SNP discovery by amplicon sequencing and multiplex SNP genotyping in the allopolyploid species Brassica napus.

Oilseed rape (Brassica napus) is an allotetraploid species consisting of two genomes, derived from B. rapa (A genome) and B. oleracea (C genome). The presence of these two genomes makes single nucleotide polymorphism (SNP) marker identification and SNP analysis more challenging than in diploid species, as for a given locus usually two versions of a DNA sequence (based on the two ancestral genomes) have to be analyzed simultaneously during SNP identification and analysis. One hundred amplicons derived from expressed sequence tag (ESTs) were analyzed to identify SNPs in a panel of oilseed rape varieties and within two sister species representing the ancestral genomes. A total of 604 SNPs were identified, averaging one SNP in every 42 bp. It was possible to clearly discriminate SNPs that are polymorphic between different plant varieties from SNPs differentiating the two ancestral genomes. To validate the identified SNPs for their use in genetic analysis, we have developed Illumina GoldenGate assays for some of the identified SNPs. Through the analysis of a number of oilseed rape varieties and mapping populations with GoldenGate assays, we were able to identify a number of different segregation patterns in allotetraploid oilseed rape. The majority of the identified SNP markers can be readily used for genetic mapping, showing that amplicon sequencing and Illumina GoldenGate assays can be used to reliably identify SNP markers in tetraploid oilseed rape and to convert them into successful SNP assays that can be used for genetic analysis.

FUSCA3 encodes a protein with a conserved VP1/AB13-like B3 domain which is of functional importance for the regulation of seed maturation in Arabidopsis thaliana.

Conditionally lethal mutant alleles of the FUSCA3 (FUS3) gene of Arabidopsis thaliana are specifically defective in the gene expression program responsible for seed maturation. FUS3 was isolated by map-based cloning and expression of the FUS3 cDNA resulted in complementation of the Fus3- phenotype. In the predicted FUS3 gene product, a continuous stretch of more than 100 amino acids shows significant sequence similarity to the B3 domains of the polypeptides encoded by ABI3 (Arabidopsis) and VP1 (maize). FUS3 transcription was detected mainly in siliques and was found to be developmentally regulated during embryogenesis. Transcripts of abnormal sizes were observed in fus3 mutants due to aberrant splicing caused by point mutations at intron termini. Sequence analysis of mutant and wild-type FUS3 alleles, as well as sequencing of fus3 cDNAs, revealed small inframe deletions at two different sites of the coding region. While a deletion between B3 and the C-terminus of the predicted polypeptide was found in conjunction with normal FUS3 function, another deletion located within the conserved B3 domain (as well as truncations therein) were associated with the Fus3- phenotype. It is apparent, therefore, that an intact B3 domain is essential for the regulation of seed maturation by FUS3.

The nucleotide sequence of boar transition protein 2 (TNP2) cDNA and haploid expression of the gene during spermatogenesis.

A cDNA clone coding for boar transition protein 2 (TNP 2) was isolated from a randomly primed cDNA library of boar testis. Sequence analysis revealed an open reading frame of 414 bp (corresponding to 138 amino acids), 33 bp of the 5' untranslated and about 300 bp of the 3' untranslated region. As compared to TNP 2 of mouse and rat, similarity with TNP 2 of the boar is approximately 70% at the nucleotide level and only about 40% on the basis of amino acid sequence. The similarity between boar and bull TNP2 is 77% and 64%, respectively. Northern blot experiments with RNA of different boar tissues and in situ hybridization on mature boar testis sections revealed testis-specific expression of the TNP 2 gene which is restricted to haploid germ cells. Hybridization experiments of boar TNP2 cDNA with testicular RNA of boar, bull, rat and mouse revealed decreasing intensities of the hybridization signals. With human testicular RNA no hybridization could be obtained.

Characterization of a gene encoding a basic protein of the spermatid nucleus, TNP2, and its close linkage to the protamine genes in the bull.

During elongation and condensation of the spermatid nucleus, histones are replaced by spermatid-specific transition proteins (TNP). TNP1 is well characterized at the cDNA and at the genomic level and was found to be highly conserved during mammalian evolution (similarity between 83 to 98%). We here describe for the first time the nucleotide sequence and organization of the gene for TNP2. The gene was isolated from a bull cosmid library and was found to contain a single intron of 910 bp. The coding sequence consists of 390 bp and has a similarity of about 70% to that of the TNP2 cDNAs of mouse and rat. At the basis of amino-acid sequences, the bull TNP2 is 14 and 15 amino acids longer than that of mouse and rat, respectively, and the similarity is only 45% between bull and mouse and 42% between bull and rat. However, the evolutionary divergence has not occurred at the cost of basic amino acids which are of functional importance in DNA-protein interaction in the condensing spermatid nucleus. The TNP2 gene is closely linked to the protamine genes in the bull genome.

Nucleotide sequence of the gene for human transition protein 1 and its chromosomal localization on chromosome 2.

Transition protein 1 (TNP1) is a highly basic nuclear protein of 54 amino acids that is found in haploid spermatogenic cells during the period of transition of histones to protamines. Using the cDNA clone for human TNP1, we have isolated the gene encoding human TNP1 from human genomic libraries. The gene contains an intron of 200 bp; 1104 bp of the 5'- and 276 bp of the 3'-noncoding region have been sequenced. Comparison with the rat TNP1 gene yielded a similarity of 77% over the region between the transcription start point and the polyadenylation signal. The gene contains typical CAAT and TATAA boxes at conventional distances from the transcriptional start site. Using a series of human-rodent somatic cell hybrids containing variant complements of human chromosomes, the TNP1 gene was found to cosegregate with human chromosome 2. By in situ hybridization, the gene was assigned to the q35 and q36 bands of the long arm of chromosome 2. This chromosomal region encodes several genes, including TNP1, that are located on murine chromosome 1.

Nucleotide sequences and expression of cDNA clones for boar and bull transition protein 1 and its evolutionary conservation in mammals.

During spermatogenesis, the nucleoproteins undergo several dramatic changes as the germinal cells differentiate to produce the mature sperm. With nuclear elongation and condensation, the histones are replaced by basic spermatidal transition proteins, which are themselves subsequently replaced by protamines. We have isolated cDNA clones for one of the transition proteins, namely for TP1, of bull and boar. It turned out that TP1 is a small, but very basic protein with 54 amino acids (21% arginine, 19% lysine) and is highly conserved during mammalian evolution at the nucleotide as well as at the amino-acid level. Gene expression is restricted to the mammalian testis, and the message first appears in round spermatids. Thus production of TP1 is an example of haploid gene expression in mammals. The size of the mRNA for TP1 was found to be identical in 11 different mammalian species at around 600 bp. Hybridization experiments were done with cDNAs from boar and bull, respectively. The positive results in all mammalian species give further evidence for the conservation of the TP1 gene during mammalian evolution and its functional importance in spermatid differentiation.