不同发育时期菊芋块茎DNA提取效果比较研究

为筛选菊芋块茎DNA提取的适宜生育时期,以“青芋1号”菊芋品种为试材,用改良CTAB法对不同发育时期菊芋块茎DNA进行提取,经琼脂糖凝胶电泳及全波长分光光度计检测总DNA的纯度、浓度及质量,选用4条ISSR引物进行PCR验证.结果表明:不同发育时期提取菊芋块茎DNA效果较好,DNA浓度呈现单峰曲线变化,峰值出现在第9周,与琼脂糖凝胶电泳检测呈现的亮度结果一致,PCR验证结果显示,提取的DNA能够满足后续相关分子生物学的要求.     

Sunflower mutant containing high levels of palmitic acid in high oleic background

A new sunflower mutant, CAS-12, was obtained, which has both high palmitic (≈30%) and high oleic acid contents, and also a substantial amount of palmitoleic acid (≈7%). The mutant was selected after X-ray irradiation of dry seeds of the inbred line BSD-2-423, which had normal palmitic (≈3%) and high oleic (≈88%) acid levels. The increase of palmitic and palmitoleic acids occurred at the expense of the oleic acid content, which decreased to around 55% in respect to the original line. Linoleic acid content is always under 5%. Palmitic and palmitoleic acid levels were similar to those of the high palmitic mutant CAS-5 obtained in a previous programme from a low oleic line isogenic to BSD-2-423 using a similar mutagenic treatment. In that previous programme we also selected three high stearic acid mutants using chemical mutagenic treatment on the same sunflower line (RDF-1-532). We attempted to obtain mutants in other lines but were unsuccessful. The isolation of similar mutants in isogenic parental lines illustrates the importance of the genetic background in the development of specific mutants with an altered seed oil fatty acid composition. The oil of this mutant will increase the range of potential uses of sunflower oil. This revised version was published online in July 2006 with corrections to the Cover Date.     

A new source of cytoplasmic male sterility in sunflower

Summary Interspecific substitutions of the nucleus of Helianthus annuus (2n=34) cv. Saturn into the cytoplasm of H. petiolaris (2n=34) by successive backcrossing, resulted in progenies with indehiscent anthers containing white, rather than normal yellow, pollen. Further backcrossing by cv. Saturn failed to restore pollen shed, suggesting that the male sterility was cytoplasmic. In vivo germination tests of pollen from 23 such plants from eight BC₅ lines, indicated complete pollen sterility for 14 plants, but normal seed set, suggesting that female fertility was not affected. Meiosis in all plants examined was normal.Crosses between seven sources of pollen-fertility restorer, one collection of wild H. annuus, and an existing source of cytoplasmic male sterility, resulted in a high frequency of plants with normal pollen shed in all F₁ progenies. However, no normal pollen shed was evident in F₁ progenies for similar crosses between BC₅ male-steriles and three of the seven restorer sources, nor for the single wild H. annuus evaluated. The foregoing suggests that the backcross substitution lines are a new source of cytoplasmic male sterility. The inheritance of restoration of pollen shed was complex and not fully elucidated. Some data suggested that two independent, complementary, dominant genes were required, but others indicated two to three independent, dominant genes.     

Characterization for agronomic use of cytoplasmic male-sterility in sunflower (Helianthus annuus L.) introduced from H. resinosus Small

A backcrossing programme was carried out both to assess the stability of a cytoplasmic male‐sterility (CMS) source from Helianthus resinosus, designated RES1, and to incorporate it into inbred sunflower lines (HA89, RHA271, RHA801). All the progenies, grown in different environments, were completely male‐sterile. This suggests that the expression of this cytoplasm is stable. Female‐fertility of lines HA89, RHA271 and RHA801 carrying CMS RES1 were compared with those of the corresponding fertile inbred lines. There were no differences in the number of seeds per head. This indicates that female‐fertility is not affected by RES1 cytoplasm. Cytological studies showed that meiosis proceeds normally until the tetrad stage; consequently, the absence of pollen is caused by alterations that take place during postmeiotic stages. With the aim of identifying male‐fertility restorer genotypes, crosses were made between HA89 (CMS RES1) plants and different annual diploid and perennial hexaploid Helianthus species. All the diploid germplasm evaluated behaved as a CMS RES1 maintainer. However, the hexaploid species, H. resinosus, H. x laetiflorus, H. pauciflorus and H. tuberosus, restored pollen fertility in CMS RES1 plants.     

Inheritance of reduced plant height in the sunflower line Dw 89

The objective of the present research was to study the inheritance of reduced plant height in the sunflower line Dw 89. Plants of the cytoplasmic male sterile version of this line, cmsDw 89 (mean plant height of 47.4 cm) were crossed with plants of the restorer line RHA 271 (mean of 120.9 cm). F₁ plants averaged 120.4 cm, which indicated dominance of standard over reduced plant height. F₂ plants followed a segregation pattern of 1 : 15 (reduced : normal height), suggesting that reduced plant height in Dw 89 is controlled by alleles at two loci, designated Dw1 and Dw2. Class assignment in the F₂ was confirmed through the evaluation of the F₃ generation. Backcrosses to Dw 89 segregated with 1 : 3 (reduced : normal height) ratios, which confirmed the digenic inheritance of the trait. The evaluation of plant height distributions in F₃ families suggested possible genetic interaction between the Dw1 and Dw2 loci.     

Resistance to Sclerotinia sclerotiorum of 'high oleic' sunflower inbred lines

Cultivation of sunflower (Helianthus annuus L.) is strongly affected by Sclerotinia sclerotiorum. To identify new sources of genetic diversity for sunflower breeding 25 sunflower inbred lines were analysed using eight Amplified Fragment Length Polymorphism (AFLP) primer combinations and their genetic similarities (GS) were estimated. Data were used to develop a Unweighted Pair Group Method using Arithmetic Averages (UPGMA) dendrogram. GS values of 0.58‐0.98 were observed but with no separate groupings dependant on oil quality. The inbred lines were screened for their reaction to inoculation with Sclerotinia sclerotiorum (Lib.) de Bary. Sunflower heads were artificially inoculated with S. sclerotiorum in three environments. Head infection was monitored after 1 week (lesion length) and 2 weeks (head rot). The F5 generation of a cross between a resistant (SWS‐B‐04) and a susceptible inbred line (SWS‐B‐01) was also tested for sclerotinia reaction across three environments. Significant differences in sclerotinia resistance, moderate heritabilities and a high correlation between the two assessments were observed. Inbred lines with a high level of resistance could be identified. These lines can be used for further breeding to improve sunflower sclerotinia resistance and to develop superior new hybrids.     

Construction and characterization of a BAC library for sunflower (Helianthus annuus L.)

A bacterial artificial chromosome (BAC) library was constructed using the sunflower (Helianthus annuus L.) restorer line RHA325, which carries the restorer gene Rf1 and the Pl2-gene conferring resistance to downy mildew. High molecular weight DNA was prepared from nuclei using leaf material from two-week old seedlings. The library was constructed using the HindIII site of pBeloBAC11. The current BAC library comprises 104,736 clones. The insert size of the clones varied between 20 and 270 kb, with an average insert size of 60 kb. The whole 1.9× sunflower BAC library was spotted in duplicate on four high-density filters, each carrying 55,296 clones. The content of organellar DNA, which was estimated by colony hybridisation against the mitochondrial probe coxI and the chloroplast probe rbcL, proved to be less than 0.03 and 0.1%, respectively. BAC pools, allowing PCR-based screening, were made and used to identify positive BAC clones for the markers OP-K13_454, closely linked to the restorer gene Rf1. The PCR-based screening was verified by the results obtained for this marker by colony hybridisation.     

Accumulation of soluble phenolic compounds in sunflower capitula correlates with resistance to Sclerotinia sclerotiorum

Disease symptoms and total soluble phenolics content have been analysed in four sunflower (Helianthus annuus L.)lines with different resistance levels(from highly susceptible to resistant) to head rot caused by Sclerotinia sclerotiorum (Lib.) de Bary. At the beginning of the flowering stage, capitula were inoculated by spraying with a water suspension of ascospores, and disease symptoms were evaluated from day 6 to day14 after inoculation. The most susceptible genotypes showed all their ovaries to be necrosed and abundant lesions in corollas, bracts and receptacle. In the resistant line, the ovary and corolla were only partially necrosed with no symptoms in the bracts or the receptacle. Total soluble phenolics were extracted and quantified from different parts of the capitulum in both inoculated and non-inoculated plants. The amount of phenolic compounds depended on the sunflower line, the time after inoculation, and the tissue. Higher constitutive and induced phenolic content as well as phenylalanine ammonia-lyase activity were present in the most resistant line, these differences correlated with the absence/presence of disease symptoms. This revised version was published online in July 2006 with corrections to the Cover Date.     

Inheritance of high palmitic acid content in the sunflower mutant CAS-12 and its relationship with high oleic content

CAS‐12 is a sunflower mutant with increased levels of palmitic (C16: 0 = 30%) and oleic (C18: 1 = 55%) acids in its seed oil, hence it has a reduced linoleic acid content (C18: 2 < 5%). This study was conducted to determine the inheritance of high C16: 0 content and its relationship with high C18: 1 content in CAS‐12. Reciprocal crosses involving CAS‐12, CAS‐5 (high C16: 0 content), HAOL‐9 (high C18: 1 content) and HA‐89 (standard fatty acid profile) were made. The F₁, F₂ and BC₁F₁ generations were obtained. The genetic control of the high C16: 0 trait in CAS‐12 was partially recessive and gametophytic. In all cases, this character segregated in the ratio 19: 38: 7 (low: intermediate: high C16: 0 content) in the F₂ generation. These results, together with the lack of segregation for C16: 0 content in crosses between CAS‐12 and CAS‐5, indicated that the genetic control of the high C16: 0 trait in CAS‐12 was similar to that in CAS‐5 in being controlled by partially recessive alleles (p1, p2, and p3) at three loci. Crosses between HA‐89 and CAS‐12, and HAOL‐9 and CAS‐5 (segregating for C16: 0 and C18: 1) demonstrated that the high C16: 0 and the high C18: 1 traits were independently inherited. However, C18: 1 segregation in these crosses exhibited reversal of dominance. Apparently, the low C18: 1 parental lines carried modifier genes causing the deviation.     

Genetic variability in plants regenerated from in vitro culture of sunflower (Helianthus annum L.)

In vitro regenerated sunflower (Helianthus annuus L.) plants (R₁) were self-fertilized and the R₂ generation was evaluated for qualitative traits. A broad range of phenotypic variation was observed and mutation frequencies were calculated. Some in vitro induced variant phenotypes were similar to known spontaneous or induced mutations in sunflower, while others were new. Chlorophyll and carotenoid deficiencies, chimaerical variegation, fasciated stem and capitulum, abnormal shoot development, and other morphological variations, were noted. Substitution of anthers with petaloid structures in a disk-floret mutant indicates a possible homeotic mutation induced by in vitro tissue culture.