不结球白菜同源四倍体Pol CMS及其保持系花药发育的解剖学研究

以秋水仙素人工诱变获得的不结球白菜同源四倍体Pol CMS及其保持系为材料,采用石蜡切片法研究其花药发育过程.结果显示:四倍体Pol CMS与保持系花药发育差异明显,不育系花药发育受阻于孢原细胞分化期,不形成药室,属无花粉囊型.极少数温敏型花药能产生1~2个花粉囊,但四分体时期绒毡层提早退化,导致成熟小孢子数目减少.四倍体保持系花药发育过程与二倍体基本一致,同一时期的可育小孢子四倍体比二倍体大,二倍体花粉粒都为3个萌发孔,而四倍体多为4个萌发孔.四倍体保持系同一花粉囊中花粉粒发育有不同步现象,花粉粒败育频率比二倍体高.研究表明,不结球白菜同源四倍体Pol CMS比其二倍体Pol CMS不育更彻底,更具应用性.     

白菜OguCMS相关MYB家族新基因BcMYBogu的克隆与特征分析

为研究CMS核质互作的分子机理, 将甘蓝型油菜(Brassica napus L.)和白菜(B. campestris L. ssp. chinensis Makino)杂交并连续回交6代获得白菜OguCMS, 在与保持系花药细胞学比较的基础上, 运用cDNA-AFLP筛选得到白菜OguCMS早、中期花蕾提早表达的MYB-like差异片断, 利用RACE克隆得到该片断的cDNA全长, 命名为BcMYBogu(GenBank 登录号: EF127861), 对其氨基酸序列和表达特征进行研究。结果表明, 白菜OguCMS绒毡层在四分体后增生, 高度液泡化, 导致小孢子花粉外壁异常, 细胞质同外壁分离并降解; 花药变白; BcMYBogu具有典型的MYB DNA结合域—W残基和SH[AL]QKY[RF]基序; 系统进化分析显示BcMYBogu与AtMYB26, AtMYB32和AtMYB4等聚类在同一分枝; RT-PCR分析表明BcMYBogu在莲座叶、花茎和花蕾中均有表达, 但在OguCMS花蕾中表达量显著上升。由此推测BcMYBogu是一个新的与白菜OguCMS相关的MYB家族新成员。     

利用RNA指纹技术分析大白菜胞质雄性不育相关基因的差异表达

采用改进的RNA指纹技术(RAP-PCR)对两套大白菜胞质雄性不育材料及其杂种F1花蕾的总RNA进行了分析, 通过对186条随机引物的筛选, 获得4个重复性较好的差异片段S47-412, S93-622, S176-343, S199-904, 并对其克隆、测序。根据各差异条带的测序结果合成长引物进行验证, S47, S93所对应的差异消失, 而S176, S199长引物验证的结果与RAP-PCR相似, 序列分析表明: S176-343, S199-904两差异片段均和油菜Polima不育胞质线粒体基因组orf224/atp6位点有着极高的同源性, 两差异片段序列部分重叠, 这说明两差异片段可能和大白菜胞质雄性不育有很高的相关性。     

Differences on the microsporogenesis and tapetal development of male fertile and cytoplasmic male sterile pepper (Capsicum annuum L.)

To clarify the time and cause of pollen abortion, differences on the microsporogenesis and tapetum development in the anthers of male fertile maintainer line and cytoplasmic male sterile (CMS) line pepper were studied using transmission electron microscopy. The results showed that CMS line anthers appeared to have much greater variability in developmental pattern than male fertile maintainer line ones. The earliest deviation from normal anther development occurred in CMS line anthers at prophase I was cytomixis in some microspore mother cells (MMCs), and vacuolisation in tapetal cells. Then, MMCs in CMS line anthers developed asynchronously and a small part of ones at the different stage degenerated in advance appearing to have typical morphological features of programmed cell death (PCD). Most MMCs could complete the meiosis, but formed non-tetrahedral tetrad microspores with irregular shape and different size and uncertain number of nuclei, and some degenerated ahead of time as well. Tapetal cells in CMS line anther degenerated during meiosis, and were crushed at the tetrad stage, which paralleled the collapse of pollens. Pollen abortion in CMS line anthers happened by PCD themselves, and the premature PCD of tapetal cells were closely associated with male sterility.     

A new cytoplasmic male sterility system for hybrid seed production in Indian oilseed mustard Brassica juncea

We report a novel cytoplasmic male sterility (CMS) system in Brassica juncea (oilseed mustard) which could be used for production of hybrid seed in the crop. A male sterile plant identified in a microspore derived doubled haploid population of re-synthesized B. napus line ISN 706 was found to be a CMS as the trait was inherited from the female parent. This CMS, designated ‘126-1’, was subsequently transferred to ten different B. juncea varieties and lines through inter-specific crosses followed by recurrent backcrossing. The F₁s of inter-specific crosses were invariably partially fertile, but irrespective of the variety/line used, the recipient lines became progressively male sterile over five to seven generations and could be maintained by crossing the male sterile lines with their normal counterparts. The male sterile lines were found to be stable for the trait under both long and short day conditions. CMS lines when crossed with lines other than the respective maintainer line were restored for fertility, implying that any variety could act as a restorer for ‘126-1’ cytoplasm in B. juncea. These unique features in maintenance and restoration of CMS lines coupled with near normal floral morphology of the CMS lines have allowed the use of ‘126-1’ cytoplasm for hybrid seed production. The uniqueness of ‘126-1’ has been further established by Southern hybridization with mitochondrial DNA probes and by a histological study of the development of male sterile anthers.     

Analysis of genetic diversity in cytoplasmic male sterility,and association of mitochondrial genes with petaloid-type cytoplasmic male sterility in tuber mustard (<Emphasis Type="Italic">Brassica juncea</Emphasis> var. <Emphasis Type="Italic">tumida</Emphasis> Tsen et Lee)

In our previous study, we bred a stable cytoplasmic male sterility (CMS) line of tuber mustard by using distant hybridization and subsequent backcrosses. In this CMS plants, all floral organs are normal except the anthers, which are transformed into petals or tubular structures. Recently, 2 mitochondrial genes—atpA and orf220—that are distinctively present in the CMS line of tuber mustard were cloned and partially characterized. In our study of genetic diversity analysis of CMS, 7 species of Brassica and Raphanus crops, which included 5 CMS lines and their respective maintainer lines, were used to compare the constitution of protein-coding genes in the mitochondrial genomes. In 4 of the 43 mitochondrial genes, namely, atpA, orf220, orf256, and orf305/orf324, polymorphisms were detected among the tuber mustard CMS line and its maintainer line. The results of a cluster analysis indicate that petaloid CMS phenotype of tuber mustard is a novel CMS type and is nearer to the nap CMS in Brassica napus at the phylogenetic level. The results of individual amplifications of these genes indicate the presence of 4 sequence-characterized amplified region (SCAR) markers, which enable rapid and reliable identification of this CMS. Expressions of the orf220 and orf256 genes were detected only in the CMS line, while expression of the orf305 gene was detected in the maintainer line. The different expression patterns of different mitochondrial-specific marker genes indicate that the quantity of mitochondrial proteins is differentially regulated during organ/tissue development in tuber mustard. The results of this study suggest that the above mentioned 4 mitochondrial genes are associated with the petaloid CMS phenotype in tuber mustard.     

Cytological and ultra-structural study on microsporogenesis of cytoplasmic male sterility in <Emphasis Type="Italic">Raphanus sativus</Emphasis>

The cytological development of microspores and tapetum in cytoplasmic male sterile (CMS) line A₁₄ and its maintainer B₁₄ in radish were studied using light- and transmission electron microscopy (LM and TEM). The microspores of the CMS line began to abort soon after they were released from tetrads in pollen sacs with light microscopy investigation, while abnormal behavior of pollen mother cells (PMC) were observed during its meiotic stage in its ultra-structural study, including degeneration of organelles and irregularity of nuclear membrane. At the same time, development of tapetal cells was similar to that of the maintainer. With further development of the anther, the tapetal cells of CMS line showed an abnormal increase in size and other appearances, such as fewer organelles and indistinct cytoplasm. The microspores of the CMS line were always distinguishable from the maintainer line with irregular structure, more osphilic deposits and abnormal exine. It is inferred that abortion of microspores is attributed to mutation of genes controlling male sterility, which further leads to hypertrophy of tapetum and destruction of ultra-structure.     

Development of a molecular marker specific to a novel CMS line in radish (Raphanus sativus L.)

In this study, we have investigated the cytoplasmic male sterility (CMS) of a novel male sterile radish line, designated NWB CMS. The NWB CMS was crossed with 16 fertile breeding lines, and all the progenies were completely male sterile. The degree of male sterility exhibited by NWB CMS is more than Ogura CMS from the Cruciferae family. The NWB CMS was found to induce 100% male sterility when crossed with all the tested breeding lines, whereas the Ogura CMS did not induce male sterility with any of the breeding lines. PCR analysis revealed that the molecular factor that influenced Ogura CMS, the orf138 gene, was absent in the NWB CMS line, and that the orf138 gene was not also expressed in this CMS line. In order to identify the cytoplasmic factors that confer male sterility in the NWB CMS line, we carried out RFLP analyses with 32 mitochondrial genes, all of which were used as probes. Fourteen genes exhibited polymorphisms between the NWB CMS line and other radish cultivars. Based on these RFLP data, intergenic primers were developed in order to amplify the intergenic regions between the polymorphic genes. Among these, a primer pair at the 3′ region of the atp6 gene (5′-cgcttggactatgctatgtatga-3′) and the 5′ region of the nad3 gene (5′-tcatagagaaatccaatcgtcaa-3′) produced a 2 kbp DNA fragment as a result of PCR. This DNA fragment was found to be specific to NWB CMS and was not present in other CMS types. It appears that this fragment could be used as a DNA marker to select NWB CMS line in a radish-breeding program.     

Gametophytically alloplasmic CMS line of rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) with variant <Emphasis Type="Italic">orfH79</Emphasis> haplotype corresponds to specific fertility restorer

For years discovery and identification of the cytoplasmic male sterility (CMS) resource in wild rice is the most intriguing events in breeding field. orfH79, a chimeric gene in mitochondria, has been suggested being the determinant for Honglian CMS in rice. In this report orfH79 gene as molecular marker to screen the wild rice, we found eight accessions with orfH79 gene in the total 42 investigated objects. Sequence analysis revealed that there were a total of nine nucleotide substitutions resulting in the change of nine amino acids in the newly identified orfH79 in wild rice, which further fell into seven haplotypes. In order to investigate the underlying relationship between orfH79 haplotypes and the corresponding fertility restorers, four accessions were selected with different orfH79 haplotype as female parents to hybridize the Honglian maintainer line, Yuetai B. After eight consecutive recurrent backcrosses, four alloplasmic CMS lines with different orfH79 haplotype were developed. Microscopic observation exhibited that their pollen grains were spherical and clear in 1% I₂–KI solution same as that of Honglian CMS line. Moreover, these four CMS lines displayed various fertility restoring model through test cross, suggesting that each orfH79 haplotye represents a new CMS type and corresponds to their specific Rf allele.     

Development of a CMS-specific marker based on chloroplast-derived mitochondrial sequence in pepper

Molecular markers developed from the flanking sequences of two cytoplasmic male sterility (CMS)-associated genes, orf456 and ψatp6-2, have been used for marker-assisted selection of CMS in pepper. However, in practice, the presence of orf456 and ψatp6-2 at substoichiometric levels even in maintainer lines hampers reliable selection of plants containing the CMS gene. In this study, we developed a novel CMS-specific molecular marker, accD-U, for reliable determination of CMS lines in pepper, and used the newly and previously developed markers to determine the cytoplasm types of pepper breeding lines and germplasms. This marker was developed from a deletion in a chloroplast-derived sequence in the mitochondrial genome of a CMS pepper line. CMS pepper lines could be unambiguously determined by presence or absence of the accD-U marker band. Application of orf456, ψatp6-2 and accD-U to various pepper breeding lines and germplasms revealed that accD-U is the most reliable CMS selection marker. A wide distribution of orf456, but not ψatp6-2, in germplasms suggests that the pepper cytoplasm containing both orf456 and ψatp6-2 has been selected as CMS cytoplasm from cytoplasm containing only orf456. Furthermore, factors other than orf456 may be required for the regulation of male sterility in pepper.     

Transcriptomic analysis of differentially expressed genes in flower-buds of genetic male sterile and wild type cucumber by RNA sequencing

Cucumber (Cucumis sativus L.) pollen development involves a diverse range of gene interactions between sporophytic and gametophytic tissues. Previous studies in our laboratory showed that male sterility was controlled by a single recessive nuclear gene, and occurred in pollen mother cell meiophase. To fully explore the global gene expression and identify genes related to male sterility, a RNA-seq analysis was adopted in this study. Young male flower-buds (1–2 mm in length) from genetic male sterility (GMS) mutant and homozygous fertile cucumber (WT) were collected for two sequencing libraries. Total 545 differentially expressed genes (DEGs), including 142 up-regulated DEGs and 403 down-regulated DEGs, were detected in two libraries (Fold Change ≥ 2, FDR < 0.01). These genes were involved in a variety of metabolic pathways, like ethylene-activated signaling pathway, sporopollenin biosynthetic pathway, cell cycle and DNA damage repair pathway. qRT-PCR analysis was performed and showed that the correlation between RNA-Seq and qRT-PCR was 0.876. These findings contribute to a better understanding of the mechanism that leads to GMS in cucumber.     

水稻红莲型细胞质雄性不育系与保持系mtRNA差异显示和差别片段的分析

将T4 RNA连接酶和AFLP技术特点相结合构建了适于mtRNA的差异显示方法 ,并比较了水稻 (OryzasativaL .)红莲型细胞质雄性不育系、保持系和杂种一代mtRNA的差异。在 4组引物对的选择扩增产物中共找到 6个差异片段 ,其中差异条带DTA为不育系仅有 ,条带DAB为不育系和保持系特有 ,而条带DBF1、DBF2 、DBF3 、DBF4 为保持系和F1杂种共有。这表明水稻红莲型不育系粤泰A与杂种一代泰优 2号mtRNA的差异大于保持系粤泰B和杂种一代泰优 2号mtRNA的差异。Northern杂交证实条带DTA在不育系、保持系和杂种一代的转录确有差异 ,表明它与红莲型细胞质雄性不育有关。条带DTA全长 2 5 9bp ,尽管未发现有同源序列和新的开放阅读框 ,但它可作为探针从cDNA文库中筛选全基因序列 ,进而寻找与细胞质雄性不育有关的开放阅读框架。