Selection and evaluation of reference genes for quantitative gene expression analysis in broomcorn millet (<Emphasis Type="Italic">Panicum miliaceum</Emphasis> L.)

The appropriate reference genes are crucial for normalization of the target gene expression in qRT-PCR analysis. Broomcorn millet (Panicum miliaceum L.) is one of the most important crops in drought areas worldwide, while the systematical investigation and evaluation of reference genes has not been investigated in this species up to now. Here, 9 commonly used reference genes were selected to detect their expressional stability in different tissues and under different stresses in broomcorn millet. ΔC_t, BestKeeper, NormFinder and GeNorm approaches were used to evaluate the potentiality of these candidate genes as the reference gene in broomcorn millet. Taken together, results found that 18S and GAPDH were the suitable reference genes for gene expression normalization in different tissues and under stress treatment in broomcorn millet. This was the first study to investigate the reference genes for qRT-PCR analysis in broomcorn millet, which will facilitate the gene expression studies and also accelerate revealing the molecular mechanism of well-adapted extreme climatic conditions.     

Ectopic expression of cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.) <Emphasis Type="Italic">CsTIR</Emphasis>/<Emphasis Type="Italic">AFB</Emphasis> genes enhance salt tolerance in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

Auxin receptors TIR1/AFBs play an essential role in a series of signaling network cascades. These F-box proteins have also been identified to participate in different stress responses via the auxin signaling pathway in Arabidopsis. Cucumber (Cucumis sativus L.) is one of the most important crops worldwide, which is also a model plant for research. In the study herein, two cucumber homologous auxin receptor F-box genes CsTIR and CsAFB were cloned and studied for the first time. The deduced amino acid sequences showed a 78% identity between CsTIR and AtTIR1 and 76% between CsAFB and AtAFB2. All these proteins share similar characteristics of an F-box domain near the N-terminus, and several Leucine-rich repeat regions in the middle. Arabidopsis plants ectopically overexpressing CsTIR or CsAFB were obtained and verified. Shorter primary roots and more lateral roots were found in these transgenic lines with auxin signaling amplified. Results showed that expression of CsTIR/AFB genes in Arabidopsis could lead to higher seeds germination rates and plant survival rates than wild-type under salt stress. The enhanced salt tolerance in transgenic plants is probably caused by maintaining root growth and controlling water loss in seedlings, and by stabilizing life-sustaining substances as well as accumulating endogenous osmoregulation substances. We proposed that CsTIR/AFB-involved auxin signal regulation might trigger auxin mediated stress adaptation response and enhance the plant salt stress resistance by osmoregulation.     

The identification of QTLs associated with the <Emphasis Type="Italic">in vitro</Emphasis> response of rye (<Emphasis Type="Italic">Secale cereale</Emphasis> L.)

This study was conducted in order to identify quantitative trait loci (QTLs) for the in vitro culture response of winter rye (Secale cereale L.) immature embryos and immature inflorescences. A genetic linkage map comprising 67 SSRs, 9 ISSRs, 13 SAMPLs, 7 RAPDs, 2 SCARs and one EST marker was created based on the analyses of 102 recombinant inbred lines from the cross between lines L318 (which has a good response in tissue cultures) and L9 (which is unable to regenerate plants from somatic tissues and anthers). The map spans 979.2 cM, and the average distance between markers is 9.9 cM. Two characteristics were evaluated: callus induction (CI) and somatic embryogenesis ability (SE). They were expressed as the percentage of immature embryos/inflorescences producing callus (designated ECI/ICI) and the percentage of explants producing somatic embryos (ESE/ISE). All the analysed traits showed continuous variation in the mapping population but a non-normal frequency distribution. We identified nine putative QTLs controlling the tissue culture response of rye, explaining up to 41.6% of the total phenotypic variation: two QTLs for ECI — eci-1, eci-2; 4 for ESE — ece-1, ese-2, ese-3, ese-4; 2 for ICI — ici-1, ici2; and 1 for ISE — ise-1. They were detected on chromosomes 1R, 4R, 5R, 6R and 7R.     

Mapping of isolate-specific QTLs for clubroot resistance in Chinese cabbage (<Emphasis Type="Italic">Brassica rapa</Emphasis> L. ssp. <Emphasis Type="Italic">pekinensis</Emphasis>)

A number of clubroot resistant (CR) Chinese cabbage cultivars have been developed in Japan using resistant genes from CR European fodder turnips (B. rapa ssp. rapifera). Clubroot resistance in European fodder turnips are known to be controlled by the combined action of several dominant resistance genes. We have developed three Chinese cabbage clubroot-resistant doubled haploid (DH) lines-T136-8, K10, and C9-which express resistance in different manners against two isolates of Plasmodiophora brassicae, M85 and K04. Depending on the isolates, we identified two CR loci, CRk and CRc. CRk was identified by quantitative trait loci (QTL) analysis of an F(2) population derived from a cross between K10 and Q5. This locus showed resistance to both isolates and is located close to Crr3 in linkage group R3. The other locus, CRc was identified by QTL analysis of an F(2) population derived from a cross between C9 and susceptible DH line, 6R. This locus was mapped to linkage group R2 and is independent from any published CR loci. We developed sequence-tagged site markers linked to this locus.     

Factors affecting <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation in <Emphasis Type="Italic">Hybanthus</Emphasis><Emphasis Type="Italic">enneaspermus</Emphasis> (L.) F. Muell.

Agrobacterium tumefaciens-mediated transformation system was established for Hybanthus enneaspermus using leaf explants with the strain LBA4404 harbouring pCAMBIA 2301 carrying the nptII and gusA genes. Sensitivity of leaf explants to kanamycin was standardized (100 mg/l) for screening the transgenic plants. Transformation parameters (OD, virulence inducer, infection time, co-cultivation period, bactericidal antibiotics, etc.) influencing the gene transfer and integration were assessed in the present investigation. Fourteen-day pre-cultured explants were subjected with Agrobacterium strain LBA4404. Optimized parameters such as culture density of 0.5 OD₆₀₀, infection time of 6 min, AS concentration of 150 µM with 3 days co-cultivation revealed maximum transformation efficiency based on GUS expression assay. The presence of gusA in transgenics was confirmed by polymerase chain reaction and Southern blotting analysis. The present transformation experiment yielded 20 shoots/explant with higher transformation efficiency (28 %). The protocol could be used to introduce genes for trait improvement as well as for altering metabolic pathway for secondary metabolites production.     

<Emphasis Type="Italic">Agrobacterium-</Emphasis>mediated transformation of gypsophila (<Emphasis Type="Italic">Gypsophila paniculata</Emphasis> L.)

As a major contributor to the flower market, Gypsophila paniculata is an important target for the breeding of new varieties. However, gypsophila breeding is strongly hampered by the sterility of this species’ genotypes and the lack of a genetic-transformation procedure for this genus. Here we describe the establishment of a transformation procedure for gypsophila (Gypsophila paniculata L.) based on Agrobacterium inoculation of highly regenerative stem segments. The transformation procedure employs stem explants derived from GA₃-pretreated mother plants and a two-step selection scheme. The GA₃ treatment was crucial for obtaining high gene-transfer frequencies (75–90% GUS-expressing explants out of total inoculated explants), as shown using three different gypsophila varieties. An overall transformation efficiency of five GUS-expressing shoots per 100 stem explants was demonstrated for cv. Arbel. The applicability of the transformation system to gypsophila was further reinforced by the generation of transgenic plants expressing Agrobacterium rhizogenes rolC driven by a CaMV 35S promoter. Transgenic gypsophila plantlets exhibited extensive rooting and branching, traits that could be beneficial to the ornamental industry.     

Molecular mapping and location of QTLs for drought-resistance traits in <Emphasis Type="Italic">indica</Emphasis> rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) lines adapted to target environments

Drought is a major limitation for rice production in rainfed ecosystems. Identifying quantitative trait loci (QTLs) linked to drought resistance provides opportunity to breed high yielding rice varieties suitable for drought-prone areas. Although considerable efforts were made in mapping QTLs associated with drought-resistance traits in rice, most of the studies involved indica × japonica crosses and hence, the drought-resistance alleles were contributed mostly by japonica ecotypes. It is desirable to look for genetic variation within indica ecotypes adapted to target environment (TE) as the alleles from japonica ecotype may not be expressed under lowland conditions. A subset of 250 recombinant inbred lines (RILs) of F₈ generation derived from two indica rice lines (IR20 and Nootripathu) with contrasting drought-resistance traits were used to map the QTLs for morpho-physiological and plant production traits under drought stress in the field in TE. A genetic linkage map was constructed using 101 polymorphic PCR-based markers distributed over the 12 chromosomes covering a total length of 1,529 cM in 17 linkage groups with an average distance of 15.1 cM. Composite interval mapping analysis identified 22 QTLs, which individually explained 4.8–32.2% of the phenotypic variation. Consistent QTLs for drought-resistance traits were detected using locally adapted indica ecotypes, which may be useful for rainfed rice improvement.     

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.     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

<Emphasis Type="Italic">Organogenic callus</Emphasis> as the target for plant regeneration and transformation via <Emphasis Type="Italic">Agrobacterium</Emphasis> in soybean (<Emphasis Type="Italic">Glycine max</Emphasis> (L.) Merr.)

A regeneration and transformation system has been developed using organogenic calluses derived from soybean axillary nodes as the starting explants. Leaf-node or cotyledonary-node explants were prepared from 7 to 8-d-old seedlings. Callus was induced on medium containing either Murashige and Skoog (MS) salts or modified Finer and Nagasawa (FNL) salts and B5 vitamins with various concentrations of benzylamino purine (BA) and thidiazuron (TDZ). The combination of BA and TDZ had a synergistic effect on callus induction. Shoot differentiation from the callus occurred once the callus was transferred to medium containing a low concentration of BA. Subsequently, shoots were elongated on medium containing indole-3-acetic acid (IAA), zeatin riboside, and gibberellic acid (GA). Plant regeneration from callus occurred 90 ∼ 120 d after the callus was cultured on shoot induction medium. Both the primary callus and the proliferated callus were used as explants for Agrobacterium-mediated transformation. The calluses were inoculated with A. tumefaciens harboring a binary vector with the bar gene as the selectable marker gene and the gusINT gene for GUS expression. Usually 60–100% of the callus showed transient GUS expression 5 d after inoculation. Infected calluses were then selected on media amended with various concentrations of glufosinate. Transgenic soybean plants have been regenerated and established in the greenhouse. GUS expression was exhibited in various tissues and plant organs, including leaf, stem, and roots. Southern and T₁ plant segregation analysis of transgenic events showed that transgenes were integrated into the soybean genome with a copy number ranging from 1–5 copies.     

<Emphasis Type="Italic">S</Emphasis>-Allele characterization in self-incompatible pear (<Emphasis Type="Italic">Pyrus communis</Emphasis> L.)

Gametophytic self-incompatibility, a natural mechanism occurring in pear and other fruit-tree species, is usually controlled by the S-locus with allelic variants ( S1, S2, Sn). Recently, biochemical and molecular tools have determined the S-genotype of cultivars in various species. The present study determined the S-locus composition of ten European pear cultivars via S-PCR molecular assay, thereby obviating time-consuming fieldwork whose results are often ambiguous because of environmental effects. To verify the S-PCR assay, two putative S-allele DNA fragments of Japanese pear were isolated; their sequences proved to be identical to those reported in the databank. Six S-allele fragments of European pear were then sequenced. While field data confirmed the molecular results, fully and half-compatible field crosses were not distinguishable.     

Ancient DNA analysis of <Emphasis Type="Italic">Panicum miliaceum</Emphasis> (broomcorn millet) from a Bronze Age cemetery in Xinjiang,China

Panicum miliaceum L. (broomcorn millet) was an important cereal for Eurasian populations in prehistory. It was first domesticated in China and spread westwards into Central Asia and Europe later on. However, the domestication history and dispersal routes of this cereal are still not clear. The well desiccated P. miliaceum grains recovered from Xiaohe cemetery, a Bronze Age cemetery in Xinjiang, China, provide us with an invaluable opportunity to further explore this issue, especially considering Xinjiang’s critical geographical position at the crossroads of contact between the West and East. Here we present an analysis of ribosomal DNA (rDNA) internal transcribed spacer (ITS) and external transcribed spacer (ETS) sequences of P. miliaceum remains excavated from Xiaohe cemetery in comparison to modern landraces. The results show that the Xiaohe P. miliaceum exhibits heterogeneous characteristics in its rDNA, which are also commonly found in modern landraces. However, no exact sequence matching the Xiaohe millet was found in modern landraces so far, possibly suggesting the loss of genetic diversity through time. The most similar sequences are mainly present in landraces from India and Europe. Based on the genetic data of modern landraces and archaeological findings, we discuss the domestication of P. miliaceum and the possible prehistoric connections between northern China, Xinjiang and Europe in agricultural practices, as well as the potential routes westwards of P. miliaceum.     

Study of genetic variation in sesame (<Emphasis Type="Italic">Sesamum indicum</Emphasis> L.) using agro-morphological traits and <Emphasis Type="Italic">ISSR</Emphasis> markers

This research was conducted to study the genetic variation among eighteen genotypes of sesame (Sesamum indicum L.) collected from various agro-climatic regions of Iran along with six exotic genotypes from the Asian countries using both agro-morphological and ISSR marker traits. The results showed significant differences among genotypes for all agro-morphological traits and a relatively high genetic coefficient of variation observed for number of fruiting branches per plant, capsules per plant, plant height and seed yield per plant. Cluster analysis based on these traits grouped the genotypes into five separate clusters. Larger interthan intra cluster distances implies the presence of higher genetic variability between the genotypes of different groups. Genotypes of two clusters with a good amount of genetic divergence and desirable agronomic traits were detected as promising genotypes for hybridization programs. The 13 ISSR primers chosen for molecular analysis revealed 170 bands, of which 130 (76.47%) were polymorphic. The generated dendrogram based on ISSR profiles divided the genotypes into seven groups. A principal coordinate analysis confirmed the results of clustering. The agro-morphological traits and ISSR markers reflected different aspects of genetic variation among the genotypes as revealed by a non significant cophenetic correlation in the Mantel test. Therefore the complementary application of both types of information is recommended to maximize the efficiency of sesame breeding programs. The discordance among diversity patterns and geographical distribution of genotypes found in this investigation implies that the parental lines for hybridization should be selected based on genetic diversity rather than the geographical distribution.     

Genetics and fine mapping of a yellow-green leaf gene (<Emphasis Type="Italic">ygl</Emphasis>-<Emphasis Type="Italic">1</Emphasis>) in cabbage (<Emphasis Type="Italic">Brassica oleracea</Emphasis> var. <Emphasis Type="Italic">capitata</Emphasis> L.)

Cabbage (Brassica oleracea var. capitata L.) is one of the most popular cultivated vegetables worldwide. Cabbage has rich phenotypic diversity, including plant height, head shape, head color, leaf shape and leaf color. Leaf color plays an important role in cabbage growth and development. At present, there are few reports on fine mapping of leaf color mutants in B. oleracea. In this study, a naturally occurring yellow-green leaf cabbage mutant (YL-1), derived from the self-pollinated progenies of the hybrid ‘Hosom’, was used for inheritance analysis and gene mapping. Segregation populations including F₂ and BC₁ were generated from the cross of two inbred lines, YL-1 and 01–20. Genetic analysis with the F₂ and BC₁ populations demonstrated that the yellow-green leaf color was controlled by a single recessive nuclear gene, ygl-1. Insertion–deletion (InDel) markers, designed based on the parental re-sequencing data, were used for the preliminary mapping with BSA (bulked segregant analysis) method. A genetic map constructed with 15 InDels indicated that ygl-1 was located on chromosome C01. The ygl-1 gene is flanked by InDel markers ID2 and M8, with genetic distances of 0.4 cM and 0.35 cM, respectively. The interval distance between two markers is 167 kb. Thus, it enables us to locate the ygl-1 gene for the first time in B. oleracea. This study lays the foundation for candidate gene prediction and ygl-1gene cloning.     

QTLs for resistance to preharvest sprouting in rye (<Emphasis Type="Italic">Secale cereale</Emphasis> L.)

Grain quality of rye is often negatively affected by sprouting - a complex trait with a poorly understood genetic background and strong interaction with weather conditions. The aim of this report was to detect the main quantitative trait loci (QTLs) underlying preharvest sprouting resistance in rye, measured as a percentage of sprouted kernels after spraying spikes with water for 7 days. Simple and composite interval mapping, carried out in 3 environments on 94 F3 and F4 families of the cross between sprouting-susceptible (541) and sprouting-resistant (Ot1-3) inbred lines, revealed 5 QTLs located on chromosome arms 1RL, 2RL, 5RL, 6RL and 7RL. The significance of these QTLs was additionally proved by disruptive selection carried out on 5000 F2 plants of the 541 x Ot1-3 cross and continued to the F5 generation of recombinant inbred lines (RIL), which strongly affected allele frequencies at linked marker loci. Resistance to preharvest sprouting showed dominant inheritance except for QPhs.uas-7R.1 (recessive) and QPhs.uas-1R.1 (additive). Results of the present study suggest that introgression of 4-5 QTLs, identified in line Ot1-3, should substantially reduce sprouting risk in rye varieties.     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation of blueberry (<Emphasis Type="Italic">Vaccinium corymbosum</Emphasis> L.)

Transient expression studies using blueberry leaf explants and monitored by -glucuronidase (GUS) assays indicated Agrobacterium tumefaciens strain EHA105 was more effective than LBA4404 or GV3101; and the use of acetosyringone (AS) at 100 M for inoculation and 6 days co-cultivation was optimum compared to 2, 4, 8, 10 or 12 days. Subsequently, explants of the cultivars Aurora, Bluecrop, Brigitta, and Legacy were inoculated with strain EHA105 containing the binary vector pBISN1 with the neomycin phosphotransferase gene (nptII) and an intron-interrupted GUS gene directed by the chimeric super promoter (Aocs)₃AmasPmas. Co-cultivation was for 6 days on modified woody plant medium (WPM) plus 100 M AS. Explants were then placed on modified WPM supplemented with 1.0 mg l^–1 thidiazuron, 0.5 mg l^–1 -naphthaleneacetic, 10 mg l^–1 kanamycin (Km), and 250 mg l^–1 cefotaxime. Selection for Km-resistant shoots was carried out in the dark for 2 weeks followed by culture in the light at 30 E m^–2 s^–1 at 25°C. After 12 weeks, selected shoots that were both Km resistant and GUS positive were obtained from 15.3% of the inoculated leaf explants of cultivar Aurora. Sixty-eight independent clones derived from such shoots all tested positive by the polymerase chain reaction using a nptII primer. Eight of eight among these 68 clones tested positive by Southern hybridization using a gusA gene derived probe. The transformation protocol also yielded Km-resistant, GUS-positive shoots that were also PCR positive at frequencies of 5.0% for Bluecrop, 10.0% for Brigitta and 5.6% for Legacy.     

Mapping of the Ogura fertility restorer gene <Emphasis Type="Italic">Rfo</Emphasis> and development of <Emphasis Type="Italic">Rfo</Emphasis> allele-specific markers in canola (<Emphasis Type="Italic">Brassica napus</Emphasis> L.)

Ogura cytoplasmic male sterility (CMS) and its corresponding nuclear fertility restorer gene, Rfo, have been introduced from radish to Brassica species by interspecific crosses. Rfo restores male fertility by altering the translational expression of Orf138, a mitochondrial gene, whose expression results in the male sterile phenotype. This system has been extensively investigated and breeding restorer lines for the Ogura CMS has become a major objective for hybrid seed production in many canola breeding programs. In this study, we have sequenced genomic clones of Rfo amplified from a canola restorer line R2000, licensed from INRA, France, and a Dow AgroSciences non-restorer line Nexera 705 using primers designed from the radish Rfo sequence (GenBank accession AJ550021). Sequence alignment revealed three homologous sequences of Rfo. Two of the sequences were present in both R2000 and Nexera 705 but the third one was present only in R2000. These results suggested that the first two sequences could be the homoeologous sequences of Rfo already existing in the canola genome and the third one could be the radish Rfo introduced into canola. Based on the sequence differences between the restorer and non-restorer lines, Rfo allele-specific PCR markers were developed. We also developed a high throughput, Rfo allele-specific Invader^® assay through Third Wave Technologies. Linkage analysis revealed a co-segregation between the allele-specific marker and the phenotypes for fertility restoration. This allele-specific marker has been mapped in the linkage group N19 and proved to be very useful for direct selection of Rfo alleles for fertility restoration during marker-assisted introgression of the Ogura restorer for hybrid development in canola.