基于cDNA芯片的梨品种S基因型鉴定及新S-RNase基因进化分析

梨品种S基因型鉴定对梨栽培中授粉品种选择和遗传育种都具有重要意义。本研究利用梨S-RNase基因荧光标记的特异引物PCR扩增获得梨品种荧光标记的cDNA特异产物;进一步完善梨S-RNase基因cDNA芯片,以被检测梨品种cDNA特异序列与梨S-RNase基因cDNA芯片杂交检测不同梨品种S基因型,并发现新的S-RNase基因。结果表明:利用梨S-RNase基因cDNA芯片鉴定了泸定王皮梨、兴山24号、弥渡百合等35个未知S基因型梨品种,确定了各品种的S基因型。结合PCRRFLP及DNA克隆和测序等技术,发现了7个新的S-RNase基因资源,获得了新S-RNase基因序列。序列分析表明各新S-RNase基因均具有S-RNase基因特异区域序列的典型特征;进化分析显示7个新S-RNase基因主要属于蔷薇科苹果亚科S-RNase类群,且存在种间和属间比种内和属内进化关系更近的现象。7个新的S基因分别命名为:PpS_(53)(Pyrus pyrifolia S53)、PpS_(54)、PpS_(55)、PpS_(56)、PpS_(57)、PpS_(58)和PpS_(59),GenBank登录号分别为:KX581753、KX581754、KX581755、KX581756、KX581757、KX581751和KX581752。     

Development of a CAPS marker system for genotyping European pear cultivars harboring 17 <Emphasis Type="Italic">S</Emphasis> alleles

The full-length cDNAs of eight S ribonucleases (S-RNases) were cloned from stylar RNA of European pear cultivars that could not be characterized by the cleaved amplified polymorphic sequences (CAPS) marker system for genotyping European pear cultivars harboring nine S alleles Sa, Sb, Sd, Se, Sh, Sk, Sl, Sq, and Sr. Comparison of the nucleotide sequences between these cDNAs and six putative S-RNase alleles previously amplified by genomic PCR revealed that five corresponded to the putative Sc-, Si-, Sm-, Sn-, and Sp-RNase alleles and the other three corresponded new S-RNase alleles (designated as putative Sg-, Ss-, and St-RNase alleles). Genomic PCR with a new set of primers was used to amplify 17 S-RNase alleles: 1906 bp (Sg), 1642 bp (St), 1414 bp (Sl), ca. 1.3 kb (Sk and Sq), 998 bp (Se), 440 bp (Sb), and ca. 350 bp (Sa, Sc, Sd, Sh, Si, Sm, Sn, Sp, Sr, and Ss). Among them, S-RNase alleles of similar size were discriminated by digestion with 11 restriction endo-nucleases. The PCR amplification of 17 S-RNase alleles following digestion with the restriction endonucleases provided a new CAPS marker system for rapid S-genotyping of European pear cultivars harboring 17 S alleles. Using the CAPS analysis, Sc, Sg, Si, Sm, Sn, Sp, Ss, and St alleles were found in 32 cultivars, which were classified into 23 S-genotypes.     

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。     

Identification of Self-Incompatibility Genotypes of Apricot (<Emphasis Type="Italic">Prunus armeniaca</Emphasis> L.) by S-Allele-Specific PCR Analysis

A cDNA of 417 bp encoding an S-RNase gene, named PA S3, was isolated from apricot, Prunus aremeniaca. Nine S-alleles, S₁–S₉, were recognized by S-allele-specific PCR and confirmed by Southern blot analysis using PA S3 as probe. The S-genotypes of the six cultivars were determined and the results of self- and cross-pollination tests among the six cultivars were consistent with the predicted S-haplotypes by PCR analysis.     

24份甜樱桃材料自交不亲和S基因型鉴定

甜樱桃品种绝大部分自交不亲和,限制了甜樱桃的正确评价和合理利用,因此自交不亲和基因型的鉴定对于生产具有重要意义。以24个甜樱桃主栽品种为材料,用5对蔷薇科李属引物组合对24个甜樱桃品种进行了S等位基因的PCR扩增,克隆S基因的扩增片段,用核酸序列在Gen Bank上搜索,确定了5种S基因的核酸序列和大小。结果表明:Pru C2+Pru C4R引物组合扩增效果最好;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;5种S基因扩增片段的大小分别是S1为800 bp,S3为762 bp,S4为962bp,S5为300 bp,S6为456 bp,S9为650 bp;24个甜樱桃S基因型是红手球、早红宝石为S1S3,拉宾斯S1S4',红宝石S1S6,布鲁克斯S1S9,那翁S3S4,秦林、泰安大紫、先锋、早大果、丽珠、美早、5-106、左滕锦、桑提娜为S3S6,黑珍珠、红灯、萨米脱、秦樱为S3S9,胜利为S5S9,明珠、红蜜、雷尼、滨库为S6S9。     

Cloning and characterization of cDNAs encoding S-RNases from almond (Prunus dulcis): primary structural features and sequence diversity of the S-RNases in Rosaceae

cDNAs encoding three S-RNases of almond (Prunus dulcis), which belongs to the family Rosaceae, were cloned and sequenced. The comparison of amino acid sequences between the S-RNases of almond and those of other rosaceous species showed that the amino acid sequences of the rosaceous S-RNases are highly divergent, and intra-subfamilial similarities are higher than inter-subfamilial similarities. Twelve amino acid sequences of the rosaceous S-RNases were aligned to characterize their primary structural features. In spite of?their high level of diversification, the rosaceous S-RNases were found to have five conserved regions, C1, C2, C3, C5, and RC4 which is Rosaceae-specific conserved region. Many variable sites fall into one region, named RHV. RHV is located at a similar position to that of the hypervariable region a (HVa) of the solanaceous S-RNases, and is assumed to be involved in recognizing S-specificity of pollen. On the other hand, the region corresponding to another solanaceous hypervariable region (HVb) was not variable in the rosaceous S-RNases. In the phylogenetic tree of the T2/S type RNase, the rosaceous S-RNase fall into two subfamily-specific groups (Amygdaloideae and Maloideae). The results of sequence comparisons and phylogenetic analysis imply that the present S-RNases of Rosaceae have diverged again relatively recently, after the divergence of subfamilies.     

Primary structural features of rosaceous S-RNases associated with gametophytic self-incompatibility

We isolated cDNA clones encoding five S-RNases (S_1-,_S3- , S_5-, S_6-, S_7-RNases) from pistils of Pyrus pyrifolia (Japanese pear), a member of the Rosaceae. Their amino acid sequences were aligned with those of other rosaceous S-RNases sequenced so far. A total of 76 conserved amino acid residues were stretched throughout the sequence, but were absent from the 51–66 region which was designated the hypervariable (HV) region. The phylogenetic tree of rosaceous S-RNases showed that S-RNase polymorphism predated the divergence of Pyrus and Malus. Pairwise comparison of these S-RNases detected two highly homologous pairs, P. pyrifolia S_1- and S_4-RNases (90.0%) and P. pyrifolia S_3- and S_5-RNases (95.5%). The positions of amino acid substitutions between S_1- and S_4-RNases were spread over the entire region, but in the pair of S_3- and S_5-RNases, amino acid substitutions were found in the 21–90 region including the HV region. The substitutions in this restricted region appear to be sufficient to discriminate between S₃ and S₅ pollen and to trigger the self-incompatible reaction.     

Sequence comparison of the 5’ flanking regions of Japanese pear (Pyrus pyrifolia) S-RNases associated with gametophytic self-incompatibility

Genomic clones of 2.8 kb, 4.3 kb and 6.5 kb for the S₂-, S₃- and S₅-RNases of Japanese pear(Pyrus pyrifolia), respectively, were isolated and sequenced. Comparison of the 5’-flanking regions of these genes with the same region of the S₄-RNase gene indicated that a highly similar region of approximately 200 bp exists in the regions just upstream of the putative TATA boxes of the four Japanese pear S-RNase genes. This suggests the presence of cis-regulatory element(s) in this region. Received: 5 October 2000 / Revision accepted: 2 January 2001     

Evolutionary analysis of S-RNase genes from Rosaceae species

Eight new cDNA sequences for S-RNases were cloned and analysed from almond (Prunus dulcis) cultivars of European origin, and compared to published sequences from other Rosaceae species. Insertions/deletions of 10-20 amino acid residues were detected in the RC4 and C5 domains of S-RNases from almond and sweet cherry. The S-RNases of the Prunus species and those of the genera Malus and Pyrus formed two distinct groups on phylogenetic analysis. Nucleotide substitutions were analysed in the S-RNase genes of these species. The S-genes of almond and sweet cherry have a lower Ka/Ks value than those of apple, pear and wild apple do. The fact that there is no fixed difference between the S-RNase genes of almond and sweet cherry, or between apple and pear, suggests that nucleotide substitutions only introduce transient polymorphism into the two groups, and rarely became fixed and contribute to divergence. Through the comparative study of 17 S-RNase genes from the genus Prunus and 18 from the genera Malus and Pyrus, some fixed nucleotide differences between the two groups were identified. These differences do not appear to be the result of selection for adaptive mutations, since the number of replacement substitutions is not significantly greater than the number of synonymous substitutions. S-RNase genes of almond and sweet cherry, and of apple and pear, showed little heterogeneity in nucleotide substitution rates. However, heterogeneity was observed between the two groups of S-alleles, with the Prunus alleles exhibiting a lower rate of non-synonymous substitutions than alleles from Malus and Pyrus. The evolutionary relationships between these species are discussed.     

Identification and characterization of components of a putative petunia S-locus F-box-containing E3 ligase complex involved in S-RNase-based self-incompatibility

Petunia inflata S-locus F-box (Pi SLF) is thought to function as a typical F-box protein in ubiquitin-mediated protein degradation and, along with Skp1, Cullin-1, and Rbx1, could compose an SCF complex mediating the degradation of nonself S-RNase but not self S-RNase. We isolated three P. inflata Skp1s (Pi SK1, -2, and -3), two Cullin-1s (Pi CUL1-C and -G), and an Rbx1 (Pi RBX1) cDNAs and found that Pi CUL1-G did not interact with Pi RBX1 and that none of the three Pi SKs interacted with Pi SLF₂. We also isolated a RING-HC protein, S-RNase Binding Protein1 (Pi SBP1), almost identical to Petunia hybrida SBP1, which interacts with Pi SLFs, S-RNases, Pi CUL1-G, and an E2 ubiquitin-conjugating enzyme, suggesting that Pi CUL1-G, SBP1, and SLF may be components of a novel E3 ligase complex, with Pi SBP1 playing the roles of Skp1 and Rbx1. S-RNases interact more with nonself Pi SLFs than with self Pi SLFs, and Pi SLFs also interact more with nonself S-RNases than with self S-RNases. Bacterially expressed S₁-, S₂-, and S₃-RNases are degraded by the 26S proteasomal pathway in a cell-free system, albeit not in an S-allele–specific manner. Native glycosylated S₃-RNase is not degraded to any significant extent; however, deglycosylated S₃-RNase is degraded as efficiently as the bacterially expressed S-RNases. Finally, S-RNases are ubiquitinated in pollen tube extracts, but whether this is mediated by the Pi SLF–containing E3 complex is unknown.     

Cloning, characterization and promoter analysis of S-RNase gene promoter from Chinese pear (Pyrus pyrifolia)

The 5'-flanking region of the S(12)-, S(13)-, S(21)-RNase with a length of 854 bp, 1448 bp and 1137 bp were successfully isolated by TAIL-PCR from genomic DNA from 'Jinhua', 'Maogong' (Pyrus pyrifolia) and 'Yali' (Pyrus bretschneideri) genomic DNA. Sequence alignment and analysis of S(13)-, S(12)-, S(21)-RNase gene promoter sequences with S(2)-, S(3)-, S(4)-, S(5)-RNase 5'-flanking sequences indicated that a homology region of about 240 bp exists in the regions just upstream of the putative TATA boxes of the seven Chinese/Japanese pear S-RNase genes. Phylogenetic tree suggests that the homology region between the Chinese/Japanese pear and apple S-RNase gene promoter regions reflects the divergence of S-RNase gene was formed before the differentiation of subfamilies. Full length and a series of 5'-deletion fragments-GUS fusions were constructed and introduced into Arabidopsis thaliana plants. GUS activity were detected in S(12)-pro-(1 to 5)-GUS-pBll01.2 transgenic pistils and progressively decreased from S(12)-pro-1-GUS-pBI l01.2 to S(12)-pro-5-GUS-pBll01.2. No GUS activity was detected in S(12)-pro-6-GUS-pBll01.2 transgenic pistil and other tissues of non-transformants and all transgenic plants. The result suggested S(12)-RNase promoter is pistil specific expression promoter.     

Cloning and characterization of cDNAs encoding S-RNases from almond ( Prunus dulcis ): primary structural features and sequence diversity of the S-RNases in Rosaceae

cDNAs encoding three S-RNases of almond (Prunus dulcis), which belongs to the family Rosaceae, were cloned and sequenced. The comparison of amino acid sequences between the S-RNases of almond and those of other rosaceous species showed that the amino acid sequences of the rosaceous S-RNases are highly divergent, and intra-subfamilial similarities are higher than inter-subfamilial similarities. Twelve amino acid sequences of the rosaceous S-RNases were aligned to characterize their primary structural features. In spite of␣their high level of diversification, the rosaceous S-RNases were found to have five conserved regions, C1, C2, C3, C5, and RC4 which is Rosaceae-specific conserved region. Many variable sites fall into one region, named RHV. RHV is located at a similar position to that of the hypervariable region a (HVa) of the solanaceous S-RNases, and is assumed to be involved in recognizing S-specificity of pollen. On the other hand, the region corresponding to another solanaceous hypervariable region (HVb) was not variable in the rosaceous S-RNases. In the phylogenetic tree of the T2/S type RNase, the rosaceous S-RNase fall into two subfamily-specific groups (Amygdaloideae and Maloideae). The results of sequence comparisons and phylogenetic analysis imply that the present S-RNases of Rosaceae have diverged again relatively recently, after the divergence of subfamilies. Received: 28 May 1998 / Accepted: 13 August 1998     

确定梨自交不亲和基因型研究的技术进展

综述了运用杂交授粉试验和分子生物学方法等技术确定梨品种自交不亲和基因型研究的技术进展,分析了这些技术在确定梨品种自交不亲和基因型方面的优点和不足之处,并初步探讨了研究前景。因为HV区氨基酸的不同,不同S基因型也有所差异。因此,除了在分子生物学的水平上进行研究外,其他方法如mRNA、蛋白质和杂交授粉等水平上的研究在确定S基因型上也同样重要。     

Determination of self-locompatibility genotypes of Korean apple cultivars based on S-RNase PCR

To prevent self-fertilization, apple has a gametophytic self-incompatibility mechanism, part of a widespread intraspecific system, that is controlled by a multi-allelic locus. This attribute has been exploited in breeding programs for new cultivars. Likewise, many apple orchards depend on artificial pollination. Therefore, molecular analysis and early identification of the self-incompatibility (S) genotype could greatly improve breeding schemes and pollen donors selection. Here, we PCR-amplified the S-RNase PCR fragments from a total of 14 cultivars and parents, using new primers (ASPF3+ASPR3) common to 23 S-alleles in apple. The S-genotypes were determined for the following: ‘Hongro’ (S₁S₃), ‘Gamhong’ (S₁S₉), ‘Saenara’ (S₁S₃), ‘Chukwang’ (S₃S₉), ‘Hwahong’ (S₃S₉), ‘Seokwang’ (S₃S₃), ‘Hwarang’ (S₁S₉), ‘Sunhong’ (S₃S₉), ‘S.E.B.’ (S₁S₁₉), ‘S.G.D.’ (S₂S₃), and ‘Mollie’s Delicious’ (S₃S₇). We also confirmed the characteristics of the S-genotypes for eight Korean apple cultivars by PCR-Southern blot analysis, using seven S-RNases as probes.     

The RHV region of S-RNase in the European pear (<Emphasis Type="Italic">Pyrus communis</Emphasis>) is not required for the determination of specific pollen rejection

In the gametophytic self-incompatibility system, growth of self-pollen tubes in the style is inhibited in a haplotype-specific manner by S-RNase. The mechanism by which S-RNase confers its specificity is unknown. However, a hypervariable region (RHV in Rosaceae and HVa-HVb in Solanaceae) that differs among the many cloned S-RNase alleles has been proposed to be involved in conferring the S-haplotype specificity of the S-RNase. Region swapping experiments between S-RNases and crystallography of the enzyme support this assumption. However, the deduced amino acid sequences of Sn-RNase and Si-RNase alleles from the European pear (Pyrus communis) were recently found to have an identical RHV. In the present study it is shown that Sn-RNase does not prevent fertilization by Si-pollen haplotype, thus presenting a case in which RHV is not required for the determination of specific pollen rejection by S-RNase, and implying that other regions in the enzyme may be sufficient for this specificity.     

cDNA cloning of nine S alleles and establishment of a PCR-RFLP system for genotyping European pear cultivars

Nine full-length cDNAs of S ribonucleases (S-RNases) were cloned from stylar RNA of European pear cultivars by RT-PCR and 3′ and 5′ RACE. Comparison of the nucleotide sequences between the nine S-RNases cloned and 13 putative S alleles previously amplified by genomic PCRs revealed that seven corresponded to Sa, Sb, Sd, Se, Sh, Sk and Sl alleles, and the other two were new S alleles (designated as Sq and Sr alleles). Genomic PCR with a set of ȁ8FTQQYQȁ9 and ȁ8EP-anti-IIWPNVȁ9 primers was used to amplify nine S alleles; 1,414 bp (Sl), ca. 1.3 kb (Sk and Sq), 998 bp (Se), 440 bp (Sb) and ca. 350 bp (Sa, Sd, Sh and Sr). Among these, S alleles of similar size were discriminated by digestion with BaeI, BglII, BssHII, HindIII, EcoO109I and SphI. The PCR amplification of S allele following digestion with the restriction enzymes provided a PCR-RFLP system for rapid S-genotyping European pear cultivars harboring nine S alleles. The PCR-RFLP system assigned a total of 63 European pear cultivars to 25 genotypes. Among these, 14 genotypes were shared by two or more cultivars, which were cross-incompatible. These results suggested that the genes cloned represented the S-RNases from European pear, and that there were many cross-incompatible combinations among European pear varieties.     

Self-incompatibility (S) alleles of the rosaceae encode members of a distinct class of the T2/S ribonuclease superfamily

Stylar riboncleases (RNases) are associated with gametophytic self-incompatibility in two plant families, the Solanaceae and the Rosaceae. The self-incompatibility-associated RNases (S-RNases) of both the Solanaceae and the Rosaceae were recently reported to belong to the T2 RNase gene family, based on the presence of two well-conserved sequence motifs. Here, the cloning and characterization of S-RNase genes from two species of Rosaceae, apple (Malus × domestica) and Japanese pear (Pyrus serotina) is described and these sequences are compared with those of other T2-type RNases. The S-RNases of apple specifically accumulated in styles following maturation of the flower bud. Two cDNA clones for S-RNases from apple, and PCR clones encoding a further two apple S-RNases as well as two Japanese pear S-RNases were isolated and sequenced. The deduced amino acid sequences of the rosaceous S-RNases contained two conserved regions characteristic of the T2/S-type RNases. The sequences showed a high degree of diversity, with similarities ranging from 60.4% to 69.2%. Interestingly, some interspecific sequence similarities were higher than those within a species, possibly indicating that diversification of S-RNase alleles predated speciation in the Rosaceae. A phylogenetic tree of members of the T2/S-RNase superfamily in plants was obtained. The rosaceous S-RNases formed a new lineage in the tree that was distinct from those of the solanaceous S-RNases and the S-like RNases. The findings suggested that self-incompatibility mechanisms in Rosaceae and Solanaceae are similar but arose independently in the course of evolution.     

Self-incompatibility (S) alleles of the rosaceae encode members of a distinct class of the T2/S ribonuclease superfamily

Stylar riboncleases (RNases) are associated with gametophytic self-incompatibility in two plant families, the Solanaceae and the Rosaceae. The self-incompatibility-associated RNases (S-RNases) of both the Solanaceae and the Rosaceae were recently reported to belong to the T2 RNase gene family, based on the presence of two well-conserved sequence motifs. Here, the cloning and characterization of S-RNase genes from two species of Rosaceae, apple (Malus × domestica) and Japanese pear (Pyrus serotina) is described and these sequences are compared with those of other T2-type RNases. The S-RNases of apple specifically accumulated in styles following maturation of the flower bud. Two cDNA clones for S-RNases from apple, and PCR clones encoding a further two apple S-RNases as well as two Japanese pear S-RNases were isolated and sequenced. The deduced amino acid sequences of the rosaceous S-RNases contained two conserved regions characteristic of the T2/S-type RNases. The sequences showed a high degree of diversity, with similarities ranging from 60.4% to 69.2%. Interestingly, some interspecific sequence similarities were higher than those within a species, possibly indicating that diversification of S-RNase alleles predated speciation in the Rosaceae. A phylogenetic tree of members of the T2/S-RNase superfamily in plants was obtained. The rosaceous S-RNases formed a new lineage in the tree that was distinct from those of the solanaceous S-RNases and the S-like RNases. The findings suggested that self-incompatibility mechanisms in Rosaceae and Solanaceae are similar but arose independently in the course of evolution.     

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.     

Purification and characterization of a non-S-RNase and S-RNases from styles of Japanese pear (Pyrus pyrifolia)

In this study we biochemically characterized stylar ribonucleases (RNases) of Japanese pear (Pyrus pyrifolia), which exhibits S-RNase-based gametophytic self-incompatibility. We separated the RNase fractions NS-1, NS-2, and NS-3 from stylar extracts of the cultivar Nijisseiki (S(2)S(4)). The RNase in each fraction was purified to homogeneity through a series of chromatographic steps. Chemical analysis of the proteins revealed that the basic RNases in the NS-2 and NS-3 fractions were the S(4)- and S(2)-RNases, respectively. Five additional S-RNases were purified from other cultivars. An acidic RNase in the NS-1 fraction was also purified from other cultivars, and identified as a non-S-allele-associated RNase (non-S-RNase). The non-S-RNase is composed of 203 amino acids, is non-glycosylated and is a N-terminal-pyroglutamylated enzyme of the RNase T(2) family. The substrate specificities and optimum pH levels of the non-S-RNase and S-RNases were similar. Interestingly, the specific activity of the non-S-RNase was 7.5-221-fold higher than those of the S-RNases when tolura yeast RNA was used as the substrate. The specific activity of the S(2)-RNase was 8.8-28.6-fold lower than those of the other S-RNases. These differences in specific activities among the stylar RNases are discussed.