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.     

Molecular characterization of S-RNase genes and S-genotypes in the Japanese apricot (Prunus mume Sieb. et Zucc.)

Three partial S-RNase genes, MSRN-1, MSRN-2, and MSRN-3, in the Japanese apricot (Prunus mume Sieb. et Zucc.) were isolated from the three cultivars Nankou, Gyokuei, and Kairyouuchidaume, respectively. The structural characteristics revealed that S-RNase genes from the Japanese apricot were in the T2/SRNase-type S-RNase family with five conserved regions (C1, C2, C3, RC4, and C5) and one variable region (RHV) as reported in the other rosaceous plants. In the phylogenetic tree of T2/S SRNase-type RNases, three S-RNase genes of the Japanese apricot did not form a species-specific subgroup but the Prunus subfamily did. At least seven S-allelic genes were present in the Japanese apricot, and S-genotypes of six representative cultivars, including Nankou, Gyokuei, Kairyouuchidaume, Baigou, Kagajizou, and Oushuku were first established in this study as S ₁ S ₇, S ₂ S ₆, S ₃ S ₄, S ₃ S ₆, S ₃ S ₆ and S ₁ S ₅, respectively. An extended elucidation of the S-genotype would contribute to a more efficient breeding program of the Japanese apricot. Received: 5 September 2000 / Revision accepted: 22 December 2000     

Molecular characterization of newS-RNases (‘S31’ and ‘S32’) in apple (Malus ×domestica Borkh.)’) in apple (Malus ×domestica Borkh.)

Apple exhibits gametophytic self-incompatibility (GSI) that is controlled by the multiallelic S-locus. This S-locus encodes polymorphicS ribonuclease (S-RNase) for the pistil-part 5 determinant. Information aboutS-genotypes is important when selecting pollen donors for fruit production and breeding of new cultivars. We determined the 5-genotypes of ‘Charden’ (S₂S₃S₄), ‘Winesap’ (S₁S₂₈), ‘York Imperial’ (S₂S₃₁), ‘Stark Earliblaze’ (S₁S₂₈), and ‘Burgundy’ (S₂₀S₃₂), byS-RNase sequencing and S-allele-specific PCR analysis. Two newS-RNases, S₃₁ and S₃₂, were also identified from ‘York Imperial’ and ‘Burgundy’, respectively. These newS-alleles contained the conserved eight cysteine residues and two histidine residues essential for RNase activity. Whereas S₃₁ showed high similarity to S₂₀ (94%), S₃₂ exhibited 58% (to S₂₄) to 76% (to S₂₅) similarity in the exon regions. We designed newS-allele-specific primers for amplifying S₃₁- and S₃₂-RNasc-specific fragments; these can serve as specific gene markers. We also rearranged the apple S-allele numbers containing those newS-RNases. They should be useful, along with anS-RNase-based PCR system, in determining S-genotypes and analyzing new alleles from apple cultivars.     

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。     

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。     

RNaseI from Escherichia coli cannot substitute for S-RNase in rejection of Nicotiana plumbaginifolia pollen

Unilateral incompatibility often occurs between self-incompatible (SI) species and their self-compatible (SC) relatives. For example, SI Nicotiana alata rejects pollen from SC N. plumbaginifolia, but the reciprocal pollination is compatible. This interspecific pollen rejection system closely resembles intraspecific S-allele-specific pollen rejection. However, the two systems differ in degree of specificity. In SI, rejection is S-allele-specific, meaning that only a single S-RNase causes rejection of pollen with a specific S genotype. Rejection of N. plumbaginifolia pollen is less specific, occurring in response to almost any S-RNase. Here, we have tested whether a non-S-RNase can cause rejection of N. plumbaginifolia pollen. The Escherichia coli rna gene encoding RNaseI was engineered for expression in transgenic (N. plumbaginifolia × SC N. alata) hybrids. Expression levels and pollination behavior of hybrids expressing E. coli RNaseI were compared to controls expressing S_A2-RNase from N. alata. Immunoblot analysis and RNase activity assays showed that RNaseI and S_A2-RNase were expressed at comparable levels. However, expression of S_A2-RNase caused rejection of N. plumbaginifolia pollen, whereas expression of RNaseI did not. Thus, in this system, RNase activity alone is not sufficient for rejection of N. plumbaginifolia pollen. The results suggest that S-RNases may be specially adapted to function in pollen rejection.     

Self-incompatibility of Prunus tenella and evidence that reproductively isolated species of Prunus have different SFB alleles coupled with an identical S-RNase allele

Many species of Prunus display an S-RNase-based gametophytic self-incompatibility (SI), controlled by a single highly polymorphic multigene complex termed the S-locus. This comprises tightly linked stylar- and pollen-expressed genes that determine the specificity of the SI response. We investigated SI of Prunus tenella, a wild species found in small, isolated populations on the Balkan peninsula, initially by pollination experiments and identifying stylar-expressed RNase alleles. Nine P. tenella S-RNase alleles (S(1)-S(9)) were cloned; their sequence analysis showed a very high ratio of non-synonymous to synonymous nucleotide substitutions (K(a)/K(s)) and revealed that S-RNase alleles of P. tenella, unlike those of Prunus dulcis, show positive selection in all regions except the conserved regions and that between C2 and RHV. Remarkably, S(8)-RNase, was found to be identical to S(1)-RNase from Prunus avium, a species that does not interbreed with P. tenella and, except for just one amino acid, to S(11) of P. dulcis. However, the corresponding introns and S-RNase-SFB intergenic regions showed considerable differences. Moreover, protein sequences of the pollen-expressed SFB alleles were not identical, harbouring 12 amino-acid replacements between those of P. tenella SFB(8) and P. avium SFB(1). Implications of this finding for hypotheses about the evolution of new S-specificities are discussed.     

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.     

S-RNase triggers mitochondrial alteration and DNA degradation in the incompatible pollen tube of Pyrus pyrifolia in vitro

Pear ( Pyrus pyrifolia L.) has a S-RNase-based gametophytic self-incompatibility (SI) mechanism, and S-RNase has also been implicated in the rejection of self-pollen and genetically identical pollen. No studies, however, have examined the extent of organelle alterations during the SI response in Pyrus pyrifolia . Consequently, this study focused on the alterations to mitochondria and nuclear DNA in incompatible pollen tubes of the pear. Methylthiazolyldiphenyl-tetrazolium bromide was used to evaluate the viability of pollen tubes under S-RNase challenge. The results showed that the viability of the control and compatible pollen tubes decreased slightly, but that of the incompatible pollen and pollen tubes began to decline at 30 min. The mitochondrial membrane potential (Δ ψ _mit) was also tested with rhodamine 123 30 min after SI challenge, and was shown to have collapsed in the incompatible pollen tubes after exposure to S-RNase. Western blotting 2 h after SI challenge confirmed that the Δ ψ _mit collapse induced leakage of cytochrome c into the cytosol. Swollen mitochondria were detected by transmission electron microscopy as early as 1 h after SI challenge and the degradation of nuclear DNA was observed by both 4,6-diamidino-2-phenylindole and transferase-mediated dUTP nick-end labeling. These diagnostic features of programmed cell death (PCD) suggested that PCD may specifically occur in incompatible pollen tubes.     

植物基因转化技术在苹果遗传改良中的应用

近年来,苹果基因转化技术研究取得了较大进展,本文从转化体系,选择标记,报告基因及目的基因的遗传学行为等方面综述了苹果遗传转化研究发展现状,着重论述了其在苹果遗传改良中的应用,并对植物基因转化技术在莱果上的应用前景和应注意的几个问题进行了讨论。     

山楂叶螨在苹果不同品种上的生长发育及繁殖

室内试验结果表明 ,山楂叶螨TetrancychusvienensisZacher在苹果不同品种上的生长发育和繁殖有较大的差异。其中在红星上存活率高 ,发育速度快 ,生殖力强 ;而在秦冠上存活率低、发育速度慢、生殖力低。对山楂叶螨种群年结龄构的分析表明 ,卵在种群中所占比例较高 ,而成虫所占比例甚低 ,建议在对其进行防治时选用杀卵力强的药剂     

Identification of self-incompatibility genotypes of almond by allele-specific PCR analysis

In almond, gametophytic self-incompatibility is controlled by a single multiallelic locus (S-locus). In styles, the products of S-alleles are ribonucleases, the S-RNases. Cultivated almond in California have four predominant S-alleles (S ^a, S ^b, S ^c, S ^d). We previously reported the cDNA cloning of three of these alleles, namely S ^b, S ^c and S ^d. In this paper we report the cloning and DNA sequence analysis of the S ^a allele. The S^a-RNase displays approximately 55% similarity at the amino-acid level with other almond S-RNases (S^b, S^c, and S^d) and this similarity was lower than that observed among the S^b, S^c and S^d-RNases. Using the cDNA sequence, a PCR-based identification system using genomic DNA was developed for each of the S-RNase alleles. Five almond cultivars with known self-incompatibility (SI) geno-types were analyzed. Common sequences among four S-alleles were used to create four primers, which, when used as sets, amplify DNA bands of unique size that corresponded to each of the four almond S-alleles; S ^a (602 bp), S ^b (1083 bp), S ^c (221 bp) and S ^d (343 bp). All PCR products obtained from genomic DNA isolated from the five almond cultivars were cloned and their DNA sequence obtained. The nucleotide sequence of these genomic DNA fragments matched the corresponding S-allele cDNA sequence in every case. The amplified products obtained for the S ^a- and S ^b-alleles were both longer than that expected for the coding region, revealing the presence of an intron of 84 bp in the S ^a-allele and 556 bp in the S ^b-allele. Both introns are present within the site of the hypervariable region common in S-RNases from the Rosaceae family and which may be important for S specificity. The exon portions of the genomic DNA sequences were completely consistent with the cDNA sequence of the corresponding S-allele. A useful application of these primers would be to identify the S-genotype of progeny in a breeding program, new varieties in an almond nursery, or new grower selections at the seedling stage. Received: 21 June 1999 / Accepted: 15 November 1999     

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。     

梨花柱S-RNase对花粉管超微结构的影响

采用光学显微镜和透射电子显微镜研究了离体条件下不同品种梨花柱S—RNase对异花(亲和)及自花(不亲和)花粉萌发和花粉管生长及其超微结构的影响。结果表明,花柱S—RNase抑制不亲和花粉的萌发和花粉管的生长,对亲和花粉的萌发和花粉管的生长基本没有影响。花粉生长初期,亲和及不亲和花粉管超微结构相似;但培养24h以后,亲和花粉管中充满细胞质和细胞器,而不亲和花粉管中只有靠近花粉管前端有少量细胞质,细胞壁增厚,细胞壁与细胞质之间有一层胼胝质和电子透明区间隔。     

A tale of two continents: Baker's rule and the maintenance of self-incompatibility in Lycium (Solanaceae)

Over 50 years ago, Baker (1955, 1967) suggested that self-compatible species were more likely than self-incompatible species to establish new populations on oceanic islands. His logic was straightforward and rested on the assumption that colonization was infrequent; thus, mate limitation favored the establishment of self-fertilizing individuals. In support of Baker's rule, many authors have documented high frequencies of self-compatibility on islands, and recent work has solidified the generality of Baker's ideas. The genus Lycium (Solanaceae) has ca. 80 species distributed worldwide, and phylogenetic studies suggest that Lycium originated in South America and dispersed to the Old World a single time. Previous analyses of the S-RNase gene, which controls the stylar component of self-incompatibility, have shown that gametophytically controlled self-incompatibility is ancestral within the genus, making Lycium a good model for investigating Baker's assertions concerning reproductive assurance following oceanic dispersal. Lycium is also useful for investigations of reproductive evolution, given that species vary both in sexual expression and the presence of self-incompatibility. A model for the evolution of gender dimorphism suggests that polyploidy breaks down self-incompatibility, leading to the evolution of gender dimorphism, which arises as an alternative outcrossing mechanism. There is a perfect association of dimorphic gender expression, polyploidy, and self-compatibility (vs. cosexuality, diploidy, and self-incompatibility) among North American Lycium. Although the association between ploidy level and gender expression also holds for African Lycium, to date no studies of mating systems have been initiated in Old World species. Here, using controlled pollinations, we document strong self-incompatibility in two cosexual, diploid species of African Lycium. Further, we sequence the S-RNase gene in 15 individuals from five cosexual, diploid species of African Lycium and recover 24 putative alleles. Genealogical analyses indicate reduced trans-generic diversity of S-RNases in the Old World compared to the New World. We suggest that genetic diversity at this locus was reduced as a result of a founder event, but, despite the bottleneck, self-incompatibility was maintained in the Old World. Maximum-likelihood analyses of codon substitution patterns indicate that positive Darwinian selection has been relatively strong in the Old World, suggesting the rediversification of S-RNases following a bottleneck. The present data thus provide a dramatic exception to Baker's rule, in addition to supporting a key assumption of the Miller and Venable (2000) model, namely that self-incompatibility is associated with diploidy and cosexuality.     

苹果绵蚜对不同苹果品种春梢生长期生理指标的影响

为了筛选培育对苹果绵蚜(Eriosoma lanigerum Hausmann)的抗性品种,实现持续有效治理苹果绵蚜的目的,通过测定红富士、金帅、昭锦108、秦冠、红将军等5种不同苹果品种春梢生长期被苹果绵蚜危害前后枝条内可溶性糖、蛋白质、游离氨基酸、总酚含量以及防御性酶的活性变化,探讨苹果生理指标与抗蚜性的关系。结果表明,被害后可溶性糖含量除红将军外均有所上升,其中红富士上升达13.7%;蛋白质含量除红富士外均有所降低;氨基酸含量均有所上升,其中红将军变化明显,变化率达68.8%。酚类物质是一种重要的抗蚜物质,红富士、昭锦108、秦冠被害后总酚含量均升高,其中昭锦108、秦冠中总酚含量上升率约为红富士的2倍。红富士品种正常枝条内超氧化物歧化酶(Superoxide Dismutase,SOD)、多酚氧化酶(Polyphenol Oxidase,PPO)、过氧化物酶(Peroxidase,POD)以及过氧化氢酶(Catalase,CAT)活性均显著低于昭锦108,被害后各苹果枝条SOD活性均出现上升趋势,除红将军的PPO、POD活性降低外,其他品种均升高;金帅、红富士的CAT活性上升明显,分别为110.8%、45.5%。植物的防御性酶与其抗虫性有密切关系,苹果春梢生长期对苹果绵蚜的抗性与苹果体内的可溶性糖、游离氨基酸、SOD、PPO、POD以及CAT活性均有关,而且不同苹果品种被害后生理指标的变化也与其抗蚜性有一定关系。     

Identification of apple cultivars using RAPD markers

Summary Eleven apple cultivars were differentiated using randomly amplified polymorphic DNA (RAPD) markers obtained by the polymerase chain reaction (PCR). The variability of the technique and of the origin of the DNA extract was analyzed. A set of bands consistent in their presence or absence was chosen to create a differentiating band pattern. A key is proposed by which one can differentiate apple cultivars using commercially available prime.     

Isolation of S-RNase binding proteins from<Emphasis Type="Italic"> Solanum chacoense</Emphasis>: identification of an SBP1 (RING finger protein) orthologue

Currently, the most attractive working model of gametophytic self-incompatibility (SI) involving S-RNases postulates the presence of an inhibitor protein or complex expressed in pollen tubes that would counteract the cytotoxic effect of the ribonuclease activity of the S-RNase. Since it has been previously shown that allele-specific recognition is mediated through the hypervariable domain sequence of the S-RNase, we have targeted this region to isolate pollen-expressed interacting proteins in the yeast two-hybrid system. One of the isolated proteins corresponds to a RING finger protein highly similar to the previously isolated SBP1 protein from Petunia hybrida. This protein is postulated to be part of the RING finger E3 ligase family. The ScSBP1 gene is expressed in almost all tissues tested, suggesting a more general role than only being involved in SI. Although the ScSBP1 gene is polymorphic, linkage analysis showed that it was unlinked to the S-locus. The isolation of this S-RNase-binding protein in two different species and with four different S-RNase sequences as bait, strengthens its putative involvement in the SI response. Furthermore, comparison of the bait sequences used suggests that the SBP1 protein interacts with conserved sequences located between the HVa and HVb domains.Genbank accession numbers: ScSBP1, AY545464     

夏季苹果新梢生理指标与抗苹果绵蚜的关系

研究苹果生理指标与其对苹果绵蚜(Eriosoma lanigerum Hausmann)抗性的关系,为筛选培育抗性品种,实现持续有效治理苹果绵蚜提供依据.田间调查不同苹果品种对苹果绵蚜的抗性,测定比较各品种正常枝条生理指标,以及被苹果绵蚜危害后生理指标的变化.结果表明,正常枝条中可溶性糖(r=0.99)、蛋白质(r=0.86)含量与感蚜率呈正相关;氨基酸含量与感蚜率呈负相关(r=-0.96);酚类物质和4种酶活性与苹果感蚜率均不存在明显相关性.被害后昭锦108可溶性糖含量有所下降,红富士、金冠分别上升1.4％、7.0％;蛋白质、氨基酸、酚类物质含量均有所下降,其中红富士总酚含量明显下降,达23.5％,总酚下降率与感蚜率呈正相关(r=0.94);超氧化物歧化酶(Superoxide Dismutase,SOD)、过氧化氢酶(Catalase,CAT)活性均上升,其中CAT变化率与感蚜率存在相关性(r=-0.92),昭锦108 CAT活性明显上升,达91.2％;多酚氧化酶(Polyphenol Oxidase,PPO)、过氧化物酶(Peroxidase,POD)活性增减不一;金冠4种酶活性均上升.研究表明,对苹果绵蚜抗性较强的品种:可溶性糖、蛋白质含量较低,游离氨基酸含量较高;受害后可溶性糖含量下降,总酚含量下降率较低,游离氨基酸含量下降率较高.酶活性对抗蚜性的影响不明显.