Nonsense-mediated mRNA degradation of CtFAD2-1 and development of a perfect molecular marker for olol mutation in high oleic safflower (Carthamus tinctorius L.)

There are two types of safflower oil, high oleic (HO) with 70–75 % oleic acid and high linoleic (HL) with about 70 % linoleic acid. The original HO trait in safflower, found in an introduction from India, is controlled by a partially recessive allele ol at a single locus (Knowles and Bill 1964). In the lipid biosynthesis pathway of developing safflower seeds, microsomal oleoyl phosphatidylcholine desaturase (FAD2) is largely responsible for the conversion of oleic acid to linoleic acid. In vitro microsomal assays indicated drastically reduced FAD2 enzyme activity in the HO genotype compared to conventional HL safflower. A previous study indicated that a single-nucleotide deletion was found in the coding region of CtFAD2-1 that causes premature termination of translation in the HO genotypes, and the expression of the mutant CtFAD2-1Δ was attenuated in the HO genotypes compared to conventional HL safflower (Guan et al. 2012). In this study, we hypothesise that down-regulation of CtFAD2-1 expression in the HO genotype may be explained by nonsense-mediated RNA decay (NMD). NMD phenomenon, indicated by gene-specific RNA degradation of defective CtFAD2-1Δ, was subsequently confirmed in Arabidopsis thaliana seed as well as in the transient expression system in Nicotiana benthamiana leaves. We have developed a perfect molecular marker corresponding to the olol mutation that can facilitate a rapid screening and early detection of genotypes carrying the olol mutation for use in marker-assisted selection for the management of the HO trait in safflower breeding programmes.     

Mapping quantitative trait loci controlling oil content,oleic acid and linoleic acid content in sunflower (<Emphasis Type="Italic">Helianthus annuus</Emphasis> L.)

Sunflower oil with high oleic acid content is in great demand due to its nutritional as well as industrial benefits. The trait is mainly controlled by dominant alleles at a major gene, Ol, with other modifiers. The objectives of this research were to map the oil content, oleic acid and linoleic acid content in sunflower seeds. An F2 mapping population from cytoplasmic male-sterile line COSF 7A (33–35 % oleic acid) and high oleic acid inbred line HO 5–13 (88–90 % oleic acid) was developed and phenotyped for oil content, oleic acid and linoleic acid content at the F2 seed level. High phenotypic and genotypic coefficients of variation were recorded for oleic acid and linoleic acid content. High heritability and high genetic advance as percent of mean was recorded for oleic acid and linoleic acid content. This indicated the presence of the additive type of gene action controlling the traits oleic acid content and linoleic acid content. The Ol gene was mapped to linkage group (LG) 14 and tightly linked to the marker HO_Fsp_b. In addition, two more quantitative trait loci (QTLs) for oleic acid content were identified in LG8 and LG9. Two QTLs for oil content and two QTLs for linoleic acid content were also identified. All these QTLs explained over 10 % of phenotypic variation. A study was conducted with 13 genotypes differing in oil quality as well as quantity over three seasons to assess the reliability of the identified QTLs over seasons. It resulted in the identification of two potential QTLs for oleic acid as well as linoleic acid content with the markers ORS 762 and HO_Fsp_b. These markers explained more than 57.6–66.6 % of phenotypic variation. Hence it can be concluded that these markers/QTLs would be useful in the marker-assisted selection breeding programme to improve oil quality. The present study also indicated the presence of at least two other genomic regions controlling oleic and linoleic acid content in sunflower.     

High-stearic and High-oleic cottonseed oils produced by hairpin RNA-mediated post-transcriptional gene silencing

We have genetically modified the fatty acid composition of cottonseed oil using the recently developed technique of hairpin RNA-mediated gene silencing to down-regulate the seed expression of two key fatty acid desaturase genes, ghSAD-1-encoding stearoyl-acyl-carrier protein Delta 9-desaturase and ghFAD2-1-encoding oleoyl-phosphatidylcholine omega 6-desaturase. Hairpin RNA-encoding gene constructs (HP) targeted against either ghSAD-1 or ghFAD2-1 were transformed into cotton (Gossypium hirsutum cv Coker 315). The resulting down-regulation of the ghSAD-1 gene substantially increased stearic acid from the normal levels of 2% to 3% up to as high as 40%, and silencing of the ghFAD2-1 gene resulted in greatly elevated oleic acid content, up to 77% compared with about 15% in seeds of untransformed plants. In addition, palmitic acid was significantly lowered in both high-stearic and high-oleic lines. Similar fatty acid composition phenotypes were also achieved by transformation with conventional antisense constructs targeted against the same genes, but at much lower frequencies than were achieved with the HP constructs. By intercrossing the high-stearic and high-oleic genotypes, it was possible to simultaneously down-regulate both ghSAD-1 and ghFAD2-1 to the same degree as observed in the individually silenced parental lines, demonstrating for the first time, to our knowledge, that duplex RNA-induced posttranslational gene silencing in independent genes can be stacked without any diminution in the degree of silencing. The silencing of ghSAD-1 and/or ghFAD2-1 to various degrees enables the development of cottonseed oils having novel combinations of palmitic, stearic, oleic, and linoleic contents that can be used in margarines and deep frying without hydrogenation and also potentially in high-value confectionery applications.     

A novel <Emphasis Type="Italic">FAD2</Emphasis>-<Emphasis Type="Italic">1 A</Emphasis> allele in a soybean plant introduction offers an alternate means to produce soybean seed oil with 85% oleic acid content

The alteration of fatty acid profiles in soybean to improve soybean oil quality has been a long-time goal of soybean researchers. Soybean oil with elevated oleic acid is desirable because this monounsaturated fatty acid improves the nutrition and oxidative stability of soybean oil compared to other oils. In the lipid biosynthetic pathway, the enzyme fatty acid desaturase 2 (FAD2) is responsible for the conversion of oleic acid precursors to linoleic acid precursors in developing soybean seeds. Two genes encoding FAD2-1A and FAD2-1B were identified to be expressed specifically in seeds during embryogenesis and have been considered to hold an important role in controlling the seed oleic acid content. A total of 22 soybean plant introduction (PI) lines identified to have an elevated oleic acid content were characterized for sequence mutations in the FAD 2-1A and FAD2-1B genes. PI 603452 was found to contain a deletion of a nucleotide in the second exon of FAD2-1A. These important SNPs were used in developing molecular marker genotyping assays. The assays appear to be a reliable and accurate tool to identify the FAD 2-1A and FAD2-1B genotype of wild-type and mutant plants. PI 603452 was subsequently crossed with PI 283327, a soybean line that has a mutation in FAD2-1B. Interestingly, soybean lines carrying both homozygous insertion/deletion mutation (indel) FAD2-1A alleles and mutant FAD2-1B alleles have an average of 82–86% oleic acid content, compared to 20% in conventional soybean, and low levels of linoleic and linolenic acids. The newly identified indel mutation in the FAD2-1A gene offers a simple method for the development of high oleic acid commercial soybean varieties.     

FAD2 Gene Mutations Significantly Alter Fatty Acid Profiles in Cultivated Peanuts (Arachis hypogaea)

A panel of 55 peanut lines was analyzed for fatty acid composition with gas chromatography and also genotyped with SNP markers from the FAD2 genes by real-time PCR. Significant variation in fatty acid composition was identified, and the ratio of oleic acid to linoleic acid (O/L) ranged from 1.23 to 56.45. In terms of the FAD2 gene mutation, the assayed lines were classified into four genotypes: wild type (Ol(1)Ol(1)Ol(2)Ol(2)), single functional homozygous mutation on the A genome (ol(1)ol(1)Ol(2)Ol(2)), single functional homozygous mutation on the B genome (Ol(1)Ol(1)ol(2)ol(2)), and a double mutation on both A and B genomes (ol(1)ol(1)ol(2)ol(2)). Each genotype has a significantly different fatty acid profile. Both FAD2A and FAD2B are involved in the conversion of oleic acid to linoleic acid in peanuts. Overall, these results demonstrate the combined power of genetic analysis with biochemical analysis on peanut fatty acid research.     

Inheritance of high oleic acid content in the seed oil of soybean mutant M23

A mutant line, M23, of soybean [Glycine max (L.) Merr.] was found to have two fold increases in oleic acid content in the seed oil compared with the original variety, Bay. Our objective was to determine the inheritance of the high oleic acid content in this mutant. Reciprocal crosses were made between M23 and Bay. There were no maternal and cytoplasmic effects for oleic acid content. The F₁ seeds and F₁ plants were significantly different from either parents or the midparent value, indicating partial dominance of oleic acid content in these crosses. The oleic acid content segregated in the F₂ seeds and F₂ plants in a trimodal pattern with normal, intermediate and high classes, satisfactorily fitting a 121 ratio. The seeds of a backcross between M23 and F₁ segregated into intermediate and high classes in a ratio of 11. These results indicated that oleic acid content was controlled by two alleles at a single locus with a partial dominant effect. Thus, the allele in M23 was designated ol and the genotypes of M23 and Bay were determined to be olol and 0l0l, respectively. The oleic acid contents of the F₂ seeds and F₂ plants were inversely related with the linoleic acid content which segregated in a trimodal pattern with normal, intermediate and low classes in a 121 ratio. Thus, it was assumed that the low linoleic acid content in M23 was also controlled by the ol alleles. Because a diet with high oleic acid content reduces the content of low density lipoprotein cholesterol in blood plasma, the mutant allele, ol, would be useful in improving soybean cultivars for high oleic acid content.     

Trypanosoma cruzi oleate desaturase: molecular characterization and comparative analysis in other trypanosomatids

Trypanosoma cruzi lipids contain a high content of unsaturated fatty acids, primarily oleic acid (C18:1) and linoleic acid (C18:2). Previous data suggest that this parasite is able to convert oleic acid into linoleic acid; humans are not able to do this. Presently, we show that T. cruzi has a gene with high similarity to the delta12 (omega6)-oleate desaturase from plants. Northern blot analysis of the oleate desaturase gene from T. cruzi (OD(Tc)) indicated that this gene is transcribed in epimastigote, amastigote, and trypomastigote forms. Pulsed-field analysis showed that OD(Tc) is located at distinct chromosomal bands on distinct T. cruzi phylogenetic groups. In addition, the chromoblot analysis demonstrated the presence of homologous OD(Tc) genes in several trypanosomatids; namely, Crithidia fasciculata, Herpetomonas megaseliae, Leptomonas seymouri, Trypanosoma freitasi, Trypanosoma rangeli, Trypanosoma lewisi, Blastocrithidia sp., Leishmania amazonensis, Endotrypanum schaudinni, and Trypanosoma conorhini. The native OD(Tc) activity was detected by metabolic labeling and analysis of total fatty acids from epimastigotes and trypomastigotes of T. cruzi, coanomastigotes of C. fasciculata, and promastigotes of L. amazonensis, H. megaseliae, and L. seymouri. The fact that the enzyme oleate desaturase is not present in humans makes it an ideal molecular target for the development of new chemotherapeutic approaches against Chagas disease.     

Duplicated Genes Producing Transposable Controlling Elements for the Mating-Type Differentiation in SACCHAROMYCES CEREVISIAE

Mutation of the two homothallic genes, HML alpha/HMLa and HMRa/HMR alpha, in homothallic strains of Saccharomyces cerevisiae was studied. Of 11 mutants of the HML alpha gene, eight were due to a phenotypic mutation from HML alpha to HMLa, i.e., a mutation causing a change in function of the original HML allele to that of the other HML allele (functional mutation), and three were due to a defective mutation at the HML alpha gene, i.e., a mutation causing a nonfunctional allele (nonfunctional mutation). All 14 mutants of the HMRa gene, on the other hand, were due to a phenotypic mutation from HMRa to HMR alpha i.e., a functional mutation. Phenotypic reverse mutations, i.e., HMLa to HML alpha and HMR alpha to HMRa, were also observed in the cultivation of EMS (ethyl methanesulfonate) treated spores having the HO HMR alpha HMLa genotype. Mutation from heterothallic cells to homothallism was observed in a nonfunctional mutant of the HML alpha gene, by mutagenesis with EMS, but not in the functional mutants of the HML alpha and HMRa genes or in the authentic strains having the alpha HO HMR alpha HML alpha (alpha Hp) and a HO HMRa HMLa (a Hq) genotypes. These observations suggest that the functional mutation is not caused by the direct mutation from a homothallic allele to the opposite, but by replacement of a transposable genic element produced from a homothallic locus with a region of a different homothallic locus. These observations also support the controlling-element model and the cassette model, which have been proposed to explain the mating-type differentiation by the homothallic genes.     

High-Oleic Peanut Oils Produced by HpRNA-Mediated Gene Silencing of Oleate Desaturase

The quality of peanut oil largely depends on the quantity of oleic (18:1) and linoleic acids (18:2). These two acids comprise more than 80% of the total fatty acids in peanuts. The oleate desaturase (FAD2) gene is important for maintaining high oleic acid content. A partial conservative sequence of the FAD2 gene from peanut was selected. The sense and antisense 260-bp fragments were amplified and subcloned into pFGC1008 binary expression vectors. A total of 21 transgenic plants were obtained via Agrobacterium-mediated transformation. The resulting down-regulation of the FAD2 gene resulted in a 70% increase in oleic acid content in the seeds of transformed plants compared with a 37.93% increase in untransformed plants. The results demonstrated that the target genes were likely suppressed by hpRNA interference, a pathway capable of achieving phenotypic changes. The silencing of FAD2 enabled the development of peanut oils having novel combinations of oleic acid content that can be used in high-value applications, making this approach a reliable technique for the genetic modification of seed quality and the potential for enhancement of other traits as well.     

The Ade6-M26 Mutation of Schizosaccharomyces Pombe Increases the Frequency of Crossing over

The ade6-M26 mutation of Schizosaccharomyces pombe increases conversion frequency in comparison with the nearby mutation ade6-M375. In order to investigate the effect of ade6-M26 on crossover frequency, heteroallelic ade6 duplications were constructed by integration of plasmids carrying the marker gene ura4. One ade6 gene carries either of the mutations M26 or M375 while the other ade6 copy carries the L469 mutation in both duplications. The duplication with ade6-M26 yields Ade(+) recombinants at significantly higher frequencies in meiosis, but not in mitosis. Tetrad analysis and physical characterization of spore clones from recombination tetrads demonstrate that conversions, unequal crossovers and intrachromatid exchanges occur at higher frequencies but with unaltered proportions among them. The conversion events show a pronounced bias when M26 is involved: they take place preferentially at the M26 allele. Thus the ade6-M26 mutation not only enhances conversion frequency as demonstrated before, but also crossover frequency. It displays the properties expected for a preferred site of initiation of general meiotic recombination. The duplications also yielded new information on ectopic recombination in S. pombe: ectopic crossovers occur in the duplications at much higher frequency than among naturally dispersed homologous sequences.     

张掖市不同海拔地区5个胡麻品种品质特性比较分析

胡麻是甘肃省主要经济作物之一,海拔2 400 m以下的河西地区为甘肃高产区。为探明张掖不同海拔高度对胡麻品质特性的影响和不同胡麻品种的最佳种植区域,利用近红外品质分析仪,分析了张掖市4个不同海拔地区5个胡麻品种的常规品质指标、饱和脂肪酸和不饱和脂肪酸的含量差异。结果表明：蛋白质、粗脂肪、膳食纤维、棕榈酸、十七碳酸、豆蔻酸、二十四碳酸、油酸和α-亚麻酸含量的品种和海拔间互作效应极显著(P<0.01);山嵛酸、棕榈油酸、顺-11-二十碳一烯酸、亚油酸、γ-亚麻酸和顺-11,14,17-二十碳三烯酸含量随海拔高度不同,变化较小,稳定性好。在高含油量育种目标的基础上,兼顾高三大主要不饱和脂肪酸（α-亚麻酸、油酸和亚油酸）、低两大饱和脂肪酸（棕榈酸和硬脂酸）以及高蛋白质等其他品质育种目标的角度考虑,在张掖市甘州区、高台县巷道镇和民乐县三堡镇、民乐县六坝镇,最适宜种植的胡麻品种分别是5-9812-2-1、09-3-11、张亚2号。不同海拔高度对胡麻各品质指标具有重要影响,张掖市作为优质胡麻籽生产基地,可利用不同胡麻品种适宜种植区域不同,大力发展胡麻籽品质育种和胡麻籽综合开发等。     

花生抗青枯病种质脂肪酸组成的遗传多样性

通过对123份不同类型抗青枯病花生种质种子脂肪酸的鉴定测试,分析了抗青枯病花生种质在这些性状方面的遗传分化,并与6006份资源组成的花生基础收集品进行了比较。研究结果表明,我国抗青枯病花生资源的油酸含量平均为51.78%,显著高于基础收集品的对应值(45.64%);亚油酸含量平均为28.88%,显著低于基础收集品的对应值(34.36%);高油酸种质较多,油酸含量达61%以上的资源23份,所占比重为18.7%,显著高于基础品中的相应比重(2.65%)。标准差、变异系数以及遗传多样性指数的分析结果表明,抗青枯病资源在油酸和亚油酸含量方面的遗传分化程度高。     

河南省花生地方资源蛋白质和脂肪含量分析及其育种利用策略

对233份河南省地方花生资源进行了蛋白质含量、含油量、油酸和亚油酸含量的全面测定,并与省外和国外资源的相关性状进行了比较分析。在河南地方品种资源中,蛋白质含量中等,平均含油量和油酸含量相对较高,但缺乏蛋白质含量超过30%或含油量超过56%、油酸含量超过70%的突出材料。河南省目前高油品种选育有明显进展,育成了一批高油花生品种,但育成品种蛋白质含量普遍偏低。提出了充分利用现有地方品种资源,积极采用远缘杂交、诱变、分子标记辅助选择技术及现代基因工程技术创制优良种质,选育优质专用品种的育种策略。     

Is there a second fragrance gene in rice?

Aromatic rice is highly prized by most rice consumers, and many countries cultivate traditional and improved aromatic varieties. 2-Acetyl-1-pyrroline (2AP) is the major aromatic compound in rice, and is believed to accumulate because of an eight-base-pair (8-bp) deletion in an allele at the fragrance locus. In this study, 2AP was quantified and the presence or absence of the fragrance allele ( fgr ) was determined in 464 samples of traditional varieties of rice from the T.T. Chang Genetic Resources Centre at the International Rice Research Institute. It was shown that a number of aromatic varieties, primarily from South and South-East Asia, do not carry the 8-bp deletion, but 2AP was identified in both raw and cooked rice of these varieties. We suggest that the 8-bp deletion in fgr is not the only cause of aroma, and at least one other mutation drives the accumulation of 2AP. The amount of 2AP in most uniform fgr genotypes was not significantly different from that in aromatic n fgr genotypes, but several fgr genotypes, primarily from South Asia, reproducibly accumulated exceptionally large amounts of 2AP. We suggest that the mutation leading to 2AP in aromatic n fgr varieties possibly originated several times and, through either domestication or evolution, the fgr gene and other alleles leading to 2AP have combined in South Asia, leading to several highly aromatic traditional varieties. The identification of multiple mutations for 2AP will enable rice breeding programmes to select actively for multiple genetic sources of 2AP, leading to the development of highly aromatic and, consequently, high-quality varieties of rice.