ,将约 75 0bpPCR产物克隆到 pUCm T载体上 ,得到重组质粒 pUCm Ter。pUCm Ter上插入片段经M 13通用引物双向测序 ,其中ORF5 5 5核酸序列的 2 83～ 36 2bp部分与PseudomonasphageD3orf2基因的 1385～ 14 6 4bp部分有 86 %相同碱基。由ORF5 5 5编码 184aa蛋白序列的 1～ 16 5aa部分与PseudomonasphageD3orf2基因编码terminase的 2 2 9～ 393aa部分具有 6 6 %相同序列。因此 ,克隆到的ORF5 5 5核酸序列可能是编码嗜麦芽黄单胞菌terminase like蛋白的基因序列。     

一个新的苹果自交不亲和基因的分离

以从日本引进的苹果新品种斗南为试验材料,根据保守氨基酸序列"FTQQYQ"和"anti-1/WⅠPNV"设计苹果自交不亲和基因引物,P1:5′-TTTACGCAGCAATATCAG-3′;P2:5′-ACGTTCGGCCAAATA/CATT-3′。利用PCR-RFLP分析和目的片段测序方法得到了一个新的苹果自交不亲和基因-S33,其片段长度为348bp,包括P1、C2区和HV(含147bp的Intron)区。     

中国梨2个自交不亲和新等位基因(S等位基因)的分子鉴定

自交不亲和是显花植物的一种重要生殖生理现象,为探明中国梨的自交不亲和特性,对‘锦香’(Pyrus bretschneideri cv. Jinxiang)和‘鹅酥’(Pyrus bretschneideri cv. Esu)2个中国梨品种进行了基因组PCR特异扩增、S基因序列分析及田间杂交授粉试验。结果确定它们各含1个新S-RNA酶基因,分别命名为S37-和S38-RNase,GenBank序列号为DQ839238和DQ839239。生物信息学分析结果表明,S37-和S38-RNA酶的推导氨基酸序列与S1-至S36-RNA酶36个梨S基因具有相同的、高度保守的C1和C2区,但其高变区与S1-至S36-RNA酶差异较大,其中与S15的差异最小,只有3个氨基酸不同。在推导的氨基酸水平上,S37与S38有96%的序列相似性,但两者与S15的相似性更高,皆为98%,与S32的相似性最低,都只有63%;S37和S38的内含子较大,分别为786bp和723bp,与S15的777bp大小接近。最后,经分析验证确定‘锦香’和‘鹅酥’的S基因型分别为S34S37和S15S38。     

由基因序列开发番茄枯萎病抗性基因Ⅰ-2的共显性分子标记

根据I-2的基因序列设计特异扩增引物对I-2/5F和I-2/5R, 扩增I-2基因3 132～3 765 bp之间片段, 基因型为I-2 / I-2的材料03F-7可扩增出633 bp的条带, 而基因型为i-2/ i-2的材料Moneymaker可扩增出693 bp的条带, 杂合型材料可扩增出以上2个条带。通过这两个特异扩增片段的克隆和测序证明, 抗病材料扩增的633 bp片段为I-2基因的3 132～3 765 bp之间的序列, 而感病等位基因中出现大量的碱基突变和60 bp片段插入。利用引物对I-2/5F和I-2/5R, 可区分纯合抗病材料、杂合抗病材料和纯合感病材料, 从而建立了I-2基因的共显性分子标记。在此基础上, 利用该标记对16个主要番茄品种进行基因型鉴定, 8个品种含有I-2基因, 其中1个品种基因型为I-2 / I-2, 其他品种为I-2 / i-2。通过一次PCR和一次HindⅢ酶切建立了I-2和Tm-22双基因检测体系, 为多基因鉴定及标记辅助选择提供了有力工具。     

血红密孔菌(Pycnoporussanguineus)漆酶基因的克隆与序列分析

为克隆血红密孔菌 (Pycnoporussanguineus)漆酶基因 ,根据真菌漆酶氨基酸序列保守区设计了 1对简并引物 .以血红密孔菌基因组DNA为模板 ,PCR扩增出长 12 2 7bp的漆酶基因片段 .以此序列为基础 ,通过 5′及 3′RACE技术克隆出漆酶全长cDNA序列 ,序列长为 190 2bp ,其 5′端和 3′端非编码区长分别为 5 1bp和 2 97bp ,开放阅读框长 15 5 4bp ,编码 5 18个氨基酸的蛋白 .该蛋白具有 4个铜离子结合区域 ,预测其相对分子量为 5 6 313 2 ,等电点为 5 5 9,其氨基酸序列与Pycnoporuscinnabarinus漆酶 (lcc3 2 )的同源性最高 ,为 96 % .以该cDNA编码区的两端序列为引物 ,PCR扩增得到漆酶的长度为 2 15 4bp的全长DNA序列 ,序列中包括 10个内含子序列 ,长为 5 2～ 70bp     

由基因序列开发番茄枯萎病抗性基因I-2的共显性分子标记

根据I-2的基因序列设计特异扩增引物对I-2/5F和I-2/5R, 扩增I-2基因3 132～3 765 bp之间片段, 基因型为I-2 / I-2的材料03F-7可扩增出633 bp的条带, 而基因型为i-2/ i-2的材料Moneymaker可扩增出693 bp的条带, 杂合型材料可扩增出以上2个条带。通过这两个特异扩增片段的克隆和测序证明, 抗病材料扩增的633 bp片段为I-2基因的3 132～3 765 bp之间的序列, 而感病等位基因中出现大量的碱基突变和60 bp片段插入。利用引物对I-2/5F和I-2/5R, 可区分纯合抗病材料、杂合抗病材料和纯合感病材料, 从而建立了I-2基因的共显性分子标记。在此基础上, 利用该标记对16个主要番茄品种进行基因型鉴定, 8个品种含有I-2基因, 其中1个品种基因型为I-2 / I-2, 其他品种为I-2 / i-2。通过一次PCR和一次HindⅢ酶切建立了I-2和Tm-22双基因检测体系, 为多基因鉴定及标记辅助选择提供了有力工具。     

Determination of self-incompatibility groups of sweet cherry genotypes from Turkey

Determination of S-allele combinations of sweet cherry genotypes and cultivars has importance for both growers and breeders. We determined S-allele combinations of 40 local Turkish sweet cherry genotypes using a PCR-based method. Ten different S-alleles were detected. Although the most common S-allele was S3, as also found in Western genotypes and cultivars, there were some differences in the frequencies of some S-alleles between Turkish and Western sweet cherry genotypes. According to their S-allele compositions, 30 local Turkish sweet cherry genotypes were assigned to 10 previously identified incompatibility groups. For the remaining genotypes, whose S-allele combinations did not fit to any previous incompatibility groups, three more incompatibility groups, XLII, XLIII and XLIV, were proposed. Results obtained from this study will help both sweet cherry growers and breeders to better manage these local Turkish sweet cherry genotypes in their orchards.     

Characterization of Phytoplasmas Related to 'Candidatus Phytoplasma asteris' and Peanut WB Group Associated With Sweet Cherry Diseases in Iran

Symptoms suggestive of phytoplasma diseases were observed in infected sweet cherry trees growing in the central regions of Iran. Phytoplasmas were detected in symptomatic trees by the nested polymerase chain reaction (nested PCR) using phytoplasma universal primer pairs (P1/Tint, PA2F/R, R16F2/R2 and NPA2F/R). Restriction fragment length polymorphism analyses of 485 bp DNA fragments amplified in nested PCR revealed that different phytoplamas were associated with infected trees. Sequence analyses of phytoplasma 16S rRNA gene and 16S-23S intergenic spacer region indicated that the phytoplasmas related to ' Ca. Phytoplasma asteris ' and peanut WB group infect sweet cherry trees in these regions. This is the first report of the presence of phytoplasmas related to ' Ca. Phytoplasma asteris' and peanut WB group in sweet cherry trees.     

Characterization of microsatellites in wild and sweet cherry (Prunus avium L.)--markers for individual identification and reproductive processes.

Nuclear microsatellites were characterized in Prunus avium and validated as markers for individual and cultivar identification, as well as for studies of pollen- and seed-mediated gene flow. We used 20 primer pairs from a simple sequence repeat (SSR) library of Prunus persica and identified 7 loci harboring polymorphic microsatellite sequences in P. avium. In a natural population of 75 wild cherry trees, the number of alleles per locus ranged from 4 to 9 and expected heterozygosity from 0.39 to 0.77. The variability of the SSR markers allowed an unambiguous identification of individual trees and potential root suckers. Additionally, we analyzed 13 sweet cherry cultivars and differentiated 12 of them. An exclusion probability of 0.984 was calculated, which indicates that the seven loci are suitable markers for paternity analysis. The woody endocarp was successfully used for resolution of all microsatellite loci and exhibited the same multilocus genotype as the mother tree, as shown in a single seed progeny. Hence, SSR fingerprinting of the purely maternal endocarp was also successful in this Prunus species, allowing the identification of the mother tree of the dispersed seeds. The linkage of microsatellite loci with PCR-amplified alleles of the self-incompatibility locus was tested in two full-sib families of sweet cherry cultivars. From low recombination frequencies, we inferred that two loci are linked with the S locus. The present study provides markers that will significantly facilitate studies of spatial genetic variation and gene flow in wild cherry, as well as breeding programs in sweet cherry.     

甜樱桃品种SSR-PCR反应体系的优化

以甜樱桃品种那翁为试材,研究了樱桃SSR技术中PCR反应体系的主要成分对SSR扩增结果的影响,并比较了采用聚丙稀酰胺凝胶及琼脂糖电泳检测扩增产物多态性的差异.结果表明:在PCR反应体系中,DNA最适浓度30～45 ng;Mg2+的最适浓度范围为1.5～3.0 mmol/L;dNTP最适浓度为0.2～0.3 mmol/L;引物的最适浓度为0.3～0.4 μmol/L;Taq聚合酶在20 μl反应体系中宜加入0.5 U.利用此反应体系,对24份樱桃代表资源进行了SSR反应,用6%的非变性聚丙稀酰胺凝胶电泳检测,扩增产物在100～250 bp之间,不同品种间DNA谱带多态性丰富.琼脂糖电泳检测的DNA多态性不如聚丙稀酰胺凝胶丰富.     

Determination of S-genotypes of pear (Pyrus pyrifolia) cultivars by S-RNase sequencing and PCR-RFLP analyses

The pear (Pyrus pyrifolia) has gametophytic self-incompatibility (GSI). To elucidate the S-genotypes of Korean-bred pear cultivars, whose parents are heterozygotes, the PCR amplification using S-RNase primers that are specific for each S-genotype was carried out in 15 Korean-bred pear cultivars and 5 Japanese-bred pear cultivars. The difference of the fragment length was shown in the following order: S6 (355 bp) < S7 (360 bp) < S1 (375 bp) < S4 (376 bp) < S3 and S5 (384 bp) < S8 (442 bp) < S9 (1,323 bp) < S2 (1,355 bp). We analyzed the sequence of the S-RNase gene, which had introns of various sizes in the hypervariable (HV) region between the adjacent exons with a fairly high homology. The sizes of the introns were as follows: S1 = 167 bp, S2 = 1,153 bp, S3 = 179 bp, S4 = 168 bp, S5 = 179 bp, S6 = 147 bp, S7 = 152 bp, S8 = 234 bp, S9 = 1,115 bp. There were five conservative and five hypervariable regions in the introns of S1, S3, S4, S5, S6 and S-RNases. A pairwise comparison of these introns of S-RNases revealed homologies as follows: 93.7% between S1- and S4-RNases, 93.3% between S3- and S5-RNases and 78.9% between S6- and S7-RNases. PCR-RFLP and S-RNases sequencing determined the S-genotypes of the pear cultivars. The S-genotypes were S4S9 for Shinkou, S3S9 for Niitaka, S3S5 for Housui, S1S5 for Kimizukawase, S1S8 for Ichiharawase, S3S5 for Mansoo, S3S4 for Shinil, S3S4 for Whangkeumbae, S3S5 for Sunhwang, S3S5 for Whasan, S3S5 for Mihwang, S5S? for Chengsilri, S3S5 for Gamro, S3S4 for Yeongsanbae, S3S4 for Wonhwang, S3S5 for Gamcheonbae, S3S5 for Danbae, S3S4 for Manpoong, S3S4 for Soowhangbae and S4S6 for Chuwhangbae. The information on the S-genotypes of pear cultivars will be used for the pollinizer selection and breeding program.     

Characterization of the S-RNase promoters from sweet cherry (Prunus avium L.)

Genomic DNA fragments containing the S(3)-, S(4)-, and S(6)-RNase genes were isolated from the sweet cherry (Prunus avium L.) and sequenced. Comparison of the 5'-flanking sequences of these three S-RNases indicated that a highly conserved region (designated CR) existed just upstream from the putative TATA boxes. We postulate that CR contains cis-regulatory element(s) involved in pistil expression. To examine the activity of the isolated S-RNase promoters of sweet cherry in the pistil, we transiently introduced approximately 650-bp fragments of the S(4)- and S(6)-RNase promoters fused to beta-glucuronidase (GUS) gene into the pistil of the petunia using a particle bombardment technique. Histochemical analysis showed that the 5'-flanking region of each S-RNase was active in the pistil. This suggests that cis-regulatory element(s) for pistil-specific expression may exist(s) within the 650-bp region upstream from the TATA box in the sweet cherry S-RNase promoter.     

Wild and Rare Self-Incompatibility Allele S17 Found in 24 Sweet Cherry (Prunus avium L.) Cultivars

The pollination of self-incompatible diploid sweet cherry is determined by the S-locus alleles. We resolved the S-alleles of 50 sweet cherry cultivars grown in Estonia and determined their incompatibility groups, which were previously unknown for most of the tested cultivars. We used consensus primers SI-19/20, SI-31/32, PaConsI, and PaConsII followed by allele-specific primers and sequencing to identify sweet cherry S-genotypes. Surprisingly, 48% (24/50) of the tested cultivars, including 17 Estonian cultivars, carry the rare S-allele S₁₇, which had initially been described in wild sweet cherries in Belgium and Germany. The S₁₇-allele in Estonian cultivars could originate from ‘Leningradskaya tchernaya’ (S₆|S₁₇), which has been extensively used in Estonian sweet cherry breeding. Four studied cultivars carrying S₁₇ are partly self-compatible, whereas the other 20 cultivars with S₁₇ have not been reported to be self-compatible. The recommended pollinator of seven self-incompatible sweet cherries is of the same S-genotype, including four with S₁₇-allele, suggesting heritable reduced effectiveness of self-infertility. We classified the newly genotyped sweet cherry cultivars into 15 known incompatibility groups, and we proposed four new incompatibility groups, 64–67, for S-locus genotypes S₃|S₁₇, S₄|S₁₇, S₅|S₁₇, and S₆|S₁₇, respectively, which makes them excellent pollinators all across Europe. Alternatively, the frequency of S₁₇ might be underestimated in Eastern European populations and some currently unidentified sweet cherry S-alleles might potentially be S₁₇.

     

Assessment of genetic diversity of Czech sweet cherry cultivars using microsatellite markers

We analyzed 24 sweet and wild cherry genotypes collected in Czech Republic to determine genetic variation, using previously described 16 SSR primers to adapt a fast, reliable method for preliminary screening and comparison of sweet cherry germplasm collections. All SSRs were polymorphic and they were able all together to distinguish unambiguously the genotypes. These SSR primers generated 70 alleles; the number of alleles per primer ranged from 2 to 7, with a mean of 4.4 putative alleles per primer combination. The primer UDP-98-412 gave the highest number of polymorphic bands (totally 7), while Empa2 and Empa3 gave the lowest number (2). The allele frequency varied from 2.1% to 87.5%. We observed 10% of unique alleles at different loci. The observed heterozygosity value ranged from 0.25 to 0.96 with an average of 0.72 while expected heterozygosity value varied from 0.22 to 0.75 with an average of 0.59. The PIC value ranged from 0.21 to 0.71 with a mean value of 0.523. Cluster analysis separated the investigated cultivars in two groups. High level of genetic diversity obtained in the collection and proved to be sufficiently genetically diverse and therefore these genotypes would be useful to breeders for the development of new cherry cultivars.