A novel multiplex quantitative DNA array based PCR (MQDA-PCR) for quantification of transgenic maize in food and feed

We have developed a novel multiplex quantitative DNA array based PCR method (MQDA-PCR). The MQDA-PCR is general and may be used in all areas of biological science where simultaneous quantification of multiple gene targets is desired. We used quantification of transgenic maize in food and feed as a model system to show the applicability of the method. The method is based on a two-step PCR. In the first few cycles bipartite primers containing a universal 5′ ‘HEAD’ region and a 3′ region specific to each genetically modified (GM) construct are employed. The unused primers are then degraded with a single-strand DNA-specific exonuclease. The second step of the PCR is run containing only primers consisting of the universal HEAD region. The removal of the primers is essential to create a competitive, and thus quantitative PCR. Oligo nucleotides hybridising to internal segments of the PCR products are then sequence specifically labelled in a cyclic linear signal amplification reaction. This is done both to increase the sensitivity and the specificity of the assay. Hybridisation of the labelled oligonucleotides to their complementary sequences in a DNA array enables multiplex detection. Quantitative information was obtained in the range 0.1–2% for the different GM constructs tested. Seventeen different food and feed samples were screened using a twelve-plex system for simultaneous detection of seven different GM maize events (Bt176, Bt11, Mon810, T25, GA21, CBH351 and DBT418). Ten samples were GM positive containing mainly mixtures of Mon810, Bt11 and Bt176 DNA. One sample contained appreciable amounts of GA21. An eight-plex MQDA-PCR system for detection of Mon810, Bt11 and Bt176 was evaluated by comparison with simplex 5′ nuclease PCRs. There were no significant differences in the quantifications using the two approaches. The samples could, by both methods, be quantified as containing >2%, between 1 and 2%, between 0.1 and 1%, or <0.1% in 43 out of 47 determinations. The described method is modular, and thus suited for future needs in GM detection.     

Detection of transgenes in three genetically modified rice lines by fluorescence in situ hybridization

Fluorescence in situ hybridization (FISH) using T-DNA probes was applied to localize transgenes onto specific chromosomes and confirm the steady integration of transferred genes in three genetically modified (GM) rice lines, LS28 (event LS30-32-20-1), Cry1Ac1 (event C7-1-9-1) and LS28×Cry1Ac1 (event L/C1-1-3-1), which are a rice leaf blast-resistant single trait GM line, a leaf folder-resistant single trait GM line, and a rice leaf blast-resistant and leaf folder-resistant stacked GM hybrid line, respectively. The FISH signals were clearly detected on the arms of one homologous chromosome pair for LS28, and on the arms of another chromosome pair for Cry1Ac1 when using the transformation vector pSBM AtCK containing the rice leaf blast-resistant gene (LS28) and pMJ-RTB containing the leaf folder-resistant gene (mCry1Ac1) as a probe, respectively. As expected, we detected two pairs of FISH signals, each on the arms of different chromosome pairs in the stacked GM rice line LS28×Cry1Ac1 when using both pSBM AtCK and pMJ-RTB as probes. These results indicate that the transgenes are located at specific homologous loci and show position stability among generations in both single trait and stacked GM rice lines. The usefulness and the necessity of FISH to detect inserted genes in transformed plants will be discussed for the purpose of future studies to develop breeding programs and conduct risk assessment of GM plants.     

Mortality impact of Bt transgenic maize roots expressing eCry3.1Ab, mCry3A, and eCry3.1Ab plus mCry3A on western corn rootworm larvae in the field

Mortality of the western corn rootworm, Diabrotica virgifera virgifera LeConte, larvae due to feeding on maize, Zea mays L., expressing Bacillus thuringiensis Berliner (Bt) was evaluated in five Missouri sites in 2007, 2008, and 2009. Specifically, eCry3.1Ab (5307), mCry3A (MIR604), and eCry3.1Ab plus mCry3A proteins relative to survivorship on maize with the same genetic background without these genes (isoline maize) was evaluated. An average of 890.8 +/- 152.3 beetles emerged from isoline plots, whereas average beetle emergence from 5307, MIR604, and 5307 x MIR604 was 1.9 +/- 0.6, 19.3 +/- 6.3, and 0.8 +/- 0.3, respectively, when averaged across 22 replications in five environments. Overall, 66, 50, 61, and 51% of beetles recovered from 5307, MIR604, 5307 x MIR604, and isoline maize, respectively, were female, and there was no significant difference between the number of male and female beetles that emerged from any of these treatments. Mortality due to 5307, MIR604, and 5307 x MIR604 was 99.79, 97.83, and 99.91%, respectively. There was an 8.0-d delay in time to 50% beetle emergence from 5307 compared with isoline maize, which was significantly later than to the other three maize lines. The average delay to 50% emergence from MIR604 and 5307 x MIR604 averaged 4.1 and 4.6 d, respectively later than 50% emergence from isoline maize. Female beetles had a significant delay in time to 50% emergence compared with male beetles from all treatments with the exception of 5307 x MIR604. Data are discussed in terms of insect resistance management in relation to other control measures for western corn rootworm.     

MDA技术在转基因检测中的研究与应用

PCR技术是转基因植物及其产品检测的主要方法,但是因为其对模板纯度和质量有较高要求,对于低质量模板难以获得稳定的检测结果。本研究利用MDA技术可将低至10 copies的微量DNA样品扩增至可用PCR方法稳定检出;进一步利用MDA技术对转基因玉米有证标准物质Bt11粉末的单颗粒痕量样品释出DNA进行扩增,结合PCR扩增检测,玉米粉末颗粒中内源参照基因的检出率达到70%以上,转基因特异性序列检出与内源参照基因检出的比值,与标准物质标称值基本相符,为建立基于MDA技术转基因植物及其产品粉末颗粒等痕量样品检测方法奠定了基础,同时也为加工产品中复合性状转基因作物的检测提供了潜在的解决方案。     

商业化种植的六种基因改良玉米品系的鉴定检测方法

以PCR方法鉴定检测六种商业化种植的基因改良玉米 (geneticallymodifiedmaize,简称GM 玉米 )。针对Mon810 (Monsanto公司 )、Bt11(Novartis公司 )、Event176 (Novartis公司 )、CBH 35 1(AgrEvo公司 )、T14/T2 5Liberty (AgrEvo公司 )及GA2 1(Monsanto公司 )GM 玉米转入的外源基因质粒图谱 ,设计具有品系特异性的引物进行PCR检测 ,建立了GM 玉米品系鉴定检测的方法。     

The examinations of grain yield and yield components in new white maize varieties using line × tester analysis method

Line × tester set was obtained by crossing 10 inbred lines with 2 testers in maize. 20 F1`s along with 12 parents (10 S6 lines and 2 testers) and two standard white checks (TWC321 and TWC324) were evaluated randomized complete block Design (RCBD) with three replications during summer season 2016 to detect the best new white crosses for the potential comercial crosses. Data were recorded on cob length, ear diameter row no. per ear, no. of kernels per row, 100-kernel weight and grain yield. The results of mean squares showed highly significant differences in the all examined traits. For mean grain yield of the crosses P5 × SC24, P6 × SC21, P6 × SC24, P7 × SC21, P7 × SC24 and P10 × SC21 had significant in all traits and their combined data was higher in case of TWC321 and TWC324.Hence, it could be concluded that these crosses may be useful for improving grain yield of maize. Crosses P1 × SC21, P1 × SC24, P3 × SC24, P5 × SC24, P6 × SC21, P6 × SC24, P7 × SC21 and P7 × SC24 had highest significant and positively higher percentages over check varieties TWC321 and TWC324 for grain yield and some of yield components. These lines were having promising performance which could be used in future maize breeding programs for hybrid production. Based on the overall performance of the hybrids and parental lines, some of the lines could be used as parents of hybrids of maize with high grain yield potential. Thus, these crosses could be commercially exploited after critical evaluation for its superiority in performance as maize hybrids.     

Excretion into feces of asialo GM1 in the murine digestive tract and <Emphasis Type="Italic">Lactobacillus johnsonii</Emphasis> exhibiting binding ability toward asialo GM1. A possible role of epithelial glycolipids in the discharge of intestinal bacteria

In the digestive tract of mice (HR-1, 5 months old, ♀), asialo GM1 (GA1) exhibiting receptor activity toward several intestinal bacteria was preferentially expressed in the small intestine. Also, less than 10% of GA1 in the small intestine was converted into fucosylated and sulfated derivatives, but it was completely converted to fucosyl GA1 (FGA1) in the stomach, cecum and colon. Among the lipid components in these tissues, glycolipids other than Forssman antigen and cholesterol sulfate (CS) were present in the digestive tract contents. However, sulfated GA1, sulfatide and fucosyl GM1 in the gastro-intestinal contents were not present in the cecal and colonic contents, in which the major glycolipids were ceramide monohexoside (CMH), GA1 and FGA1. The total amount of GA1 in the whole contents was 20% of that in the tissues. Thus, glycolipids were stable during the process of digestion, and excreted from the body together with cholesterol and CS. On the other hand, Lactobacillus johnsonii (LJ), whose receptor is GA1, was detected in the cecal and colonic contents on sequential analysis of 16S-ribosomal RNA and TLC-immunostaining of antigenic glycolipids with anti-LJ antiserum. LJ was found to comprise 20% of the total bacteria cultured in the lactobacillus medium under aerobic conditions, and to be present in the cecal and colonic contents, 9.8 × 10⁷ cells versus 37 μg GA1 and 1.4 × 10⁸ cells versus 49 μg GA1, respectively. Thus, GA1 in the contents might facilitate the discharge of intestinal bacteria by becoming attached them to prevent their irregular diffusion.     

Impact of genetic structures on haploid genome-based quantification of genetically modified DNA: theoretical considerations, experimental data in MON 810 maize kernels (Zea mays L.) and some practical applications

Real-time Polymerase Chain Reaction (PCR) based assays are widely used to estimate the content of genetically modified (GM) materials in food, feed and seed. It has been known that the genetic structures of the analyte can significantly influence the GM content expressed by the haploid genome (HG) % estimated using real-time PCR assays; this kind of influence is also understood as the impact of biological factors. The influence was first simulated at theoretical level using maize as a model. We then experimentally assessed the impact of biological factors on quantitative results, analysing by quantitative real-time PCR six maize MON 810 hybrid kernels with different genetic structures: (1) hemizygous from transgenic male parent, (2) hemizygous from transgenic female parent and (3) homozygous at the transgenic locus. The results obtained in the present study showed clear influences of biological factors on GM DNA quantification: 1% of GM materials by weight (wt) for the three genetic structures contained 0.39, 0.55 and 1.0% of GM DNA by HG respectively, from quantitative real-time PCR analyses. The relationships between GM wt% and GM HG% can be empirically established as: (1) in the case of the presence of a single GM trait: GM HG% = GM wt% × (0.5 ± 0.167Y), where Y is the endosperm DNA content (%) in the total DNA of a maize kernel, (2) in the case of the presence of multiple GM traits: GM HG% = N × GM wt% × (0.5 ± 0.167Y), where N is the number of GM traits (stacked or not) present in an unknown sample. This finding can be used by stakeholders related to GMO for empirical prediction from one unit of expression to another in the monitoring of seed and grain production chains. Practical equations have also been suggested for haploid copy number calculations, using hemizygous GM materials for calibration curves.     

Stacking transgenic event DAS‐Ø15Ø7‐1 alters maize composition less than traditional breeding

The impact of crossing (‘stacking’) genetically modified (GM) events on maize‐grain biochemical composition was compared with the impact of generating nonGM hybrids. The compositional similarity of seven GM stacks containing event DAS‐Ø15Ø7‐1, and their matched nonGM near‐isogenic hybrids (iso‐hybrids) was compared with the compositional similarity of concurrently grown nonGM hybrids and these same iso‐hybrids. Scatter plots were used to visualize comparisons among hybrids and a coefficient of identity (per cent of variation explained by line of identity) was calculated to quantify the relationships within analyte profiles. The composition of GM breeding stacks was more similar to the composition of iso‐hybrids than was the composition of nonGM hybrids. NonGM breeding more strongly influenced crop composition than did transgenesis or stacking of GM events. These findings call into question the value of uniquely requiring composition studies for GM crops, especially for breeding stacks composed of GM events previously found to be compositionally normal.     

Effect of deep stacking and ensiling broiler litter on chemical composition and pathogenic organisms

The feasibility of processing broiler litter by deep stacking and ensiling was evaluated prior to its use as feed ingredient. Fresh broiler litter was collected from the peri-urban area of Islamabad, Pakistan. Broiler litter was deep stacked at 15%, 25% and 35% moisture; ensiled at 40% moisture, alone or with 5% added sugarcane molasses; and ensiled with rumen contents at 60:40 and 50:50, wet basis. Deep stacking was done in 1.2×1.2×1.2 m bins and ensiling was done in 210 l metal drums double lined with polyethylene. Broiler litter deep stacked at 15% moisture showed a lower rise in temperature than litter deep stacked at 25% and 35% moisture. Maximum temperature was recorded at 40 cm depth for litter stacked with 25% moisture. Overall, deep stacking had no effect on the chemical composition of broiler litter. Deep stacked litter was devoid of lactic acid, but the processing was effective in the destruction of pathogens. Desirable fermentation was achieved in all the silages, with significant reductions (P<0.05) in pH and water-soluble carbohydrates, and increase (P<0.05) in lactic acid. The highest pH and lowest lactic acid concentration was recorded for silages containing broiler litter and rumen contents, 60:40, wet basis. No pathogenic microbes were observed in the ensiled mixtures.     

Relevance of Bt toxin interaction studies for environmental risk assessment of genetically modified crops

In recent years, different Bacillus thuringiensis (Bt) toxin‐encoding genes have been combined or ‘stacked’ in genetically modified (GM) crops. Synergism between Bt proteins may occur and thereby increase the impact of the stacked GM event on nontarget invertebrates compared to plants expressing a single Bt gene. On the basis of bioassay data available for Bt toxins alone or in combination, we argue that the current knowledge of Bt protein interactions is of limited relevance in environmental risk assessment (ERA).     

Transportability of non‐target arthropod field data for the use in environmental risk assessment of genetically modified maize in Northern Mexico

In country, non‐target arthropod (NTA) field evaluations are required to comply with the regulatory process for cultivation of genetically modified (GM) maize in Mexico. Two sets of field trials, Experimental Phase and Pilot Phase, were conducted to identify any potential harm of insect‐protected and glyphosate‐tolerant maize (MON‐89Ø34‐3 × MON‐88Ø17‐3 and MON‐89Ø34‐3 × MON‐ØØ6Ø3‐6) and glyphosate‐tolerant maize (MON‐ØØ6Ø3‐6) to local NTAs compared to conventional maize. NTA abundance data were collected at 32 sites, providing high geographic and environmental diversity within maize production areas from four ecological regions (ecoregions) in northern Mexico. The most abundant herbivorous taxa collected included field crickets, corn flea beetles, rootworm beetles, cornsilk flies, aphids, leafhoppers, plant bugs and thrips while the most abundant beneficial taxa captured were soil mites, spiders, predatory ground beetles, rove beetles, springtails (Collembola), predatory earwigs, ladybird beetles, syrphid flies, tachinid flies, minute pirate bugs, parasitic wasps and lacewings. Across the taxa analysed, no statistically significant differences in abundance were detected between GM maize and the conventional maize control for 69 of the 74 comparisons (93.2%) indicating that the single or stacked insect‐protected and herbicide‐tolerant GM traits generally exert no marked adverse effects on the arthropod populations compared with conventional maize. The distribution of taxa observed in this study provides evidence that irrespective of variations in overall biodiversity of a given ecoregion, important herbivore, predatory and parasitic arthropod taxa within the commercial maize agroecosystem are highly similar indicating that relevant data generated in one ecoregion can be transportable for the risk assessment of the same or similar GM crop in another ecoregion.     

Identification of sources of resistance to common bacterial blight in common bean in Ethiopia

Common bacterial blight (CBB) caused by Xanthomonas axonopodis pv. phaseoli and X. axonopodis pv. phaseoli var. fuscans is one of the major biotic constraints limiting common bean (Phaseolus vulgaris L.) production and productivity in Ethiopia. The objective of this study was to identify new sources of CBB resistance from a diverse panel of genotypes to develop CBB-resistant common bean varieties. One hundred and ten diverse accessions were evaluated for CBB resistance at three hotspot sites (Melkassa, Arsi Negelle and Mieso) for two seasons (2017 and 2018) in Ethiopia. Data on mean disease severity on leaf (SL) and mean disease severity on pod (SP), the area under disease progress curve (AUDPC), number of pods per plant (PP), number of seeds per pod (SPP) and grain yield (GY) were collected. Data were subjected to standard analysis of variance and principal component analysis. The genotype × site interaction (G × E) had significant (p < .05) effect on all assessed traits. This indicated the presence of marked variation among tested genotypes in CBB resistance across the testing sites. Genotypes including SEC21, SEC23, SMC21, VAX6, SEC12, SEC25, SMC22, VAX5, SEC20, SEC22, SEC24, SEC26, SMC16 SMC24, VAX6, SEC25, SEC21, SEC23 and SMC21 exhibited lower values of SL, SP and AUDPC which are useful genetic resources for future CBB resistance breeding programmes. Nasir provided a grain yield of 3.45 ton/ha followed by VAX1 (2.86 ton/ha) and Hawassa Dume (2.83 ton/ha). Further, CBB-resistant and high yielding genotypes had the higher PPP and SPP making them ideal candidates for common bean breeding in Ethiopia or similar agro-ecologies emphasizing CBB resistance and enhanced agronomic traits.     

Five new species of Eimeria Schneider, 1875 from the endangered Tibetan antelope Pantholops hodgsonii (Abel) (Artiodactyla: Bovidae: Caprinae) in the Hoh Xil Nature Reserve Area of Qinghai Province,China

We examined faeces of 76 endangered Tibetan antelopes Pantholops hodgsonii (Abel) in May 2017, from the Hoh Xil Nature Reserve, Qinghai Province, China, and found 62/76 (82%) discharging oöcysts representing five new species of Eimeria Schneider, 1875. Oöcysts of Eimeria pantholopensis n. sp., found in 54/76 (71%) chiru, are subspheroidal/ellipsoidal, 15–22 × 12–19 (18.6 × 16.1) µm, with a length/width (L/W) ratio of 1.0–1.3 (1.2); micropyle cap and 1–3 polar granules are present, but oöcyst residuum is absent. Sporocysts are ovoidal, 7–11 × 4–6 (9.2 × 5.3) µm, with a L/W ratio of 1.6–2.0 (1.7); Stieda body and sporocyst residuum of small, scattered granules are present; each sporozoite contains 2 refractile bodies. Oöcysts of Eimeria wudaoliangensis n. sp. found in 52/76 (68%) chiru, are pyriform, 21–29 × 17–21 (24.9 × 19.0) µm, with a L/W ratio of 1.1–1.5 (1.3); micropyle, micropyle cap and 1–4 polar granules are present, but oöcyst residuum is absent. Sporocysts are ovoidal, 9–13 × 5–8 (11.7 × 6.7) µm, with a L/W ratio of 1.4–2.7 (1.7); Stieda body and sporocyst residuum of disbursed granules are present; sporozoites have a single large refractile body. Oöcysts of Eimeria hodgsonii n. sp. found in 20/76 (26%) chiru, are elongate-ellipsoidal, 25–32 × 18–21 (28.9 × 19.8) µm, with a L/W ratio of 1.2–1.7 (1.5); micropyle, micropyle cap and 1–3 polar granules are present, but oöcyst residuum is absent. Sporocysts are ovoidal, 11–14 × 6–7 (12.3 × 6.8) µm, with a L/W ratio of 1.7–2.1 (1.8); Stieda body and sporocyst residuum as group of large granules lying along the interface between intertwined sporozoites are present; sporozoites have 2 refractile bodies. Oöcysts of Eimeria schalleri n. sp. found in 49/76 (64.5%) chiru, are ellipsoidal, 26–36 × 19–25 (30.4 × 23.2) µm, with a L/W ratio of 1.2–1.5 (1.3); micropyle with micropyle cap and polar granules appearing as many diffuse tiny bodies are present, but oöcyst residuum is absent. Sporocysts are ovoidal, 12–16 × 7–9 (14.2 × 7.8) µm, with a L/W ratio of 1.6–2.1 (1.8); Stieda body and sporocyst residuum are present, the latter as a group of small dispersed granules between intertwined sporozoites; sporozoites with 2 refractile bodies. Oöcysts of Eimeria sui n. sp. found in 4/76 (5%) chiru, are ovoidal, 32–38 × 26–30 (36.6 × 28.6) µm, with a L/W ratio of 1.0–1.4 (1.3); micropyle and micropyle cap and 1–3 polar granules are present, but oöcyst residuum is absent. Sporocysts are ovoidal, 15–18 × 8–10 (16.7 × 8.9) µm, with a L/W ratio of 1.7–2.1 (1.9); Stieda body and sporocyst residuum are present, the latter as a group of dispersed small granules; sporozoites with 2 refractile bodies. Five of 62 faecal samples in which oöcysts were detected (8%) had a single species infection, 13 of 62 (21%) had two species, 28 of 62 (45%) had three species and 16 of 62 (26%) had four species.

     

Survival of kansas dipel-resistant European corn borer (Lepidoptera: Crambidae) on Bt and non-Bt corn hybrids

The Kansas Dipel-resistant and susceptible European corn borer, Ostrinia nubilalis (Hübner), were evaluated in the greenhouse on different Bt transgenic events expressed in corn hybrids. There were important differences in the resistance offered by the different Bt event corn hybrids. Hybrid comparison tests indicate that these Dipel-resistant first-instar European corn borer were not able to survive to adulthood on whorl-stage MON810, Bt11, or 176 Bt event corn plants. Third instars did not survive to adulthood on whorl-stage MON810 or Bt11 event corn plants but a small number of fifth instars were found on whorl-stage DBT418 plants infested with Dipel-resistant larvae. First and third instars of these Dipel-resistant European corn borers caused more leaf-feeding damage and more tunneling on whorl-stage Bt-corn plants than did the Dipel-susceptible European corn borers. However, in the single Bt corn hybrid test, there was no survival of the Dipel-resistant European corn borers on DK580BtX or MAX454 Bt plants 35 to 42 d after they had been infested with first instars. These results demonstrate that the current Kansas selection of Dipel-resistant European corn borer strain cannot establish reproducing populations in the tested Bt corn lines and hybrids.     

Introgression and pyramiding into common bean market class fabada of genes conferring resistance to anthracnose and potyvirus

Anthracnose and bean common mosaic (BCM) are considered major diseases in common bean crop causing severe yield losses worldwide. This work describes the introgression and pyramiding of genes conferring genetic resistance to BCM and anthracnose local races into line A25, a bean genotype classified as market class fabada. Resistant plants were selected using resistance tests or combining resistance tests and marker-assisted selection. Lines A252, A321, A493, Sanilac BC6-Are, and BRB130 were used as resistance sources. Resistance genes to anthracnose (Co-2 ^C , Co-2 ^A252 and Co-3/9) and/or BCM (I and bc-3) were introgressed in line A25 through six parallel backcrossing programs, and six breeding lines showing a fabada seed phenotype were obtained after six backcross generations: line A1258 from A252; A1231 from A321; A1220 from A493; A1183 and A1878 from Sanilac BC6-Are; and line A2418 from BRB130. Pyramiding of different genes were developed using the pedigree method from a single cross between lines obtained in the introgression step: line A1699 (derived from cross A1258 × A1220), A2438 (A1220 × A1183), A2806 (A1878 × A2418), and A3308 (A1699 × A2806). A characterization based on eight morpho-agronomic traits revealed a limited differentiation among the obtained breeding lines and the recurrent line A25. However, using a set of seven molecular markers linked to the loci used in the breeding programs it was possible to differentiate the 11 fabada lines. Considering the genetic control of the resistance in resistant donor lines, the observed segregations in the last backcrossing generation, the reaction against the pathogens, and the expression of the molecular markers it was also possible to infer the genotype conferring resistance in the ten fabada breeding lines obtained. As a result of these breeding programs, genetic resistance to three anthracnose races controlled by genes included in clusters Co-2 and Co-3/9, and genetic resistance to BCM controlled by genotype I + bc-3 was combined in the fabada line A3308.     

Genetic behavior of earliness and yield traits of some rice (Oryza sativa L.) genotypes

Rice (Oryza sativa L.) is a critical staple food crop that provides more than half of the world's population with its primary nutritional source. Breeders and growers of rice would profit from robust genotypes with improved morphological and yield-related characteristics. The aim of this work is to determine the nature and magnitude of gene action on yield quantity and quality, to define the best combinations of earliness and yield characters, develop hybrids that perform better on yield quantity and quality. Three replications were used in the experiment's randomized complete block design (RCBD). During the 2016 season, seven different parents, namely Sakha 101, Sakha 104, Sakha 105, Giza 177, Black rice 1, Black rice 2, and Black rice 3, were crossed using A 7 × 7 half-diallel set analysis without reciprocals to generate 21 F1 crosses. The results indicated that genotype-dependent mean squares were very significant for main characteristics. Significant combining ability SCA variance estimates were more considerable than general combining ability (GCA) variance for all characters except days to 50% flowering. It demonstrated that both additive and non-additive genetic variance played a role in expressing the attributes investigated. The Parents, Black rice, Sakha 105, and Sakha 101, were recognized as the best general combiner for most growth and yield attributes. Sakha105 × Black Rice 1, Sakha105 × Black Rice 2, Sakha101 × Sakha104, Sakha105 × Giza 177, and Sakha101 × Giza 177 all demonstrated non-additive gene activity for the majority of maturity and yield traits. Heterosis breeding would be most efficient for qualities where high performance was determined by dominance and dominance gene effects. The increased yield of crosses results from parents with a diverse genetic background and genetic diversity.     

Effect of feeding genetically modified Bt MON810 maize to ∼40-day-old pigs for 110 days on growth and health indicators

A total of 72 male weaned pigs were used in a 110-day study to investigate the effect of feeding genetically modified (GM) Bt MON810 maize on selected growth and health indicators. It was hypothesised that in pigs fed Bt maize, growth and health are not impacted compared with pigs fed isogenic maize-based diets. Following a 12-day basal period, pigs (10.7 ± 1.9 kg body weight (BW); ∼40 days old) were blocked by weight and ancestry and randomly assigned to treatments: (1) non-GM maize diet for 110 days (non-GM), (2) GM maize diet for 110 days (GM), (3) non-GM maize diet for 30 days followed by GM maize diet up to day 110 (non-GM/GM) and (4) GM maize diet for 30 days followed by non-GM maize diet up to day 110 (GM/non-GM). BW and daily feed intake were recorded on days 0, 30, 60 and 110 (n = 15). Body composition was determined by dual energy X-ray absorptiometry (n = 10) on day 80. Following slaughter on day 110, organs and intestines were weighed and sampled for histological analysis and urine was collected for biochemical analysis (n = 10). Serum biochemistry analysis was performed on days 0, 30, 60, 100 and 110. Growth performance and serum biochemistry were analysed as repeated measures with time and treatment as main factors. The slice option of SAS was used to determine treatment differences at individual time points. There was no effect of feeding GM maize on overall growth, body composition, organ and intestinal weight and histology or serum biochemistry on days 60 and 100 and on urine biochemistry on day 110. A treatment × time interaction was observed for serum urea (SU; P < 0.05), creatinine (SC; P < 0.05) and aspartate aminotransferase (AST; P < 0.05). On day 30, SU was lower for the non-GM/GM treatment compared with the non-GM, GM and GM/non-GM treatments (P < 0.05). On day 110, SC was higher for the non-GM/GM and GM/non-GM treatments compared with non-GM and GM treatments (P < 0.05). Overall, serum total protein was lower for the GM/non-GM treatment compared with the non-GM/GM treatment (P < 0.05). The magnitude of change observed in some serum biochemical parameters did not indicate organ dysfunction and the changes were not accompanied by histological lesions. Long-term feeding of GM maize to pigs did not adversely affect growth or the selected health indicators investigated.     

加工产品中转基因玉米Bt11成分实时荧光PCR定量(性)检测

实验在玉米自身基因和外源基因的边界序列之间设计了具有品种和品系特异性的引物和探针 ,并以实时荧光PCR技术 ,建立了加工产品中转基因玉米Bt1 1成分品系鉴定检测和定量检测的方法。实验对加热条件和时间对检测转基因成分的影响作了探讨 ,并检测了部分市售食品和饲料。检测结果发现 ,加热时间温度越高、时间越长 ,对转基因成分定量检测的影响越大 ;在所检测的样品中可以检测出转基因玉米Bt1 1成分 ,有些样品还同时检出其他转基因成分。本研究实验建立的方法 ,可以用于加工产品中转基因成分的定量检测 ,也可以用于定性检测 ,或作为常规PCR定性检测后的确证实验方法。     

Optimized breeding strategies for multiple trait integration: I. Minimizing linkage drag in single event introgression

From a breeding standpoint, multiple trait integration (MTI) is a four-step process of converting an elite variety/hybrid for value-added traits (e.g. transgenic events) using backcross breeding, ultimately regaining the performance attributes of the target hybrid along with reliable expression of the value-added traits. In the light of the overarching goal of recovering equivalent performance in the finished conversion, this study focuses on the first step of MTI, single event introgression, exploring the feasibility of marker-aided backcross conversion of a target maize hybrid for 15 transgenic events, incorporating eight events into the female hybrid parent and seven into the male parent. Single event introgression is conducted in parallel streams to convert the recurrent parent (RP) for individual events, with the primary objective of minimizing residual non-recurrent parent (NRP) germplasm, especially in the chromosomal proximity to the event (i.e. linkage drag). In keeping with a defined lower limit of 96.66 % overall RP germplasm recovery (i.e. ≤120 cM NRP germplasm given a genome size of 1,788 cM), a breeding goal for each of the 15 single event conversions was developed: <8 cM of residual NRP germplasm across the genome with ~1 cM in the 20 cM region flanking the event. Using computer simulation, we aimed to identify optimal breeding strategies for single event introgression to achieve this breeding goal, measuring efficiency in terms of number of backcross generations required, marker data points needed, and total population size across generations. Various selection schemes classified as three-stage, modified two-stage, and combined selection conducted from BC1 through BC3, BC4, or BC5 were compared. The breeding goal was achieved with a selection scheme involving five generations of marker-aided backcrossing, with BC1 through BC3 selected for the event of interest and minimal linkage drag at population size of 600, and BC4 and BC5 selected for the event of interest and recovery of the RP germplasm across the genome at population size of 400, with selection intensity of 0.01 for all generations. In addition, strategies for choice of donor parent to facilitate conversion efficiency and quality were evaluated. Two essential criteria for choosing an optimal donor parent for a given RP were established: introgression history showing reduction of linkage drag to ~1 cM in the 20 cM region flanking the event and genetic similarity between the RP and potential donor parents. Computer simulation demonstrated that single event conversions with <8 cM residual NRP germplasm can be accomplished by BC5 with no genetic similarity, by BC4 with 30 % genetic similarity, and by BC3 with 86 % genetic similarity using previously converted RPs as event donors. This study indicates that MTI to produce a ‘quality’ 15-event-stacked hybrid conversion is achievable. Furthermore, it lays the groundwork for a comprehensive approach to MTI by outlining a pathway to produce appropriate starting materials with which to proceed with event pyramiding and trait fixation before version testing.