甘蓝自交不亲和反应的细胞信号转导

S受体激酶(S—receptor kinase,SRK)和S位点富含半胱氨酸(S-locus cysteine-rich,SCR)分别是甘蓝柱头和花粉中导致自交不亲和反应的决定性蛋白质因子。本文就SRK、SCR的结构和功能加以综述,阐明两者在细胞信号转导中的作用。     

Commonality of self-recognition specificity of S haplotypes between Brassica oleracea and Brassica rapa

We have identified several interspecific pairs of S haplotypes having highly similar SRK and SP11/SCR sequences between Brassica oleracea and Brassica rapa. The recognition specificities of S haplotypes in these pairs were examined with three different methods. Stigmas of interspecific hybrids between an S-32 homozygote in B. oleracea and an S-60 homozygote in B. rapa, which were produced to avoid the interspecific incompatibility between the two species, showed incompatibility to the pollen of an S-8 homozygote in B. rapa and to the pollen of an S-15 homozygote in B. oleracea, while it showed compatibility to the pollen of other S haplotypes, suggesting B. oleracea S-32 and B. rapa S-60 have the same recognition specificity as B. rapa S-8 and B. oleracea S-15. Pollen grains of transgenic S-60 homozygous plants in B. rapa carrying a transgene of SP11-24 from B. oleracea were incompatible to B. rapa S-36 stigma, indicating that B. oleracea S-24 and B. rapa S-36 have the same recognition specificity. Application of the SP11 protein of B. rapa S-41 and S-47 onto the surface of B. oleracea S-64 stigmas and S-12 stigmas, respectively, resulted in the incompatibility reaction to pollen grains of another S haplotype, but application onto the stigmas of other S haplotypes did not, suggesting that B. oleracea S-64 stigmas and S-12 stigmas recognized the B. rapa SP11-41 and SP11-47 proteins as self SP11 proteins, respectively. Besides having evolutionary implications, finding of many interspecific pairs of S haplotypes can provide insight into the molecular mechanism of self-recognition. Comparing deduced amino-acid sequences of SP11 proteins and SRK proteins in the pairs, regions of SP11 and SRK important for self-recognition are discussed.     

Gene conversion from SLG to SRK resulting in self-compatibility in Brassica rapa

Self-compatible S-54 homozygotic plants were found in progenies of an F(1) hybrid cultivar in Chinese cabbage. Pollination tests revealed that this self-compatibility is controlled by the S locus and caused by the loss of the recognition function of the stigma. SRK, the gene for the recognition molecule in the stigma, was normally transcribed and translated in the self-compatible plants. The 1034-bp region in the receptor domain of SRK in the self-compatible plants was 100% identical to SLG in S-54, while that in self-incompatible S-54 homozygotic plants was 95.1% identical. These results suggest that the self-compatibility of the S-54 homozygotes is due to amino-acid changes caused by gene conversion from SLG to SRK.     

Towards an in vitro bioassay for the self-incompatibility response in Brassica oleracea

Summary Eluates of stigmas of Brassica oleracea that were known to contain S locus-specific glycoproteins (SLSG) discriminated between self and cross pollen in vitro in three different media. Discrimination was equally evident in experiments that were the in vitro equivalents of reciprocal pollinations. In a TAPS-buffered medium, self eluates depressed pollen germination in a dose-dependent manner. TAPS medium allowed a bioassay of the effects of SLSG in eluates because it optimized germination in a way that eliminated the complicating features of the stimulatory substances in the eluates. Stigma eluates affected percentage pollen germination and optimum calcium concentrations in vitro whether or not SLSG were present in the eluates, but differently in different media, and depending on whether the eluates were cross or self with respect to the pollen tested. Thus, the effect of stigma eluates on the in vitro germination of pollen in Brassica depends on the balance of stimulatory versus inhibitory substances in the eluates.     

Breakdown of self-incompatibility in the perennial Arabidopsis lyrata (Brassicaceae) and its genetic consequences

Mating systems in plants are known to be highly labile traits, with frequent transitions from outcrossing to selfing. The genetic basis for breakdown in self-incompatibility (SI) systems has been studied, but data on variation in selfing rates in species for which the molecular basis of SI is known are rare. This study surveyed such variation in Arabidopsis lyrata (Brassicaceae), which is often considered an obligately outcrossing species, to examine the causes and genetic consequences of changes in its breeding system. Based on controlled self-pollinations in the greenhouse, three populations from the Great Lakes region of North America included a minority of self-compatible (SC) individuals, while two showed larger proportions of SC individuals and all populations contained some individuals capable of setting selfed seeds. Loss of SI was not associated with particular haplotypes at the S-locus (as estimated by alleles amplified at the SRK locus, the gene controlling female specificity) and all populations contained similar numbers of SRK alleles, suggesting that some other genetic factor is responsible for modifying the SI reaction. The loss of SI has resulted in an effective shift in the mating system, as the two populations with a high frequency of SC individuals showed significantly lower microsatellite-based multilocus outcrossing rates and higher inbreeding coefficients than the other populations. Based on microsatellites, observed heterozygosities and genetic diversity were also significantly depressed in these populations. These findings provide the unique opportunity to examine in detail the consequences of mating system changes within a species with a well-characterized SI system.     

Effect of hexane and humidity on self-incompatibility in Brassica oleracea

Summary The effects of hexane, high humidity, flower age and temperature in overcoming the self-incompatibility of Brassica oleracea were studied using three plants, each of which was homozygous for a different dominant S-allele. Hexane had a significant effect in all cases, but the size of the effect varied considerably. In one plant there was a marked interaction between the effect of hexane, humidity and flower age, but temperature had relatively little effect. In another plant high humidity alone gave a very much greater response than hexane alone. This plant gave as many self-seeds from the high humidity treatment as from bud selfing, indicating that the incompatibility reaction was almost completely overcome by the high humidity. The results are discussed in the light of current views of the mechanism of incompatibility in Brassica.     

Evolutionary genetics of self-incompatibility in the Solanaceae

The self-incompatibility (S) gene in flowering plants has long been appreciated as an example of extreme allelic polymorphism maintained by frequency-dependent selection. Recent studies of population samples of S-allele sequences obtained by RT-PCR from five species of Solanaceae now reveal a picture of conspicuous inter-specific variation in both S-allele number and age. Explanations for this variation are examined with reference to current theory. We propose that changes in species' effective population size, particularly those associated with the evolution of different life histories, best account for interspecific differences in both the number and average age of S alleles.     

Transformation of Arabidopsis with a Brassica SLG/SRK region and ARC1 gene is not sufficient to transfer the self-incompatibility phenotype

Self-incompatibility (SI) promotes outbreeding in flowering plants, and in Brassica SI is genetically controlled by the S locus. Self-incompatible Brassica and self-fertile Arabidopsis belong to the same crucifer family. In addition, a comparative analysis reveals a high degree of microsynteny between the B. campestris S locus and its homologous region in Arabidopsis– with the notable exception that the Brassica SI genes, SLG and SRK, are missing. Brassica ARC1 encodes a component of the SRK signal transduction pathway leading to self-pollen rejection, and no closely related ARC1 homolog has been identified in Arabidopsis. The purpose of the research reported here was to introduce Brassica SI components into Arabidopsis in an attempt to compensate for the missing genes and to investigate whether the SI phenotype can be transferred. Inserts of approximately 40 kb from the fosmid clones F20 and F22, which span the B. napus W1 SLG-SRK region, were cloned into the plant transformation vector pBIBAC2. Transgenic plants were generated that expressed the Brassica SI genes in the flower buds. In addition, the endogenous, SLG-like, gene AtS1 was not co-suppressed by the Brassica SLG transgene. No SI phenotype was observed among the T1 BIBAC2-F20 and BIBAC2-F22 transgenic plants. When the ARC1 gene was transformed into BIBAC2-F20 or BIBAC2-F22 plants, the resulting BIBAC2-F20-ARC1 and BIBAC2-F22-ARC1 plants still set seeds normally, and no rejection response was observed when self-incompatible B. napus W1 pollen was placed on BIBAC2-F20-ARC1 or BIBAC2-F22-ARC1 Arabidopsis stigmas. Taken together, our results suggest that complementing Arabidopsis genome with Brassica SLG, SRK and ARC1 genes is unlikely to be sufficient to transfer the SI phenotype. Received: 11 November 1999 / Accepted: 14 February 2000     

High‐throughput multiplex cpDNA resequencing clarifies the genetic diversity and genetic relationships among Brassica napus,Brassica rapa and Brassica oleracea

Brassica napus (rapeseed) is a recent allotetraploid plant and the second most important oilseed crop worldwide. The origin of B. napus and the genetic relationships with its diploid ancestor species remain largely unresolved. Here, chloroplast DNA (cpDNA) from 488 B. napus accessions of global origin, 139 B. rapa accessions and 49 B. oleracea accessions were populationally resequenced using Illumina Solexa sequencing technologies. The intraspecific cpDNA variants and their allelic frequencies were called genomewide and further validated via EcoTILLING analyses of the rpo region. The cpDNA of the current global B. napus population comprises more than 400 variants (SNPs and short InDels) and maintains one predominant haplotype (Bncp1). Whole‐genome resequencing of the cpDNA of Bncp1 haplotype eliminated its direct inheritance from any accession of the B. rapa or B. oleracea species. The distribution of the polymorphism information content (PIC) values for each variant demonstrated that B. napus has much lower cpDNA diversity than B. rapa; however, a vast majority of the wild and cultivated B. oleracea specimens appeared to share one same distinct cpDNA haplotype, in contrast to its wild C‐genome relatives. This finding suggests that the cpDNA of the three Brassica species is well differentiated. The predominant B. napus cpDNA haplotype may have originated from uninvestigated relatives or from interactions between cpDNA mutations and natural/artificial selection during speciation and evolution. These exhaustive data on variation in cpDNA would provide fundamental data for research on cpDNA and chloroplasts.     

Expression level of the SLG gene is not correlated with the self-incompatibility phenotype in the class II S haplotypes of Brassica oleracea

In Brassica, the S-locus glycoprotein (SLG) gene has been strongly implicated in the self-incompatibility reaction. Several alleles of this locus have been sequenced, and accordingly grouped as class I (corresponding to dominant S-alleles) and class II (recessive). We recently showed that a self-compatible (Sc) line of Brassica oleracea expressed a class II-like SLG (SLG-Sc) gene. Here, we report that the SLG-Sc glycoprotein is electrophoretically and immunochemically very similar to the recessive SLG-S15 glycoprotein, and is similarly expressed in stigmatic papillae. Moreover, by seed yield analysis, we observe that both alleles are associated with a self-compatibility response, in contrast with the other known recessive S haplotypes (S2 and S5). By genomic DNA blot analysis, we show the existence of molecular homologies between the Sc and S15 haplotypes, but demonstrate that they are not identical. On the other hand, we also report that the S2 haplotype expresses very low amounts of SLG glycoproteins, although it exhibits a self-incompatible phenotype. These results strongly question the precise role of the SLG gene in the molecular mechanisms that control the self-incompatibility reaction of Brassica.     

Conservation of S-locus for self-incompatibility in Brassica napus (L.) and Brassica oleracea (L.)

 Self-incompatibility (SI) in Brassica is a sporophytic system, genetically determined by alleles at the S-locus, which prevents self-fertilization and encourages outbreeding. This system occurs naturally in diploid Brassica species but is introduced into amphidiploid Brassica species by interspecific breeding, so that in both cases there is a potential for yield increase due to heterosis and the combination of desirable characteristics from both parental lines. Using a polymerase chain reaction (PCR) based analysis specific for the alleles of the SLG (S-locus glycoprotein gene) located on the S-locus, we genetically mapped the S-locus of B. oleracea for SI using a F₂ population from a cross between a rapid-cycling B. oleracea line (CrGC-85) and a cabbage line (86-16-5). The linkage map contained both RFLP (restriction fragment length polymorphism) and RAPD (random amplified polymorphic DNA) markers. Similarly, the S-loci were mapped in B. napus using two different crosses (91-SN-5263×87-DHS-002; 90-DHW-1855-4×87-DHS-002) where the common male parent was self-compatible, while the S-alleles introgressed in the two different SI female parents had not been characterized. The linkage group with the S-locus in B. oleracea showed remarkable homology to the corresponding linkage group in B. napus except that in the latter there was an additional locus present, which might have been introgressed from B. rapa. The S-allele in the rapid-cycling Brassica was identified as the S₂₉ allele, the S-allele of the cabbage was the S ₅ allele. These same alleles were present in our two B. napus SI lines, but there was evidence that it might not be the active or major SI allele that caused self-incompatibility in these two B. napus crosses. Received: 7 June 1996/Accepted: 6 September 1996     

A glycoprotein associated with the acquisition of the self-incompatibility system by maturing stigmas of Brassica oleracea

Iso-electric focusing of extracts derived from stigmatic homogenates of Brassica oleracea reveals that the mature stigma possesses large quantities of a glycoprotein not present in earlier stages of development in the bud. Pollen germination experiments carried out in parallel with the biochemical tests suggest that the appearance of this glycoprotein, which has an isoelectric point of pH 5.8, is coincident with the development of the self-incompatibility response. The site of this protein, and the role it may play in pollen-stigma interactions are discussed in terms of current models of the self-incompatibility system in Brassica.Abbreviations PAS periodic acid-schiffs - PEG polyethylene glycol - SI system self-incompatibility system     

Comparative pangenome analyses provide insights into the evolution of Brassica rapa resistance gene analogues (RGAs)

Brassica rapa is grown worldwide as economically important vegetable and oilseed crop. However, its production is challenged by yield-limiting pathogens. The sustainable control of these pathogens mainly relies on the deployment of genetic resistance primarily driven by resistance gene analogues (RGAs). While several studies have identified RGAs in B. rapa, these were mainly based on a single genome reference and do not represent the full range of RGA diversity in B. rapa. In this study, we utilized the B. rapa pangenome, constructed from 71 lines encompassing 12 morphotypes, to describe a comprehensive repertoire of RGAs in B. rapa. We show that 309 RGAs were affected by presence-absence variation (PAV) and 223 RGAs were missing from the reference genome. The transmembrane leucine-rich repeat (TM-LRR) RGA class had more core gene types than variable genes, while the opposite was observed for nucleotide-binding site leucine-rich repeats (NLRs). Comparative analysis with the B. napus pangenome revealed significant RGA conservation (93%) between the two species. We identified 138 candidate RGAs located within known B. rapa disease resistance QTL, of which the majority were under negative selection. Using blackleg gene homologues, we demonstrated how these genes in B. napus were derived from B. rapa. This further clarifies the genetic relationship of these loci, which may be useful in narrowing-down candidate blackleg resistance genes. This study provides a novel genomic resource towards the identification of candidate genes for breeding disease resistance in B. rapa and its relatives.     

Polymorphism of the kinase domain of the S-locus receptor kinase gene (SRK) in Brassica oleracea L.

 DNA polymorphism of the S-locus receptor kinase gene (SRK) participating in self-incompatibility in Brassica was analyzed by PCR-RFLP and nucleotide sequencing. In the screening of primers for specific amplification of polymorphic DNA fragments of SRK, the best combination was that of a forward primer (PK1) having the nucleotide sequence of the second exon of S6 SRK and a reverse primer (PK4) having the complementary nucleotide sequence of the fifth exon of S6 SRK. PCR using this primer pair amplified DNA fragments of 0.9–1.0 kb from 36 S haplotypes out of 42 tested. These DNA fragments showed high polymorphism in polyacrylamide-gel electrophoresis after digestion with restriction endonuclease(s): 25 types were found in a double digestion with MboI and AfaI. Nucleotide sequencing of the DNA fragments amplified from five S haplotypes showed that the third, fourth, and fifth exons of SRK are highly conserved, and that there are high variations of the second and third introns of SRK, which produced polymorphism of the band pattern in PCR-RFLPs. Another forward primer (PK5) having the nucleotide sequence of the second exon, which is derived from S2 SRK, amplified DNA fragments of almost the same region of SRK from 27 S haplotypes in combination with PK4. Although SRK alleles of the class-II S haplotypes were not amplified, all of the class-I S-haplotypes were amplified with a primer mixture of PK1, PK4 and PK5. The DNA fragments of both SRK alleles in S heterozygotes, or a 1 : 1 mixture of the genomic DNA of different S homozygotes, were amplified without exception, suggesting the usefulness of these primers for the identification of S heterozygotes. The DNA fragment sizes obtained by digestion with restriction endonucleases served as markers for the identification of S haplotypes. Received: 15 December 1996 / Accepted: 14 February 1997     

中国沿海日本囊对虾线粒体16S rRNA基因序列变异及遗传分化

对中国东南沿海4个日本囊对虾地理群体16S rRNA基因片段进行了序列测定和分析.在获得的467bp 序列中,共检测到45个变异位点,多态位点比例为9.64%.具有10种单倍型,三亚群体单倍型多样性最高(0.900±0.161).各群体的核苷酸多样性(π)为0.0007～0.0082,其中三亚群体核苷酸多样性最为丰富,远高于湛江、北海和海口群体.群体间遗传分化指数(F_(st))为0.000～0.945.结合已报道的16S rRNA基因序列,构建了NJ分子系统树,中国海域日本囊对虾可分为3支.香港至台湾沿岸所有单倍型聚集为一分支,湛江、海口和北海群体内的所有单倍型聚集成另一分支,两分支分别属于已报道的变种Ⅰ和变种Ⅱ,但三亚群体内的4个单倍型单独聚集成一分支,与前两分支的核苷酸序列差异大(7.95%～8.35%),遗传分化明显,可能为一亲缘关系很远的新种(或变种).研究结果可为日本囊对虾的资源管理和遗传选育提供参考.     

Transformation of Brassica oleracea with an S-locus gene from B. campestris changes the self-incompatibility phenotype

Summary An SLG gene derived from the S-locus and encoding and S-locus-specific glycoprotein of Brassica campestris L. was introduced via Agrobacterium-mediated transformation into B. oleracea L. A self-incompatible hybrid and another with partial self-compatibility were used as recipients. The transgenic plants were altered in their pollen-stigma interaction and were fully compatible upon self-pollination. Reciprocal crosses between the transgenic plants and untransformed control plants indicated that the stigma reaction was changed in one recipient strain while the pollen reaction was altered in the other. Due to interspecific incompatibility, we could not demonstrate whether or not the introduced SLG gene confers a new allelic specificity in the transgenic plants. Our results show that the introduced SLG gene perturbs the self-incompatibility phenotype of stigma and pollen.     

白菜和甘蓝基因组转座子表达及其对基因调控的潜在影响

转座子或转座元件是大多数真核生物基因组的主要组成成分。甘蓝(Brassica oleracea)基因组比白菜(B. rapa)大主要是转座子的扩增差异造成的。然而, 这两个芸薹属近缘物种转座子表达水平以及对基因的调控和功能的影响目前还不清楚。文章对白菜和甘蓝叶、根、茎3个器官的转录组数据进行了初步分析。结果显示, 转座子的表达量很低, 转录组reads中有1%来自转座子的转录本; 转座子的表达存在器官差异, 且不同类别和家族的转座子表达量相差很大, 相同类别和同一家族的转座子在白菜和甘蓝基因组中的表达活性也不相同。进一步鉴定到转录读出的LTR反转座子, 其与下游基因距离小于2 kb的有41个, 小于100 bp的有9个, 这些LTR的转录读出很可能通过正义或反义的转录本激活或干扰下游基因的表达。同时, 具有转录读出的intact LTR比solo LTR具有更强的读出活性。通过深入分析转座子的插入位点发现, 白菜基因组中转座子插入基因内部的频率比甘蓝基因组中的高; 与反转座子相比, DNA转座子更偏向于插入或保留在基因的内含子当中。这些结果为认识转座子对其他蛋白编码基因的影响提供了基础。