| Plant cytoplasmic male sterility (CMS) was not only a basis for hybrid seed production but also the ideal experimental material to research the mechanism of nucleo-cytoplasmic interaction. Studing on CMS molecular mechanism was hotspot and difficulty both the domestic and foreign research for a long time.Kenaf (Hibiscus cannabinus L.) was an important annual herbal bast fibre crop. It was widely used in manufacturing paperboard products and synthetic fibres. Heterosis utilization was extremely attractive for kenaf breeders with the aim of stalks harvest and bast fibers production and advantage in its vegetative growth in kenaf. The first male sterile plant of kenaf was found in wild species UG93 during winter breeding in Haiman provience in March 2001 by professor Zhou and his innovation team. Seven CMS lines and four "hongyou" three-line hybrid seeds had been breeded, which was used as honor of cytoplasm. Heterosis utilization of kenaf had been reached to the international leading position in our country.The most fertile plants in UG93 group of kenaf could maintain the features of CMS in recently study. UG93 A and its maintain line UG93B had been breeded by using the fertile plants as donor of cell nucleus. Because of both them were derived from the same species group, it was speculated that the UG93A was the CMS mutation of UG93B, the latter which was the wild type. Becase of the cytoplasmic near-isogenic lines between UG93A and UG93B, which provided very valuable research materials for studing the molecular mechanism of CMS in kenaf.The CMS line UG93A, its maintainer line UG93B and F1(UG93 A/992), which was the descendant of restorer 992 hybrized with UG93A, had been used to analysis the differences in mitochondrial genomes by High-throughput genome sequencing technology and Northern Blot, the main conclusions as follows:1 The complete mitochondrial genome sequence has been determined in UG93B of kenaf through single molecular real-time sequencing.67,152 reads had been obtained, with the average length of 5.4kb and sequencing depth of 605×. The longest read was up to 32kb. Short sequences thought to seed reads were aligned to long seeds in order to inprove the accuracy of sequences. Totally, the data of high quanlity and long reads sequences were 12.1Mb, with the average length of 6.7kb and the sequence quantity was as high as Q59.2 The UG93B mitochondrial genome was a circular molecule, with the length of 569,912 bp and GC content of 44.9% after assembleing the mitochondrial sequencing data, and the whole mitochondrial genome was mapped in UG93B of kenaf.59 mitochondrial genes were annotated, including 36 protein-coding genes,3 rRNA genes,20 tRNA genes, all of which only account for 7.1% of the whole mitochondrial genomic sequences.375 orfs was predict with length greater than 100 bp, which occupatied 26% of the mitochondrial genome.3 There were 50 repeat sequences range from 65 bp to 7782 bp in UG93B mtDNA of kenaf, the detected repeats only occupied 3.4% of the mt genome in total. Of the 50 repeats, 56%(28 repeats) were larger than 100bp, only three repeats larger than 1 OOObp. Copy number for the larger repeats (>= 100 bp) varied narrowly from two (22 repeats) to three (3 repeats) copies. The repeats sequence were mainly in the form of direct repeats and palindrome repeats.4 The resequencing of UG93A mt genome was aligned to the reference sequence of UG93B.398 SNPs and 230 InDels variation locuses had been identified in UG93A. In which, 362 SNPs scattered in the intergenic and intron region, occuping 90% of total SNPs. Only 36 SNPs scattered in the coding region, occuping 10% of total SNPs. of which 15 SNPs were synonymous mutation, resulting in a change in amino acid composition of six mt genes. Small fragments InDels sequences(1-5 bp) were the mainly variation in 230 InDels variation locuses, which occupied 77.4% of the total InDels. Only 22.6% was occupied among 6-31 bp InDels. Most of the InDels was scattered among the intergenic and intron region, only 11 of them (4.8%) scattered in gene encoding region which was in accordance with the SNPs variation.5 36 protein-coding genes had been used to analysis the transcript differences among UG93A,UG93B and F1(UG93A/992) by Northern Blot. Six different transcripts had been detected between sterile cytoplasm and fertile cytoplasm, which were atpl、atp4%、atp6、atp9、 cox3 and sdh4, respectively. In five of which, the different transcrips were only present in sterile line and fertile line, including atpl、atp4、atp6、cox3 and sdh4, but the transcript were the same as UG93A in F1(UG93A/992), excepting atp9. which indicated that these genes was not a direct to cause CMS of kenaf.6 The transcript of atp9 in UG93A were smaller than that of UG93B and F1 (UG93A/992), eventhough UG93A and F1(UG93A/992) possessed the same cytoplasm. The fertile plant F1(UG93/992),which contained the restore gene (Rfs) altered the expression of atp9, which was a further suggestion that atp9 was closely related to CMS of kenaf.7 The atp8 gene was cloned from the cytoplasmic male sterile (CMS) line UG93A and its maintainer line UG93B in kenaf. Its DNA sequence analysis showed that atp8 contained 480 bp, encoding 159 amino acid residues, and a 9 bp insertion was found at the 3’flanking sequence in UG93A compared with UG93B. The cDNA sequence of atp8 analyzed by RT-PCR indicated that there were five loci edited, but six loci edited in UG93B. The editing frequencies were higher in sterile cytoplasm UG93 A than in fertile cytoplasms UG93B. The relative expression of atp8 analyzed by real-time PCR, showed that the expressed level of atp8 in UG93A was lower than that of its maitainer UG93B, and its F1 hybrid UG93 A/992 (a restore line). Furthermore, based on the difference of the 9 bp differences at the 3’flanking sequence of atp8 between UG93A and UG93B, a molecular marker specific to male sterile cytoplasm was developed, which can be used for indentificating whether any germplasm of kenaf is male sterile cytoplasm or male fertile cytoplasm. |
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