Assessment of genetic diversity and DNA profiling of linseed (<Emphasis Type="Italic">Linum usitatissimum</Emphasis> subsp. <Emphasis Type="Italic">usitatissimum</Emphasis> L.) germplasm using SSR markers

Access to genetic diversity is essential for any progress in adapting linseed (Linum usitatissimum subsp. usitatissimum L.) cultivation to changing environmental conditions or to the changing market needs. An attempt has been made in the present study to assess genetic diversity in 96 genotypes of linseed including varieties, landraces and exotic material. A total of 38 SSR primers amplified 153 alleles with 4.0 alleles per marker locus. The number of alleles ranged from 2 to 15 and the observed polymorphism ranged from 50 to 100%. Average genetic dissimilarity ranged from 2 to 50%. In order to analyze the efficiency for unambiguous identification of linseed germplasm, various statistical measures, viz., number of genotyping patterns, polymorphism information content, resolving power, discrimination power, probability of identity and probability of random identity, identified a set comprising of primers LU7, LU27, LU25, LU20 and LU31 (or LU637) for DNA fingerprinting of linseed germplasm. UPGMA cluster analysis showed that all genotypes could be grouped into four main clusters. Cluster 2 was the largest consisting of mainly landraces, whereas, Cluster 4 was the smallest. Cluster 1 consisted of mainly the released cultivars. Cluster 3 and Cluster 4 were smaller clusters and consisted of exotic genotypes. Principal co-ordinate analysis further substantiated the UPGMA clustering patterns of the observed genetic relationship. To explain 70–80% variability, 17–23 PCOs were needed, whereas 70 components were needed to explain the whole variability in the linseed material under study. Analysis of molecular variance indicated that most of the genetic variation is owing to the individuals within single population, whereas grouping of linseed material into varieties, landraces and exotics accounted for nearly 10% of the total genetic variation. The utility of SSR markers in diversity assessment and cultivar identification is discussed.     

Sonication assisted <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation enhances the transformation efficiency in flax (<Emphasis Type="Italic">Linum usitatissimum</Emphasis> L<Emphasis Type="Italic">.</Emphasis>)

A sonication-assisted, Agrobacterium-mediated, co-cultivation technique was used in an attempt to increase the transformation efficiency of flax. Hypocotyls and cotyledons excised from about 10-day-old flax seedlings grown in vitro were placed into a 10 mM MgSO₄ solution, and inoculated with an A. tumefaciens vector bearing the mgfp5-ER gene driven by the CaMV 35S promoter. The explants were subjected to pulses of ultrasound delivered by a sonicator apparatus (35 kHz) for 0–150 s and co-cultivated for 2 h at 27°C. The dried hypocotyls and cotyledons were grown on a selective MS medium to promote shoot regeneration. An electron microscopic study showed that the sonication treatment resulted in thousands of microwounds on and below the surface of the explants. A stereo microscope Leica MZ 12 equipped with a GFP adaptor was used to assess the infection and transformation of plant tissues in real time. After only 48 h and for at least 30 days after bacteria elimination, signs of transgene expression could be seen as a bright fluorescence. Our results show that treatment with ultrasound facilitates an enhanced uptake of plasmid DNA into the cells of flax hypocotyls and cotyledons and that its efficiency depends on the duration of the treatment and the frequency used. SAAT could be a promising tool for enhancing transformation efficiency in flax.     

Regeneration of flax (<Emphasis Type="Italic">Linum usitatissimum</Emphasis> L.) plants from anther culture and somatic tissue with increased resistance to<Emphasis Type="Italic"> Fusarium oxysporum</Emphasis>

The aim of this study was to establish a protocol for the efficient production of flax plants of microspore origin. The results were compared to those obtained for plants regenerated from somatic explants from hypocotyls, cotyledons, leaves, stems and roots. All the plants obtained during the experiments were regenerated from callus that was grown for periods from a few weeks to a few months before the regeneration was achieved. Anther cultures were less effective in plant regeneration than somatic cell cultures. However, regenerants derived from anther cells showed valuable breeding features, including increased resistance to fungal wilt. The age of the donor plants and the season they grew in had a noticeable effect on their anther callusing and subsequent plant regeneration. Low temperature had a negative effect and dark pre-treatment a positive effect on callusing and plant regeneration. Different media were most effective for callus induction, shoot induction and rooting. For callus induction two carbon sources (2.5% sucrose and 2.5% glucose) were most effective; for shoots, only sucrose at lower concentration (2%) was effective. Rooting was most efficient in 1% sucrose and reduced (50%) mineral concentration in the medium. It was found that the length of in vitro cultivation significantly increases the ploidy and affects such features as regenerant morphological characteristics, petal colour, and resistance to Fusarium oxysporum-induced fungal wilt. The established plant regeneration system provides a basis for the creation of transgenic flax.Abbreviations BAP 6-Benzyl-aminopurine - IAA Indole-3-acetic acid - MS Murashige and Skoog medium - NAA -Naphthalene-acetic acidCommunicated by H. Lörz     

Effect of over-expression of <Emphasis Type="Italic">Linum usitatissimum</Emphasis> PINORESINOL LARICIRESINOL REDUCTASE (<Emphasis Type="Italic">LuPLR</Emphasis>) gene in transgenic <Emphasis Type="Italic">Phyllanthus amarus</Emphasis>

Shoot tip explants of Phyllanthus amarus were cocultivated with Agrobacterium tumefaciens strain LBA 4404 carrying plasmid pCAMBIA 2301 harbouring genes coding for betaglucuronidase (gus), kanamycin (kan), and neomycin phosphotransferase II (nptII) along with a gene coding for Linum usitatissimum PINORESINOL LARICIRESINOL REDUCTASE (Lu-PLR). Transformed shoot tip explants were maintained in a Murashige and Skoog (MS) medium containing TDZ 1.54 mg l^?1, kan 50 mg l^?1 and cephotaxime 62.5 mg l^?1. The optimum medium for regeneration of multiple shoots was MS supplemented with TDZ 1.54 mg l^?1, kan 50 mg l^?1. Efficient and effective rooting of plantlets was achieved by culturing the in vitro regenerated shoots on liquid ½ MS medium containing 0.7 mg l^?1 indole 3-butyric acid (IBA) and 5 mg l^?1 kan. Rooted plants were acclimatized in the mixtures of vermiculite and soil. The transformation of kan-resistant plantlets regenerated from shoot-tip explants was confirmed by GUS and polymerase chain reaction (PCR) analysis. Southern blot and reverse transcribed PCR (RT-PCR) analysis confirmed successful integration and expression of Lu-PLR gene. Quantitative analysis of phyllanthin performed on transgenic and wild plants using high-performance liquid chromatography (HPLC) revealed that transgenic lines contained higher phyllanthin content (0.3–0.81% w/w) than wild plants (0.09% w/w). The highest yield of phyllanthin was detected in transgenic lines was up to 1.16, 1.22 and 1.23 folds higher than that of wild plant. This report highlights the transgenic approach to enhance the contents of phyllanthin and hypophyllanthin.     

Genetic diversity,population structure and association analysis in linseed (<Emphasis Type="Italic">Linum usitatissimum</Emphasis> L.)

The present investigation aimed to explore the level of genetic diversity, determine the population structure in a larger set of germplasm of linseed using microsatellite marker and identify linked markers through association mapping. A total of 168 accessions of linseed were evaluated for major agro-economic traits and SSRs markers deployed for diversity assessment. A total of 337 alleles were amplified by 50 SSRs ranging from 2 to 13 with an average of 6.74 ± 2.8 alleles per loci. The neighbor joining based clustering grouped all the accessions into three major clusters that were also confirmed by scatter plot of PCoA. While model based clustering determined four sub-populations (K = 4). Further, analysis of molecular variance analysis considering three population showed that maximum variation (79%) was within the population. We identified one putative SSR marker (Lu_3043) linked with days to 50% flowering through both GLM and MLM analysis of association mapping. The results of this preliminary study revealed genetic diversity, population structure in linseed and linked marker which could be utilized in future breeding program.     

Nitric oxide promotes in vitro organogenesis in <Emphasis Type="Italic">Linum usitatissimum</Emphasis> L.

The effects of nitric oxide (NO) on caulogenesis, shoot organogenesis and rhizogenesis from hypocotyl explants of Linum usitatissimum were investigated. Exogenously supplied NO donors, 5 μM sodium nitroprusside (SNP), 2 μM S-nitroso-N-acetylpenicillamine (SNAP) and 2 μM 3-morpholinosydnonimine (SIN-1), significantly promoted shoot differentiation from the hypocotyl explants of L. usitatissimum excised from its in vitro raised seedlings. Potassium ferrocyanide, a structural analogue of SNP, lacking NO group, did not promote shoot organogenesis. Likewise, products of NO, \textNO₂ ^- {\text{NO}}_{2}^{ - } and \textNO₃ ^- {\text{NO}}_{3}^{ - } supplied as 5 μM NaNO₂ and 5 μM NaNO₃ did not enhance shoot differentiation. Another source of NO, a mixture of sodium nitrite (SN) provided along with ascorbic acid (AsA), also caused significant promotion in the average number of shoots per responding explant. SNP also augmented the rhizogenic response of the microshoots in terms of percentage of responding explants, number of roots per responding explant and average root length. The NO scavengers, 2-(4-carboxy-phenyl)-4, 4, 5, 5-tetramethylimideazoline-1-oxyl-3-oxide (cPTIO) or methylene blue (MB), provided along with SNP, SNAP, SIN-1 or SN + AsA, at concentrations equimolar to the optimum concentration of the donors, reversed the promotory influence, thereby, confirming the role of NO in promotion of in vitro morphogenesis. However, NO scavengers individually did not affect the observed morphogenic processes. Morphological and histological studies of hypocotyl segments cultured on BM or BM + SNP for 4, 8 and 12 days demonstrated that SNP enhanced shoot differentiation by inducing a higher number of shoot primordia, each of which develops into a single shoot.     

Germination kinetics and seed reserve mobilization in two flax (<Emphasis Type="Italic">Linum usitatissimum</Emphasis> L.) cultivars under moderate salt stress

Because of its high contents of protein, α-linolenic-rich oil, lignans, and fiber, demand is increasing for flax(Linum usitatissi-mum L.) and flax seed oil as a food source. In this comparative survey, we examined germination and the mobilization of seed storage products (lipids and soluble proteins) of 3-d-old seedlings from two flax cultivars (N 51 and H 52) challenged with moderate salinity (up to 200 mM NaCl). At the highest salt concentration, germination appeared to be cultivar-dependent, with that of ‘N 51’ being less impaired and delayed than in ’H 52’. Sodium chloride inhibited germination via osmotic and toxic effects, so that seed viability was altered, especially in ‘H 52’. At 200 mM NaCl, lipid mobilization was delayed in the earliest germination phases. This response was associated with increased proportions of linolenic acid contents in both cultivars and more linolenic acid-rich molecular species of TAGs. Irrespective of the salt level, soluble protein contents in both cultivars decreased over time, although a salt-related precocity of protein degradation occurred at 200 mM NaCl.     

SSR-based linkage map of flax (<Emphasis Type="Italic">Linum usitatissimum</Emphasis> L.) and mapping of QTLs underlying fatty acid composition traits

Flax (Linum usitatissimum L.) seeds contain nearly 50% oil which is high in linolenic acid (an omega-3 fatty acid). In this study, a genetic linkage map was constructed based on 114 expressed sequence tag-derived simple sequence repeat (SSR) markers in addition to five single nucleotide polymorphism markers, five genes (fad2A, fad2B, fad3A, fad3B and dgat1) and one phenotypic trait (seed coat color), using a doubled haploid (DH) population of 78 individuals generated from a cross between SP2047 (a yellow-seeded Solin™ line with 2–4% linolenic acid) and UGG5-5 (a brown-seeded flax line with 63–66% linolenic acid). This map consists of 24 linkage groups with 113 markers spanning ~833.8 cM. Quantitative trait locus (QTL) analysis detected two major QTLs each for linoleic acid (LIO, QLio.crc-LG7, QLio.crc-LG16), linolenic acid (LIN, QLin.crc-LG7, QLin.crc-LG16) and iodine value (IOD, QIod.crc-LG7, QIod.crc-LG16), and one major QTL for palmitic acid (PAL, QPal.crc-LG9). The mutant allele of fad3A, mapped to the chromosomal segment inherited from the parent SP2047, underlies the QTL on linkage group 7 and was positively associated with high LIO content but negatively associated with LIN and IOD. This fad3A locus accounted for approximately 34, 25 and 29% of the phenotypic variation observed in this DH population for these three traits, respectively. The QTL localized on linkage group 16 explained approximately 20, 25 and 13% of the phenotypic variation for these same traits, respectively. For palmitic acid, QPal.crc-LG9 accounted for ~42% of the phenotypic variation. This first SSR-based linkage map in flax will serve as a resource for mapping additional markers, genes and traits, in map-based cloning and in marker-assisted selection.     

Cloning of a Novel Omega-6 Desaturase from Flax (<Emphasis Type="Italic">Linum usitatissimum</Emphasis> L.) and Its Functional Analysis in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The Δ12 desaturase represents a diverse gene family in plants and is responsible for conversion of oleic acid (18:1) to linoleic acid (18:2). Several members of this family are known from plants like Arabidopsis and Soybean. Using primers from conserved C- and N-terminal regions, we have cloned a novel Δ12 desaturase gene amplified from flax genomic DNA, denoted as LuFAD2-2. This intron-less gene is 1,149-base pair long encoding 382 amino acids—putative membrane-bound Δ12 desaturase protein. Sequence comparisons show that the novel sequence has 85% similarity with previously reported flax Δ12 desaturase at amino acid level and shows typical features of membrane-bound desaturase such as three conserved histidine boxes along with four membrane-spanning regions that are universally present among plant desaturases. The signature amino acid sequence ‘YNNKL’ was also found to be present at the N terminus of the protein, which is necessary and sufficient for ER localization of enzyme. Neighbor-Joining tree generated from the sequence alignment grouped LuFAD2-2 among the other FAD2 sequences from Ricinus, Hevea, Jatropha, and Vernicia. When LuFAD2-2 and LuFAD2 were expressed in Saccharomyces cerevisiae, they could convert the oleic acid to linoleic acid, with an average conversion rate of 5.25 and 8.85%, respectively. However, exogenously supplied linoleic acid was feebly converted to linolenic acid suggesting that LuFAD2-2 encodes a functional FAD2 enzyme and has substrate specificity similar to LuFAD2.     

The estimation of productivity and yield of linseed (<Emphasis Type="Italic">Linum usitatissimum</Emphasis> L.) using the growth analysis

In three year field experiments (2001 – 2003) the growth, yield and productivity of 8 flax cultivars were compared. Cultivars ‘AC Linora’, ‘Flanders’, ‘Linola™ 947’, ‘Norlin’ and ‘Omega’ were obtained from Canada, ‘Barbara’ and ‘Hungarian Gold’ from Hungary and ‘Opal’ from Poland. Apart from the estimation of the yield of aboveground parts dry matter and seed yield the determinations of the primary index value of growth analysis were done and on their basis the indices LAI, LAD, RGR, CGR and HI were calculated. The obtained yield results of the examined flax cultivars show significant genotypic — environmental relationships pertaining to the dynamics of dry matter accumulation and the amount of seed yield. Meteorological conditions in the successive years significantly influenced the particular phases of growth and development of cultivars and the factor which increased the amount of dry matter was the air temperature during the period of plant emergence — budding. During the vegetative season with a large amount of rainfall the average seed yield was about 40 % lower than compared with a year of average precipitation and a warm second part of the second period of flax vegetation. Among the analyzed cultivars a stable yield in all the years was characteristic for cultivars ‘Flanders’, ‘Barbara’ and ‘AC Linora’ (that cultivar, however, during a wet year yielded at a low level). The assimilation leaf surface of the linseed quickly increased during the period from budding to flowering and the accumulation of dry matter of the aboveground parts lasted up to the green maturity. In the successive years of the experiment there were observed significant (linear or logarithmic regressions) relationship between the yield of dry matter and the indices of growth analysis. The biggest values of the CGR indicator were observed for the period from budding to flowering. The maintaining of a high CGR value after plant flowering in the year with a favourable course of climatic parameters was beneficial for a better yield of all flax cultivars. The low values of the RGR index after flowering of cultivar ‘Hungarian Gold’ and ‘Opal’ strictly corresponded to their low yield of seed and straw biomass.     

Fine mapping of a <Emphasis Type="Italic">Phytophthora</Emphasis>-resistance gene <Emphasis Type="Italic">RpsWY</Emphasis> in soybean (<Emphasis Type="Italic">Glycine max</Emphasis> L.) by high-throughput genome-wide sequencing

Key message

Using a combination of phenotypic screening, genetic and statistical analyses, and high-throughput genome-wide sequencing, we have finely mapped a dominant Phytophthora resistance gene in soybean cultivar Wayao.

Abstract

Phytophthora root rot (PRR) caused by Phytophthora sojae is one of the most important soil-borne diseases in many soybean-production regions in the world. Identification of resistant gene(s) and incorporating them into elite varieties are an effective way for breeding to prevent soybean from being harmed by this disease. Two soybean populations of 191 F₂ individuals and 196 F_7:8 recombinant inbred lines (RILs) were developed to map Rps gene by crossing a susceptible cultivar Huachun 2 with the resistant cultivar Wayao. Genetic analysis of the F₂ population indicated that PRR resistance in Wayao was controlled by a single dominant gene, temporarily named RpsWY, which was mapped on chromosome 3. A high-density genetic linkage bin map was constructed using 3469 recombination bins of the RILs to explore the candidate genes by the high-throughput genome-wide sequencing. The results of genotypic analysis showed that the RpsWY gene was located in bin 401 between 4466230 and 4502773 bp on chromosome 3 through line 71 and 100 of the RILs. Four predicted genes (Glyma03g04350, Glyma03g04360, Glyma03g04370, and Glyma03g04380) were found at the narrowed region of 36.5 kb in bin 401. These results suggest that the high-throughput genome-wide resequencing is an effective method to fine map PRR candidate genes.

     

<Emphasis Type="Italic">Drosophila melanogaster</Emphasis> gene <Emphasis Type="Italic">Merlin</Emphasis> interacts with the clathrin adaptor protein gene <Emphasis Type="Italic">lap</Emphasis>

The protein Merlin is involved in the regulation of cell proliferation and differentiation in the eyes and wings of Drosophila and is a homolog of the human protein encoded by the Neurofibromatosis 2 (NF2) gene whose mutations cause auricular nerve tumors. Recent studies show that Merlin and Expanded cooperatively regulate the recycling of membrane receptors, such as the epidermal growth factor receptor (EGFR). By performing a search for potential genetic interactions between Merlin (Mer) and the genes important for vesicular trafficking, we found that ectopic expression in the wing pouch of the clathrin adapter protein Lap involved in clathrin-mediated receptor endocytosis resulted in the formation of extra vein materials. On the one hand, coexpression of wild-type Merlin and lap in the wing pouch restored normal venation, while overexpression of a dominant-negative mutant Mer ^DBB together with lap enhanced ectopic vein formation. Using various constructs with Merlin truncated copies, we showed the C-terminal portion of the Merlin protein to be responsible for the Merlin-lap genetic interaction. Furthermore, we showed that the Merlin and Lap proteins colocalized at the cortex of the wing imaginal disc cells.     

WRKY gene family evolution in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

The Arabidopsis thaliana WRKY proteins are characterized by a sequence of 60 amino acids including WRKY domain. It is well established that these proteins are involved in the regulation of various physiological programs unique to plants including pathogen defense, senescence and response to environmental stresses, which attracts attention of the scientific community as to how this family might have evolved. We tried to satisfy this curiosity and analyze reasons for duplications of these gene sequences leading to their diversified gene actions. The WRKY sequences available in Arabidopsis thaliana were used to evaluate selection pressure following duplication events. A phylogenetic tree was constructed and the WRKY family was divided into five sub-families. After that, tests were conducted to decide whether positive or purified selection played key role in these events. Our results suggest that purifying selection played major role during the evolution of this family. Some amino acid changes were also detected in specific branches of phylogeny suggesting that relaxed constraints might also have contributed to functional divergence among sub-families. Sites relaxed from purifying selection were identified and mapped onto the structural and functional regions of the WRKY1 protein. These analyses will enhance our understanding of the precise role played by natural selection to create functional diversity in WRKY family.     

Molecular evolution of the clustered <Emphasis Type="Italic">MIC</Emphasis>-<Emphasis Type="Italic">3</Emphasis> multigene family of <Emphasis Type="Italic">Gossypium</Emphasis> species

The Gossypium MIC-3 (Meloidogyne Induced Cotton-3) gene family is of great interest for molecular evolutionary studies because of its uniqueness to Gossypium species, multi-gene content, clustered localization, and root-knot nematode resistance-associated features. Molecular evolution of the MIC-3 gene family was studied in 15 tetraploid and diploid Gossypium genotypes that collectively represent seven phylogenetically distinct genomes. Synonymous (d_S) and non-synonymous (d_N) nucleotide substitution rates suggest that the second of the two exons of the MIC-3 genes has been under strong positive selection pressure, while the first exon has been under strong purifying selection to preserve function. Based on nucleotide substitution rates, we conclude that MIC-3 genes are evolving by a birth-and-death process and that a ‘gene amplification’ mechanism has helped to retain all duplicate copies, which best fits with the “bait and switch” model of R-gene evolution. The data indicate MIC-3 gene duplication events occurred at various rates, once per 1 million years (MY) in the allotetraploids, once per ~2 MY in the A/F genome clade, and once per ~8 MY in the D-genome clade. Variations in the MIC-3 gene family seem to reflect evolutionary selection for increased functional stability, while also expanding the capacity to develop novel “switch” pockets for responding to diverse pests and pathogens. Such evolutionary roles are congruent with the hypothesis that members of this unique resistance gene family provide fitness advantages in Gossypium.     

Genome-wide identification and resistance expression analysis of the <Emphasis Type="Italic">NBS</Emphasis> gene family in <Emphasis Type="Italic">Triticum urartu</Emphasis>

As the largest class of resistant genes, the nucleotide binding site (NBS) has been studied extensively at a genome-wide level in rice, sorghum, maize, barley and hexaploid wheat. However, no such comprehensive analysis has been conducted of the NBS gene family in Triticum urartu, the donor of the A genome to the common wheat. Using a bioinformatics method, 463 NBS genes were isolated from the whole genome of T. urartu, of which 461 had location information. The expansion pattern and evolution of the 461 NBS candidate proteins were analyzed, and 118 of them were duplicated. By calculating the lengths of the copies, it was inferred that the NBS resistance gene family of T. urartu has experienced at least two duplication events. Expression analysis based on RNA-seq data found that 6 genes were differentially expressed among Tu38, Tu138 and Tu158 in response to Blumeria graminis f.sp.tritici (Bgt). Following Bgt infection, the expression levels of these genes were up-regulated. These results provide critical references for further identification and analysis of NBS family genes with important functions.