Check of Gene Number during the Process of rDNA Magnification

THE multiple sequences of rDNA (DNA complementary to ribosomal RNA) of the Drosophila genome are localized at the bobbed locus, located in the X chromosome, position 66 and in the short arm of the Y chromosome^1,2. Wild bobbed (bb⁺) is that locus which, without a partner, gives rise to a normal phenotype. That locus which in similar conditions is incapable of giving rise to a normal phenotype is called a bobbed mutation (bb) and contains fewer genes for rRNA. The number of genes for rRNA in different individuals can vary considerably. One mechanism for rDNA variation is unequal crossing over³. Another mechanism, described by Tartof⁴, becomes apparent when individual flies, carrying only one bobbed locus, are constructed and only if such a locus is on the X chromosome; that is, if one constructs Xbb⁺/O males (and also Xbb/O males) or Xbb⁺/X_NO- females. Such individuals show a higher rDNA content than expected from the analysis of the same locus in Xbb⁺/Xbb⁺ females or in Xbb⁺/Ybb⁺ males. The increase of rDNA in this case is not inheritable⁴.     

Protective Effects of <Emphasis Type="Italic">Mekabu</Emphasis> Aqueous Solution Fermented by <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> Sanriku-SU7 on Human Enterocyte-Like HT-29-luc Cells and DSS-Induced Murine IBD Model

Most wakame Undaria pinnatifida, a brown algae, products are made from the frond portion. In this study, the polysaccharide content and antioxidant property of aqueous extract solutions (AESs) of the four parts (frond: wakame, stem of the frond: kuki-wakame, sporophyll: mekabu, and kuki-mekabu) of wakame were investigated. Polysaccharide content was high in both the wakame and mekabu. Superoxide anion (O₂ ^?) radical-scavenging capacities were high in the mekabu. These AESs could be fermented by Lactobacillus plantarum Sanriku-SU7. The O₂ ^? radical-scavenging activity of the kuki-wakame, mekabu, and kuki-mekabu were increased by the fermentation. Fermented mekabu clearly showed a protective effect on human enterocyte-like HT-29-luc cells and in a mouse model of dextran sodium sulphate-induced inflammatory bowel disease (IBD). These results suggest that the mekabu fermented by L. plantarum Sanriku-SU7 has anti-IBD effect related to O₂ ^? radical-scavenging.     

Genome wide association studies (GWAS) of spot blotch resistance at the seedling and the adult plant stages in a collection of spring barley

Spot blotch (SB) in barley is caused by the fungal pathogen Cochliobolus sativus and considered one of the major constraints to successful barley production. Resistance to C. sativus was evaluated, using a barley collection of 336 genotypes (AM-2014), at the seedling and adult stages. Seedling resistance was evaluated by using a mixture of 19 virulent isolates in Morocco. Virulent isolates prevalent in Uttar Pradesh were used for phenotyping resistance at the adult stage in India. The AM-2014 panel was genotyped with 9-K single-nucleotide polymorphism (SNP) markers using iSelect Illumina Infinium. Genome wide association studies (GWAS) were carried out using SNP markers, infection responses, disease severity, and area under the disease progress curve (AUDPC). The mixed linear model was employed in TASSEL using principal component analysis (PCA) and Kinship matrix (K) as covariates. Higher SB severity, 82.3?±?13.5 (mean?±?SD), was recorded at the Banaras Hindu University (BHU) compared to 47.6?±?15.0 at the Narendra Dev University of Agriculture and Technology (NDUAT). Nine QTL, Rcs-qtl-1H-126.9, Rcs-qtl-2H-148.16, Rcs-qtl-3H-25.27, Rcs-qtl-5H-80.35, Rcs-qtl-6H-58.24, Rcs-qtl-7H-29.62, Rcs-qtl-7H-29.72, Rcs-qtl-7H-32.81, and Rcs-qtl-7H-34.74, were detected for SB resistance at the seedling stage. For SB severity at the adult stage, a QTL, Rcs-qtl-7H-32.81, was detected at BHU while seven QTL, Rcs-qtl-2H-91.09, Rcs-qtl-3H-145.64, Rcs-qtl-4H-14.43, Rcs-qtl-6H-6.49, Rcs-qtl-7H-114.43, Rcs-qtl-7H-151.66, and Rcs-qtl-7H-150.36, were found for SB severity at NDUAT. Three QTL, Rcs-qtl-4H-18.61, Rcs-qtl-4H-67.91, and Rcs-qtl-5H-110.25, were significant for AUDPC of SB at BHU. The QTLs reported in this study are important to advance marker-assisted selection and gene pyramiding of SB resistance in South Asia and North Africa in future.     

Quantitative trait locus mapping of genes associated with vacuolation in the adrenal X-zone of the DDD/Sgn inbred mouse

Background

Adrenal gland of mice contains a transient zone between the adrenal cortex and the adrenal medulla: the X-zone. There are clear strain differences in terms of X-zone morphology. Nulliparous females of the inbred mouse DDD strain develop adrenal X-zones containing exclusively vacuolated cells, whereas females of the inbred mouse B6 strain develop X-zones containing only non-vacuolated cells. The X-zone vacuolation is a physiologic process associated with the X-zone degeneration and is tightly regulated by genetic factors. Identification of the genetic factors controlling such strain differences should help analyze the X-zone function. In this study, a quantitative trait locus (QTL) analysis for the extent of X-zone vacuolation was performed for two types of F₂ female mice: F₂A ^y mice (F₂ mice with the A ^y allele) and F₂ non-A ^y mice (F₂ mice without the A ^y allele). These were produced by crossing B6 females and DDD.Cg-A ^y males. DDD.Cg-A ^y is a congenic mouse strain for the A ^y allele at the agouti locus and is used for this study because a close association between the X-zone morphology and the agouti locus genotype has been suggested. The A ^y allele is dominant and homozygous lethal; therefore, living A ^y mice are invariably heterozygotes.

Results

Single QTL scans identified significant QTLs on chromosomes 1, 2, 6, and X for F₂ non-A ^y mice, and on chromosomes 2, 6, and 12 for F₂A ^y mice. The QTL on chromosome 2 was considered to be because of the agouti locus, which has been suggested to be associated with X-zone vacuolation. A significant QTL that interacted with the agouti locus was identified on chromosome 8.

Conclusions

The extent of X-zone vacuolation in DDD females was controlled by multiple genes with complex interactions. The murine X-zone is considered analogous structure to the human fetal zone. Therefore, the results of this study will aid in understanding function of not only of the X-zone but also of the human fetal zone. Identifying the genes responsible for the QTLs will be essential for understanding the molecular basis of X-zone function, which is currently unclear.

     

Genome-wide association studies of net form of net blotch resistance at seedling and adult plant stages in spring barley collection

Net form of net blotch (NFNB) of barley (Hordeum vulgare L.), caused by Pyrenophora teres f. teres (Ptt) Drechsler (anamorph: Drechslera teres [Sacc.] Shoem.), is considered one of the major constraints of successful barley production in major barley growing regions of the world. Resistance to NFNB was evaluated in a barley collection of 336 genotypes (AM-2014), at seedling stage using isolates LGDPtt.19 and TD10 in the USA, and adult stage in seven hotspot environments in Morocco. The AM-2014 panel was genotyped with 9K SNP markers and genome-wide association studies (GWAS) were carried out using mixed linear model (MLM: Q?+?K) accounting for population structure (Q) and kinship (K) as covariates. Significant (P?<?0.001) marker trait associations were corrected for false discovery rate (FDR) at the q?<?0.05. Four genotypes showed an average infection response (IRs ≤ 2) to both isolates, LGDPttt.19 and TD10, at the seedling stage, and 30 genotypes showed resistance in all environments in the field while three genotypes exhibited the highest resistance at both stages. The GWAS of NFNB identified 31 distinct QTLs on all seven barley chromosomes, of which 8 with resistance at seedling stage, 21 were associated with resistance at the adult stage, and two QTLs, QRptt.2H-132.15 and QPtt.6H-54-55, conferred resistance at both stages. Of 31 resistance QTLs reported in this study, 10 QTLs coincided with previously mapped QTL while 21 are novel, thereby validating the GWAS approach used in this study. The resistance sources identified in AM-2014 and QTL mapped in this study are valuable resources for marker-assisted breeding for NFNB resistance in the future.     

Laboratory and simulated-field bioassays for assessing mixed cultures of <Emphasis Type="Italic">Lysinibacillus sphaericus</Emphasis> against <Emphasis Type="Italic">Aedes aegypti</Emphasis> (Diptera: Culicidae) larvae resistant to temephos

Aedes aegypti (L.) is the main vector of tropical diseases such as dengue, chikungunya and Zika. Due to the overuse of insecticides, Ae. aegypti resistant populations have increased. Biological control with Lysinibacillus sphaericus (Ahmed) has been used against Culex sp. and Anopheles sp. Although Ae. aegypti is refractory to the binary toxin of L. sphaericus spores, vegetative cells have been shown to be effective against Ae. aegypti larvae. In this work, the effect of L. sphaericus vegetative cells on Ae. aegypti temephos-resistant larvae was assessed under lab and simulated field conditions. L. sphaericus caused about 90% mortality of insecticide-resistant Ae. aegypti larvae under simulated field conditions. Likewise, Ae. aegypti larvae were more sensitive to mixed cultures of L. sphaericus than to individual strains; then, the most effective mixed culture exhibited an LC₅₀ of 1.21 × 10⁵ CFU/mL with Rockefeller larvae and 8.04 × 10⁴ CFU/mL with field-collected larvae. Additionally, we found that mixed cultures composed of two L. sphaericus strains were more effective than a culture formed by the three strains. Our results suggest that mixed cultures comprising L. sphaericus vegetative cells could be useful for controlling temephos-resistant populations of Ae. aegypti, as evidenced by the effectiveness demonstrated under laboratory and simulated field conditions.     

Disruption of the gene encoding restriction endonuclease <Emphasis Type="Italic">Sua</Emphasis>I and development of a host–vector system for the thermoacidophilic archaeon <Emphasis Type="Italic">Sulfolobus acidocaldarius</Emphasis>

Sulfolobus acidocaldarius is a useful model organism for the genetic study of thermophilic archaea due to its ease of cultivation. Here we describe the development of a host–vector system for S. acidocaldarius consisting of SuaI restriction system-deficient strain SK-1 and shuttle vector pSAV2. The new host strain SK-1 was constructed by pop-out recombination based on the pyrE marker gene. Plasmid pSAV2 was constructed from the S. islandicus native plasmid pRN1, in which selectable markers and functional genes were inserted in suitable locations and orientations followed by the deletion of non-essential open reading frames. SK-1 allowed direct transformation without N⁴-methylation at SuaI restriction sites, so unmethylated vector pSAV2 could be introduced directly into SK-1 by electroporation. The transformants were selected by pyrEF complementation on xyrose–tryptone solid medium without prior liquid culturing. The transformation efficiency was approximately 1.0 × 10³/μg DNA. After replication in S. acidocaldarius, pSAV2 was successfully recovered from transformant cultures by the standard alkaline lysis method. Plasmid yield was approximately 40–50 ng/ml from late-log through stationary phase cultures. In addition, pSAV2 was maintained stably and at relatively high copy number in S. acidocaldarius.     

Polymorphism of the genes for antioxidant defense enzymes and their association with the development of chronic obstructive pulmonary disease in the population of Bashkortostan

In this study, frequencies of the polymorphic variants of the genes encoding antioxidant enzymes, GSTM1, GSTT1, GSTP1, CAT, GPX1, NQO1, SOD1, and SOD3 were examined in three ethnic groups of healthy subjects from the Republic of Bashkortostan (Russians, Tatars, and Bashkirs). An association of these markers with the development of chronic obstructive pulmonary disease (COPD) was tested. Interethnic differences relative to the distribution of the polymorphic variants of the GSTP1 locus Ile105Val and the NQO1 locus 609C/T were revealed. Relative to the genotype distribution at the Ile105Val locus of the GSTP1 gene, ethnic group of Bashkirs was found to be statistically significantly different from Tatars (χ² = 8.819; d.f. = 2; P = 0.012). Relative to the genotype frequency distribution pattern at the NQO1 locus 609C/T, the group of Bashkirs differed from Russians (χ² = 8.913; d.f. = 2; P = 0.012). An association of genotype Val/Val of the GSTP1 Ile105Val locus with the risk of COPD in Russians (χ² = 5.25; P = 0.022; P _cor = 0.044; OR = 4.09), and of the GSTP1 haplotype *D in Tatars, was demonstrated (χ² = 11.575; P = 0.0014; P _cor = 0.0042; OR = 3.178). Genotype TT of the CAT ?262C/T locus marked resistance to the COPD development in Russians (χ² = 6.82; P = 0.0098; P _cor = 0.0196; OR = 0.31; 95%CI, 0.119 to 0.77). The risk for COPD in the ethnic group of Tatars was associated with the CAT haplotype (?262)C/(1167)T (χ² = 6.038; P = 0.0147; P _cor = 0.044; OR = 1.71). Analysis of the NQO1 haplotypes at the 465C/T and 609C/T loci showed that haplotype 465C/609T was associated with COPD in Russians (χ² = 4.571; P = 0.0328; P _cor = 0.01; OR = 1.799). It was demonstrated that Gly allele of the Arg213Gly polymorphic locus of the SOD3 gene marked the risk for COPD in the ethnic group of Tatars (OR = 2.23; 95%CI, 1.22 to 4.1). Thus, GSTP1, CAT, NQO1, and SOD3 polymorphisms play an important role in the development of COPD among the population of Bashkortostan.     

Acceleration of Senescence induced in Wheat by Light

CELLS from patients with G-trisomy or E-trisomy and XXY cells from patients with XY/XXY mosaic Klinefelter's syndrome are more susceptible to transformation in vitro by SV40 than are cells from normal individuals^1–3. We have used triploid (69XXY) human cells to determine whether the presence of extra chromosomes per se increases susceptibility to transformation.     

Limited diversity associated with duplicated class II MHC-<Emphasis Type="Italic">DRB</Emphasis> genes in the red squirrel population in the United Kingdom compared with continental Europe

The red squirrel (Sciurus vulgaris) population in the United Kingdom has declined over the last century and is now on the UK endangered species list. This is the result of competition from the eastern grey squirrel (S. carolinensis) which was introduced in the 19th century. However, recent evidence suggests that the rate of population decline is enhanced by squirrelpox disease, caused by a viral infection carried asymptomatically by grey squirrels but to which red squirrels are highly susceptible. Population genetic diversity provides some resilience to rapidly evolving or exotic pathogens. There is currently no data on genetic diversity of extant UK squirrel populations with respect to genes involved in disease resistance. Diversity is highest at loci involved in the immune response including genes clustered within the major histocompatibility complex (MHC). Using the class II DRB locus as a marker for diversity across the MHC region we genotyped 110 red squirrels from locations in the UK and continental Europe. Twenty-four Scvu-DRB alleles at two functional loci; Scvu-DRB1 and Scvu-DRB2, were identified. High levels of diversity were identified at both loci in the continental populations. In contrast, no diversity was observed at the Scvu-DRB2 locus in the mainland UK population while a high level of homozygosity was observed at the Scvu-DRB1 locus. The red squirrel population in the UK appears to lack the extensive MHC diversity associated with continental populations, a feature which may have contributed to their rapid decline.     

The <Emphasis Type="Italic">Adh</Emphasis> locus and adaptation of <Emphasis Type="Italic">cn</Emphasis> and <Emphasis Type="Italic">vg</Emphasis> mutants in experimental populations of <Emphasis Type="Italic">Drosophila melanogaster MEIG</Emphasis>

A complex study on the adaptation of cn and vn mutants and the allozymes of alcoholdehydrogenase (ADH) was carried out in initially pure lines, and their panmixia populations during exchange of the mutant genotype with that of wild-type flies (C-S) and D) through saturating crossings. The relative adaptation of the genotypes was estimated by their effect on reproductive efficiency in the experimentally obtained population. Fecundity, lifespan, and the resistance of the studied genotypes to hyperthermia were investigated individually. It was shown that the high level of adaptation of the cn mutants and the low level of adaptation of the vg mutants was correlated with the presence of different ADH allozymes. In the studied population, the F-allozyme of ADH accompanied the vg mutation, while the S-allozyme of the enzyme was detected in cn mutants. Saturating crossings of C-S(Adh ^S)×vg(Adh ^F) and D(Adh ^F) × cn(Adh ^S), along with the parallel determination of the allele composition of the Adh locus, demonstrated that the complete substitution of the F-allozyme of ADH in the vg mutants by the S-allozyme in D flies, as well as the substitution of the S-allozyme of ADH in the cn mutants by the F-allozyme in D flies was realized only after the 15th–20th backcrosses. These results favor the coadaptation of cn and vg marker genes with alleles of the Adh locus and indicate the important role of the latter in the adaptation of genotypes. In the studied population, selection acted primarily against the vg mutants, which were inferior to the cn mutants, and heterozygote genotypes in indices of the main adaptation components.     

Development of an <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation method for <Emphasis Type="Italic">Taxus</Emphasis> suspension cultures

The FDA-approved anti-cancer compound paclitaxel is currently produced commercially by Taxus plant cell suspension cultures. One major limitation to the use of plant cell culture as a production platform is the low and variable product yields. Therefore, methods to increase and stabilize paclitaxel production are necessary to ensure product security, especially as the demand for paclitaxel continues to rise. Although a stable transformation method for Taxus suspension cultures has been developed, stable transformant yields are low (around 1% of experiments) and the method does not translate to the Taxus cuspidata Siebold and Zucc. and Taxus canadensis Marshall cell lines utilized in this study. Therefore, a new method for Agrobacterium-mediated transformation of Taxus callus and suspension cultures was developed through identification of the optimal Agrobacterium strain, inclusion of an anti-necrotic cocktail (silver nitrate, cysteine, and ascorbic acid) and increased recovery time for cells after cocultivation, the time following infection with Agrobacterium tumefaciens. Application of the increased recovery time to transformation of T. cuspidata line PO93XC resulted in 200 calluses staining positive for GUS. Additionally, two transgenic lines have been maintained with stable transgene expression for over 5 yr. This method represents an improvement over existing transformation methods for Taxus cultures and can be applied for future metabolic engineering efforts.     

Enantioconvergent hydrolysis of racemic styrene oxide at high concentration by a pair of novel epoxide hydrolases into (<Emphasis Type="Italic">R</Emphasis>)-phenyl-1,2-ethanediol

Objectives

To prepare (R)-phenyl-1,2-ethanediol ((R)-PED) with high enantiomeric excess (ee _p) and yield from racemic styrene oxide (rac-SO) at high concentration by bi-enzymatic catalysis.

Results

The bi-enzymatic catalysis was designed for enantioconvergent hydrolysis of rac-SO by a pair of novel epoxide hydrolases (EHs), a Vigna radiata EH3 (VrEH3) and a variant (AuEH2^A250I) of Aspergillus usamii EH2. The simultaneous addition mode of VrEH3 and AuEH2^A250I, exhibiting the highest average turnover frequency (aTOF) of 0.12 g h^?1 g^?1, was selected, by which rac-SO (10 mM) was converted into (R)-PED with 92.6% ee _p and 96.3% yield. Under the optimized reaction conditions: dry weight ratio 14:1 of VrEH3-expressing E. coli/vreh3 to AuEH2^A250I-expressing E. coli/Aueh2 ^A250I and reaction at 20 °C, rac-SO (10 mM) was completely hydrolyzed in 2.3 h, affording (R)-PED with 98% ee _p. At the weight ratio 0.8:1 of rac-SO to two mixed dry cells, (R)-PED with 97.4% ee _p and 98.7% yield was produced from 200 mM (24 mg/ml) rac-SO in 10.5 h.

Conclusions

Enantioconvergent hydrolysis of rac-SO at high concentration catalyzed by both VrEH3 and AuEH2^A250I is an effective method for preparing (R)-PED with high ee _p and yield.

     

Validation of reference genes for gene expression analysis of response to anthocyanin induction in cell cultures of <Emphasis Type="Italic">Vitis davidii</Emphasis> (Rom. Caill.) Foëx

Real-time quantitative polymerase chain reaction (RT-qPCR) is an effective method for detecting changes of gene expression in plant cell metabolic regulation. A set of 15 reference gene candidates were selected for the present study of anthocyanin biosynthesis regulation, and stability. The suitability of their expression was evaluated in eight different experimental treatments in spine grape (Vitis davidii [Rom. Caill.] Foëx.) cell cultures. The results indicated that SAND family protein (SAND) and V-type proton ATPase subunit G (VAG) were the most stable reference genes for culture duration, tubulin alpha-3/alpha-5 chain (α-tubulin) and tubulin beta-1 chain (β-tubulin) for illumination conditions, ubiquitin-conjugating enzyme E2-17 kDa (UBQ) and VAG for UVB treatment, VAG and 60S ribosomal protein L18-2 (60SRP) for temperature treatment, AP47, clathrin adaptor complex subunit mu (AP-2) and 60SRP for cinnamic acid treatment, α-tubulin and UBQ for chitosan treatment, actin and alcohol dehydrogenase 2 (ADH2) for kinetin treatment, and β-tubulin and elongation factor 1-α (EF1-α) for cell line. Finally, the reliability of the selected reference genes was confirmed by investigating the expression profiles of the target gene dihydroflavonol 4-reductase (DFR) in spine grape cell cultures. The results of the present study offer the most robust platform for the most precise and broad application of RT-qPCR to investigate gene expression associated with anthocyanin biosynthesis in spine grape cell cultures.     

Generation of an <Emphasis Type="Italic">Rtel1</Emphasis>-<Emphasis Type="Italic">CreERT2</Emphasis> knock-in mouse model for lineage tracing RTEL1+ stem cells during development

Regulator of telomere length 1 (RTEL1) DNA helicase has been demonstrated to be essential for the maintenance of telomeres and genomic stability. This function of RTEL1 could be required for protecting stem cells from genomic mutations as suggested by its selective expression in stem cell-zones, as well as by RTEL1 mutations identified in Hoyeraal-Hreidarsson syndrome, a severe dyskeratosis congenita that targets primarily stem cell compartments. As a first step to establish a role of RTEL1 in stem cells, we generated an Rtel1^CreERT2 mouse allele in which a tamoxifen-inducible Cre (CreERT2) cDNA was specifically knocked into the Rtel1 genomic locus and controlled by the endogenous Rtel1 regulatory elements. By crossing with a Cre-dependent LacZ reporter mouse strain (R26R^LacZ), we further demonstrated that Cre activity in Rtel1^CreERT2 mice could be specifically induced by tamoxifen, which allowed the fate of RTEL1+ cells to be traced at various stages of development. Using this tracing assay, we showed for the first time that RTEL1+ cells in the intestine and the testis can act as stem cells that have the capacity to self-renew and differentiate into progeny cells. Therefore, the Rtel1^CreERT2 mice generated in this study will be a valuable transgenic tool to explore the function of RTEL1 in stem cells.     

Effects of Colistin and Bacteriocins Combinations on the In Vitro Growth of <Emphasis Type="Italic">Escherichia coli</Emphasis> Strains from Swine Origin

Escherichia coli strains from swine origin, either susceptible or resistant to colistin, were grown under planktonic and biofilm cultures. After which, they were treated with antibacterial agents including nisin and enterocin DD14 bacteriocins, colistin and their combinations. Importantly, the combination of colistin, enterocin DD14 and nisin eradicated the planktonic and biofilm cultures of E. coli CIP54127 and the E. coli strains with colistin-resistance phenotype such as E. coli 184 (mcr-1 ⁺) and E. coli 289 (mcr-1 ^?), suggesting therefore that bacteriocins from lactic acid bacteria could be used as agents with antibiotic augmentation capability.     

Allelic differentiation at the <Emphasis Type="Italic">Sg-1</Emphasis> locus for the terminal sugar of the C-22 position of group A saponin in Chinese wild soybean (<Emphasis Type="Italic">Glycine soja</Emphasis> Sieb. &amp; Zucc.)

Group A saponins are thought to be the cause of bitter and astringent tastes in processed foods of soybean (Glycine max), and the elimination of group A saponins is an important breeding objective. The group A saponins include two main Aa and Ab types, controlled by codominant alleles at the Sg-1 locus that is one of several key loci responsible for saponin biosynthesis in the subgenus Glycine soja. However, A0 mutant lacking group A saponin is a useful gene resource for soybean quality breeding. Here, eight Chinese wild soybean A0 accessions were sequenced to reveal the mutational mechanisms, and the results showed that these mutants were caused by at least three kinds of mechanisms involving four allelic variants (sg-1^0-b2, sg-1^0-b3, Sg-1^b-0, and Sg-1^b-01). The sg-1^0-b2 had two nucleotide deletions at positions +?72 and +?73 involving in the 24th and 25th amino acids. The sg-1^0-b3 contained a stop codon (TGA) at the 254th residue. The Sg-1^b-0 and Sg-1^b-01 were two novel A0-type mutants, which likely carried normal structural alleles, and nevertheless did not encode group A saponin due to unknown mutations beyond the normal coding regions. In addition, to reveal the structural features, allelic polymorphism, and mechanisms of the abiogenetic absence of group A (i.e., A0 phenotype), nucleotide sequence analysis was performed for the Sg-1 locus in wild soybean (Glycine soja). The results showed that Sg-1 alleles had a lower conservatism in the coding region; as high as 18 sequences were found in Chinese wild soybeans in addition to the Sg-1^a (Aa) and Sg-1^b (Ab) alleles. Sg-1^a and Sg-1^b alleles were characterized by eight synonymous codons and nine amino acid substitutions. Two evolutionarily transitional allelic sequences (Sg-1^a7 and Sg-1^b2) from Sg-1^a toward Sg-1^b were detected.     

Mapping of novel salt tolerance QTL in an Excalibur × Kukri doubled haploid wheat population

Key message

Novel QTL for salinity tolerance traits have been detected using non-destructive and destructive phenotyping in bread wheat and were shown to be linked to improvements in yield in saline fields.

Abstract

Soil salinity is a major limitation to cereal production. Breeding new salt-tolerant cultivars has the potential to improve cereal crop yields. In this study, a doubled haploid bread wheat mapping population, derived from the bi-parental cross of Excalibur?×?Kukri, was grown in a glasshouse under control and salinity treatments and evaluated using high-throughput non-destructive imaging technology. Quantitative trait locus (QTL) analysis of this population detected multiple QTL under salt and control treatments. Of these, six QTL were detected in the salt treatment including one for maintenance of shoot growth under salinity (QG_(1–5).asl-7A), one for leaf Na⁺ exclusion (QNa.asl-7A) and four for leaf K⁺ accumulation (QK.asl-2B.1, QK.asl-2B.2, QK.asl-5A and QK:Na.asl-6A). The beneficial allele for QG_(1–5).asl-7A (the maintenance of shoot growth under salinity) was present in six out of 44 mainly Australian bread and durum wheat cultivars. The effect of each QTL allele on grain yield was tested in a range of salinity concentrations at three field sites across 2 years. In six out of nine field trials with different levels of salinity stress, lines with alleles for Na⁺ exclusion and/or K⁺ maintenance at three QTL (QNa.asl-7A, QK.asl-2B.2 and QK:Na.asl-6A) excluded more Na⁺ or accumulated more K⁺ compared to lines without these alleles. Importantly, the QK.asl-2B.2 allele for higher K⁺ accumulation was found to be associated with higher grain yield at all field sites. Several alleles at other QTL were associated with higher grain yields at selected field sites.

     

High-efficient <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated <Emphasis Type="Italic">in planta</Emphasis> transformation in black gram (<Emphasis Type="Italic">Vigna mungo</Emphasis> (L.) Hepper)

In vitro culture and genetic transformation of black gram are difficult due to its recalcitrant nature. Establishment of gene transfer procedure is a prerequisite to develop transgenic plants of black gram in a shorter period. Therefore, genetic transformation was performed to optimize the factors influencing transformation efficiency through Agrobacterium tumefaciens-mediated in planta transformation using EHA 105 strain harbouring reporter gene, bar, and selectable marker, gfp-gus, in sprouted half-seed explants of black gram. Several parameters, such as co-cultivation, acetosyringone concentration, exposure time to sonication, and vacuum infiltration influencing in planta transformation, have been evaluated in this study. The half-seed explants when sonicated for 3 min and vacuum infiltered for 2 min at 100 mm of Hg in the presence of A. tumefaciens (pCAMBIA1304– bar) suspensions and incubated for 3 days co-cultivation in MS medium with 100 µM acetosyringone showed maximum transformation efficiency (46 %). The putative transformants were selected by inoculating co-cultivated seeds in BASTA^® (4 mg l^?1) containing MS medium followed by BASTA^® foliar spray on 15-day-old black gram plants (35 mg l^?1) in green house, and the transgene integration was confirmed by biochemical assay (GUS), Polymerase chain reaction, Dot-blot, and Southern hybridisation analyses.