Registration of S alleles in Brassica campestris L by the restriction fragment sizes of SLGs

Polymorphism of SLG (the S-locus glycoprotein gene) in Brassica campestris was analyzed by PCR-RFLP using SLG-specific primers. Nucleotide sequences of PCR products from 15 S genotypes were determined in order to characterise the exact DNA fragment sizes detected in the PCR-RFLP analysis. Forty-seven lines homozygous for 27 S-alleles were used as plant material. One combination of primers, PS5 + PS 15, which had a nucleotide sequence specific to a class-I SLG, gave amplification of a single DNA fragment of approximately 1.3kb from the genomic DNA of 15 S genotypes. All the DNA fragments showed different electrophroetic profiles from each other after digestion with MboI or MspI. Different lines having the same S genotype had an identical electrophoretic profile even between the lines collected in Turkey and in Japan. Another class-I SLG-specific primer, PS 18, gave amplification of a 1.3-kb DNA fragment from three other S genotypes in combination with PS 15, and the PCR product also showed polymorphism after cleavage with the restriction endonucleases. Genetic analysis, Southern-hybridization analysis, and determination of the nucleotide sequences of the PCR products suggested that the DNA fragments amplified with these combinations of primers are class-I SLGs. Expected DNA fragment sizes in the present PCR-RFLP condition were calculated from the determined nucleotide sequence of SLG PCR products. A single DNA fragment was also amplified from six S genotypes by PCR with a combination of primers, PS3 + PS21, having a nucleotide sequence specific to a class-II SLG. The amplified DNA showed polymorphisnm after cleavage with restriction endonucleases. The cleaved fragments were detected by Southern-hybridization analysis using a probe of S ⁵ SLG cDNA, a class-IISLG. Partial sequencing revealed a marked similarity of these amplified DNA fragments to a class-II SLG, demonstrating the presence of class-I and class-II S alleles also in B. campestris. The high SLG polymorphism detected by the present investigation suggests the usefulness of the PCR-RFLP method for the identification of S alleles in breeding lines and for listing S alleles in B. campestris.     

Development of SCAR markers linked to self-incompatibility in <Emphasis Type="Italic">Brassica napus</Emphasis> L.

‘SI1300’ is a self-incompatible Brassica napus line generated by introgressing an S haplotype from B. rapa ‘Xishuibai’ into a rapeseed cultivar ‘Huayou No. 1’. Five S-locus specific primer pairs were employed to develop cleaved amplified polymorphic sequences (CAPS) markers linked the S haplotype of ‘SI1300’. Two segregating populations (F₂ and BC₁) from the cross between ‘SI1300’ and self-compatible European spring cultivar ‘Defender’, were generated to verify the molecular markers. CAPS analysis revealed no desirable polymorphism between self-incompatible and self-compatible plants. Twenty primer pairs were designed based on the homology-based candidate gene method, and six dominant sequence characterized amplified region (SCAR) markers linked with the S-locus were developed. Of the six markers, three were derived from the SRK and SP11 alleles of class II B. rapa S haplotypes and linked with S haplotype of ‘SI1300’. The other three markers were designed from the SLG-A10 and co-segregated with S haplotype of ‘Defender’. We successfully combined two pairs of them and characterized two multiplex PCR markers which could discriminate the homozygous and heterozygous genotypes. These markers were further validated in 24 F₃ and 22 BC₁F₂ lines of ‘SI1300 × Defender’ and another two segregating populations from the cross ‘SI1300 × Yu No. 9’. Nucleotide sequences of fragments linked with S-locus of ‘SI1300’ showed 99% identity to B. rapa class II S-60 haplotype, and fragments from ‘Defender’ were 97% and 94% identical to SLG and SRK of B. rapa class I S-47 haplotype, respectively. ‘SI1300’ was considered to carry two class II S haplotypes and the S haplotype on the A-genome derived from B. rapa ‘Xishuibai’ determines the SI phenotype, while ‘Defender’ carry a class I S haplotype derived from B. rapa and a class II S haplotype from B. oleracea. SCAR markers developed in this study will be helpful for improving SI lines and accelerating marker-assisted selection process in rapeseed SI hybrid breeding program.     

Identification and classification of S haplotypes in Raphanus sativus by PCR-RFLP of the S locus glycoprotein (SLG) gene and the S locus receptor kinase (SRK) gene

Polymorphism of the S-locus glycoprotein (SLG) and S-locus receptor kinase (SRK) genes in Raphanus sativus was analyzed by PCR-RFLP using SLG- and SRK-specific primers. Twenty four inbred lines of R. sativus could be grouped into nine S haplotypes. DNA fragments of SLG alleles specifically amplified from five S haplotypes by PCR with Class-I SLG-specific primers showed different profiles upon polyacrylamide-gel electrophoresis after digestion with restriction endonucleases. The five R. sativus SLG alleles were determined for their nucleotide sequences of DNA fragments. Comparison of the amino-acid sequences with a reported Brassica SLG (S₆) showed 77-84% homology. Deduced amino-acid sequences showed 12-conserved cystein residues and three hypervariable regions which are characteristic of Brassicsa SLG. A DNA fragment was also amplified by PCR from two of each S haplotype with Class-II SLG-specific primers, and showed polymorphism when cleaved with restriction endonucleases. The nucleotide sequences of amplified DNA fragments of the Class-II SLG revealed about 60% similarity with those of the Class-I SLG. It is concluded that there exist both Class I and Class II S alleles in R. sativus, as in Brassica campestris and Brassica oleracea. PCR using SRK-specific primers amplified a DNA fragment of about 1.0 kb from seven of each S haplotype out of 24 tested. These DNA fragments showed high polymorphism in polyacrylamide-gel electrophoresis after digestion with restriction endonucleases. Nucleotide sequences of the DNA fragments amplified from the seven S haplotypes showed that the fourth and the fifth exons of SRK are highly conserved, and that there is high variation in the fifth intron, the sixth intron and seventh exon of the SRK which may be responsible for the polymorphic band patterns in PCR-RFLP analysis. The PCR-RFLP method has proven useful for the identification of S alleles in inbred lines and for listing S haplotypes in R. sativus. Phylogenic analysis of the SLG and SRK sequences from Raphanus and Brassica revealed that the Raphanus SLGs and SRKs did not form an independent cluster, but were dispersed in the tree, clustering together with Brassica SLGs and SRKs. Furthermore, SLGs and SRKs from Raphanus were both grouped into Class-I or Class-II S haplotypes. Therefore, these results suggest that the diversification of the SLG and SRK alleles occurred prior to the differentiation of the two genera Brassica and Raphanus.     

Physical distances between S-locus genes in various S haplotypes of Brassica rapa and B. oleracea

In Brassica, self-incompatibility genes SLG (for S-locus glycoprotein) and SRK (for S-receptor kinase) are located in the S-locus complex region with several other S-linked genes. The S locus is a highly polymorphic region: polymorphism has been observed not only in sequences of SLG and SRK but also in the location of the S-locus genes. In order to compare the physical location of the S-locus genes in various S haplotypes, we used six class-I S haplotypes of B. rapa and seven class-I S haplotypes of B. oleracea in this study. DNA gel blot analysis using pulsed-field gel electrophoresis (PFGE) showed that the physical distances between SLG and SRK in B. rapa are significantly shorter than those in B. oleracea and that the sizes of MluI and BssHII fragments harboring SLG and SRK are less variable within B. rapa than within B. oleracea. We concluded that several large genomic fragments might have been inserted into the S-locus region of B. oleracea after allelic differentiation of S-locus genes. Received: 20 September 1999 / Accepted: 8 October 1999     

Single-seed PCR-RFLP analysis for the identification of S haplotypes in commercial F1 hybrid cultivars of broccoli and cabbage

 Several simple methods of DNA preparation from plant tissues were evaluated for PCR-RFLP analyses of SLG and SRK alleles, which can be used for the identification of S haplotypes of breeding lines in broccoli and cabbage (Brassica oleracea L.) and in purity tests of F₁ hybrid seeds. On the five methods tested, the NaI method was found to be the most suitable for the amplification of the SLG and SRK alleles. This method enables the use of a single seed as testing material. Using this method, we identified S haplotypes of 31 broccoli and 31 cabbage cultivars. Ninety-four percent of the cultivars of broccoli and 97% of those of cabbage were-single cross F₁ hybrids. Nine and 15 S haplotypes were found in broccoli and cabbage, respectively. The small number of S haplotypes in broccoli suggests the importance of incorporating new S haplotypes in the breeding program. Received: 18 February 1999 / Revision received: 4 May 1999 / Accepted: 14 May 1999     

Molecular markers residing close to the Rhg4 locus conferring resistance to soybean cyst nematode race 3 on linkage group A of soybean

 The restriction fragment length polymorphism (RFLP) clone pBLT65 is a 450-nt soybean cDNA encoding a portion of the bifunctional enzyme aspartokinase-homoserine dehydrogenase (AK-HSDH). pBLT65 maps within 3.5 cM of the i locus, conferring a pigmented seed coat, on linkage group A; hence, it is closely linked to the Rhg ₄ locus conferring resistance to race 3 of the soybean cyst nematode. From this useful RFLP we developed a PCR reaction yielding polymorphic bands for use in marker-assisted breeding programs to select progeny containing the Rhg ₄ allele. The polymorphic bands were sequenced to determine the cause of the polymorphisms. Using primers 548 and 563, PCR amplification of DNA from the soybean cultivar Peking (Rhg ₄ ) yielded three DNA fragments, 1a (1160 bp), 1b (1146 bp) and 3 (996 bp). Amplification of DNA from the cultivar Kent (rhg ₄) yielded DNA fragments 2 (1020 bp), 3 (996 bp) and 4 (960 bp). Fragments 1a, 1b, 2 and 4 were also polymorphic between the soybean lines PI 290136 and BARC-2(Rj ₄ ). A segregating population of 80 F₂ and F₃ plants derived from the cross PI 290136×BARC-2 (Rj ₄ ) was used to confirm the map position of the PCR polymorphisms near the i locus, and hence the Rhg ₄ locus on linkage group A. The nucleotide sequences of fragments 1b, 3 and 4 were determined. Large and small deletions in the intronic region were responsible for the size differences of the different fragments, whereas the exon was well conserved. Received: 8 January 1998 / Accepted: 15 July 1998     

Single nucleotide polymorphisms in randomly selected genes among japonica rice (Oryza sativa L.) varieties identified by PCR-RF-SSCP.

DNA polymorphism of randomly selected genes in rice cultivars was analyzed by the polymerase chain reaction-restriction fragment-single strand conformation polymorphism (PCR-RF-SSCP) technique. Single DNA fragments were amplified from genomic DNA of the Nipponbare cultivar by 671 primer pairs among the 1000 primer pairs tested. PCR-RF-SSCP analysis using the 671 primer pairs detected polymorphism in 108 DNA fragments between 17 japonica paddy-rice cultivars. An average of 36.9 DNA fragments showed polymorphism between any pair of japonica paddy-rice cultivars. The nucleotide sequences of the polymorphic DNA fragments were determined for 50 alleles of 45 genes together with Nipponbare alleles. In these genes, 142 SNPs and 32 insertions/deletions were identified. Among these 174 sequence variations, 71 were in exons, 78 in introns, and 25 in unassigned regions. There were 28 alleles which had sequence variations in the exons. One allele had a 1-bp deletion in the exon causing a frame-shift mutation, 15 alleles had missense mutations, and the other 12 alleles had synonymous changes and/or sequence variations in 3' untranslated regions. The number of genes having sequence variations between the rice cultivars and the functional implications of the identified SNPs are herein discussed.     

PCR-RFLP of S locus for identification of breeding lines in cruciferous vegetables

A simple method of detecting polymorphism of S locus glycoprotein gene, SLG, in Chinese cabbage and cabbage was developed, and used for identification of breeding lines. DNA was amplified by the polymerase chain reaction (PCR) with a pair of primers having S ₆ SLG sequences from inbred lines, and digested with restriction endonucleases which recognize tetranucleotide sequences. The cleaved DNA fragments were size-fractionated by polyacrylamide gel electrophoresis and detected by silver staining. PCR with S ₆ SLG primers amplified a fragment of ca. 1.3kb in more than half of the inbred lines tested. After digestion, polyacrylamide gel electrophoresis revealed polymorphism between the amplified 1.3kb DNA fragments. These polymorphic bands were detected by Southern hybridization using a probe of S ₆ SLG cDNA, suggesting that the amplified DNA was SLG. Primers having the SLG sequences of S ₂ , a representative of recessive S alleles, were used for amplification of SLG in the lines which did not give the 1.3kb DNA fragment by the PCR with S ₆ SLG primers. Polymorphism of amplified DNA was found in these lines. However these primers also appeared to amplify an invariant SLR-2 sequence of 1.3kb in addition to the polymorphic S ₂ SLG related sequences. Although the used primer sequences still need improvement for the analysis of recessive S alleles, PCR-RFLP of SLG was considered to be useful for identification of breeding lines as well as for S allele identification in cruciferous vegetables. F₁ hybrids exhibited the sum of the bands of both parents, and, therefore, this method is expected to be used for a purity test of F₁ seeds.     

Chloroplast DNA diversity within and among populations of the allotetraploid Prunus spinosa L.

High chloroplast DNA (cpDNA) diversity was found within and among populations of Prunus spinosa sampled from seven European deciduous forests. A study of 12% of the total chloroplast genome detected 44 mutations, which were distributed over 24 haplotypes; four were common to two or more populations and the rest were unique haplotypes. The most-abundant and widely distributed haplotype was H2 (frequency = 41% approximately). Six of the seven populations were polymorphic. All of the six polymorphic populations had ”private” haplotypes (frequency < 5%) in addition to common haplotypes. The UPGMA dendrogram demonstrated a correlation between populations and their geographical locations. The total diversity was high (h_T= 0.824) and a major portion of it was within populations (h_s= 0.663). The level of population subdivision for unordered alleles was low (G_ST= 19.5%) and for ordered alleles was lower (N_ST= 13.6%). No phylogeographic structure could be demonstrated in the present geographical scale. High polymorphism in the cpDNA of P. spinosa has to be considered carefully when planning phylogenetic studies involving this species. Received: 20 September 1999 / Accepted: 10 November 1999     

Characterization of S tester lines in Brassica oleracea: polymorphism of restriction fragment length of SLG homologues and isoelectric points of S-locus glycoproteins

 Forty three S tester lines of Brassica oleracea were characterized using DNA and protein gel-blotting analyses. DNA gel-blot analysis of HindIII-digested genomic DNA with class-I and class-II SLG probes revealed that 40 lines could be classified as class-I S haplotypes while three lines could be classified as class-II S haplotypes. The band patterns in the S tester lines were highly polymorphic. Although the S tester lines typically showed two bands corresponding to SLG and SRK in the analysis with the class-I SLG probe, only one band was observed in the S ²⁴ homozygote. This band was identified as SRK, suggesting that this haplotype has no class-I SLG band. In the analysis using the class-II SLG probe, one plant yielded a different band pattern from the known class-II haplotypes, S ² , S ⁵ and S ¹⁵ . Unexpectedly, this plant was reciprocally cross-incompatible with the S ² haplotype. Therefore, it was designated as S ^2-b . We found an S ¹³ haplotype having a restriction fragment length polymorphism different from that of the S ¹³ homozygotes of the S tester line. These findings indicate that S homozygous lines with the same S specificity do not necessarily show the same band pattern in the DNA gel-blot analysis. Soluble stigma proteins of 32 S homozygotes were separated by isoelectric focusing and detected using anti-S ²² SLG antiserum. S haplotype-specific bands were detected in 27 S homozygotes but not in five S homozygotes, including the S ²⁴ homozygote. This is consistent with the observation that the S ²⁴ haplotype had no SLG band. Received: 13 July 1998 / Accepted: 29 September 1998     

Efficient detection of DNA polymorphism in cabbage and rice cultivars by PCR-RF-SSCP (PRS)

PCR (polymerase chain reaction)-RF(restriction fragment)-SSCP (single-strand conformation polymorphism) - designated here as PRS - is a combined method of SSCP and PCR-RFLP (restriction fragment length polymorphism) - designated as CAPS (cleaved amplified polymorphic sequence) - and was efficient in detecting intraspecific variation of the SLR1 gene in Brassica oleracea. One to six nucleotide changes in restriction fragments of the SLR1 gene were detected as different bands in PRS. In an analysis of randomly chosen DNA fragments in cabbage, PRS detected DNA polymorphism between different cultivars with more than 60% of the primer pairs used except for a combination of two cultivars having highly similar characteristics. In rice, no DNA polymorphism was found between two Japonica cultivars, while more than 80% of the primer pairs showed DNA polymorphism between Japonica cultivars and Indica cultivars. PRS had a 1.5- to twofold greater ability to detect DNA polymorphism in these cabbage and rice cultivars than CAPS. The present study indicated that PRS is potentially useful for the identification of crop cultivars and genetic mapping of DNA fragments including genes of interest.     

Highly repetitive DNA sequences that are restricted to the germ line in the hagfish Eptatretus cirrhatus: a mosaic of eliminated elements

The New Zealand hagfish, Eptatretus cirrhatus, is known to eliminate parts of its chromosomes during embryogenesis from presumptive somatic cells. Electrophoresis of germ line and somatic DNAs of this species, after treatment with the restriction endonucleases DraI and EcoRI, revealed three fragments of DNA that were restricted to the germ line. DNA filter hybridization experiments demonstrated that these fragments were present almost exclusively in the germ line DNA of E. cirrhatus and that they were highly and tandemly repeated. Thus, these DNA fragments appeared to be eliminated during embryogenesis. Moreover, one fragment (a DraI fragment) cross-hybridized with the germ line DNA from other species of hagfish, namely, Eptatretus okinoseanus and Paramyxine atami. Molecular cloning and sequence analysis revealed that the DraI fragment was composed mainly of closely related sequences of 85 bp in length and that this sequence was about 75% homologous to the sequence of EEEo2 (eliminated element of E. okinoseanus 2) which is a germ line-restricted and highly repetitive sequence that was isolated previously from E. okinoseanus. The other two fragments were composed of three families of closely related sequences that were 172 bp long (designated EEEc1), 61 bp long (EEEc2) and 54 bp long (EEEc3). Fluorescence in situ hybridization experiments revealed that each eliminated element was distributed on several chromosomes that are limited to germ cells. EEEo2 was dispersed on 12 C-band-positive chromosomes. EEEc1 and EEEc3 were dispersed on all C-band-positive and several C-band-negative chromosomes. By contrast, EEEc2 was located to terminal regions of several C-band-negative chromosomes. These results suggest that the eliminated chromosomes in hagfish are mosaics of highly repeated, germ line-restricted families of DNA sequences. Received: ██; in revised form: 25 October 1997 / Accepted: ██     

Sequence variability analysis on major histocompatibility complex class II DRB alleles in three felines

The variation of the exon 2 of the major histo-compatibility complex (MHC) class II gene DRB locus in three feline species were examined on clouded leopard (Neofelis nebulosa), leopard (Panthera pardus) and Amur tiger (Panthera tigris altaica). A pair of degenerated primers was used to amplify DRB locus covering almost the whole exon 2. Exon 2 encodes the β1 domain which is the most variable fragments of the MHC class II molecule. Single-strand conformational polymorphism (SSCP) analysis was applied to detect different MHC class II DRB haplotypes. Fifteen recombinant plasmids for each individual were screened out, isolated, purified and sequenced finally. Totally eight distinct haplotypes of exon 2 were obtained in four individuals. Within 237 bp nucleotide sequences from four samples, 30 variable positions were found, and 21 putative peptide-binding positions were disclosed in 79 amino acid residues. The ratio of nonsynonymous substitutions (d _N ) was much higher than that of synonymous substitutions (d _S ), which indicated that balancing selection probably maintain the variation of exon 2. MEGA neighbor joining (NJ) and PAUP maximum parsimony (MP) methods were used to reconstruct phylogenetic trees among species, respectively. Results displayed a more close relationship between leopard and tiger; however, clouded leopard has a comparatively distant relationship form the other two. __________ Translated from Zoological Research, 2006, 27(2): 181-C188 [译自:动物学研究]     

Trans-species origin of Mhc-DRB polymorphism in the chimpanzee

Trans-specific evolution of allelic polymorphism at the major histocompatibility complex loci has been demonstrated in a number of species. Estimating the substitution rates and the age of trans-specifically evolving alleles requires detailed information about the alleles in related species. We provide such information for the chimpanzee DRB genes. DNA fragments encompassing exon 2 were amplified in vitro from genomic DNA of ten chimpanzees. The nucleotide sequences were determined and their relationship to the human DRB alleles was evaluated. The alleles were classified according to their positioni in dendrograms and the presence of lineage-specific motifs. Twenty alleles were found at the expressed loci Patr-DRB1,-DRB3, -DRB4, -DRB5, and at the pseudogenes Patr-DRB6, -DRB7; of these, 13 are new alleles. Two other chimpanzee sequences were classified as members of a new lineage tentatively designated DRBX. Chimpanzee counterparts of HLA-DRB1 ^* 01 and ^* 04 were not detected. The number of alleles found at individual loci indicates asymmetrical distribution of polymorphism between humans and chimpanzees. Estimations of intra-lineage divergence times suggest that the lineages are more than 30 million year old. Predictions of major chimpanzee DRB haplotypes are made.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers M94937-M94954.     

Efficient mutagenesis method for producing the templates of single nucleotide polymorphisms

DNA templates harboring specific single nucleotide polymorphism (SNP) sites are largely needed as positive controls in practical SNP analysis and in determination of the reliability of newly developed methods in high-throughput screening assays. Here we report a one-step method to produce SNP templates by amplifying a wild-type sequence with primers having single nucleotide mismatches at or near their 3′ ends. A short amplicon harboring an EcoRI site was used to evaluate the feasibility of our strategy. Perfectly matched primers and primers with a single base mismatch occurring from the first base to the sixth base of the EcoRI site were used for primer extension. By using polymerase without a proofreading function, we kept mismatched nucleotides from occurring in extended primer products, as confirmed by EcoRI digestion and sequencing analysis. The strategy of using primers with a single mismatched base and exo- polymerase was shown to be an efficient one-step method for preparing SNP templates, either for application in the development of SNP screening assays or as positive controls in practical SNP assays.     

Rapid amplification of genomic DNA ends bynla III partial digestion and polynucleotide tailing

We report a simple and efficient method, which combines restriction endonuclease digestion and deoxynucleotide tailing, for cloning unknown genomic sequences adjacent to a known sequence. Total genomic DNA is partially digested with the frequent-cutting restriction enzymeNla III. A homo-oligomeric cytosine tail is added by terminal transferase. The tailed DNA fragments are used as the template for cloning flanking regions from all sequences of interest. A first round PCR amplification is performed with a gene-specific primer and the selective (modified polyguanine) anchor primer complementary to the cytosine tail and theNla III recognition site, with a universal amplification primer sequence at its 5′ end. This is followed by another PCR amplification with a nested gene-specific primer and the universal amplification primer. Finally, the amplified products are fractionated, cloned, and sequenced. Using this method, we cloned the upstream region of a salt-induced gene based upon a partial cDNA clone (RSC5-U) obtained from sunflower (Helianthus annuus L.).