Predictive study of the advantages of cleistogamy in oilseed rape in limiting unwanted gene flow

Summary The aim of this study was to evaluate the interest of beginning the selection process on a new genetic characteristic, cleistogamy, to manage gene flow in oilseed rape. The first step was to introduce this characteristic in an existing model of gene flow between oilseed rape populations in time and space, GeneSys-Rape. The second step was to evaluate the parameters of the model linked to this characteristic using field experimentations. Cleistogamous oilseed rape was shown to have an autogamy rate as high as 94% and to emit 10 times less pollen than an open-flowered oilseed rape in the same conditions. But the cleistogamous character was also shown to be unstable in the genotypes tested. The third step was to evaluate the interest of cleistogamy using simulations comparing several genotypes with or without cleistogamy in two different cropping systems. These simulations showed that an oilseed rape both dwarf and cleistogamous was interesting to limit gene escape and that a 99%-autogamous oilseed rape was interesting to limit both gene escape from and harvest contamination of the 99%-autogamous oilseed rape.     

Evaluation and use of a spatio-temporal model of cropping system effects on gene escape from transgenic oilseed rape varieties: Example of the GeneSys model applied to three co-existing herbicide tolerance transgenes

Gene flow between coexisting oilseed rape varieties and rape volunteers cannot be solely studied on field experiments. Previously, a model (GeneSys: [Colbach, N., Clermont-Dauphin, C., Meynard, J.M., 2001. GeneSys: A model of the inluence of croping system on gene escape from herbicide-tolerant rapeseed crops to rape volunteers. I. Temporal evolution of a population of rapeseed volunteers in a field. Agric. Ecosyst. Environ. 83, 235–253; Colbach, N., Clermont-Dauphin, C., Meynard, J.M., 2001. GeneSys: A model of the inluence of croping system on gene escape from herbicide-tolerant rapeseed crops to rape volunteers. II. Genetic exchanges among volunteer and cropped populations in a small region. Agric. Ecosyst. Environ. 83, 255–270]) was therefore developed to quantify the effects of cropping systems on gene flow from rapeseed varieties to rape volunteers in time and in space. In the present work, the model was improved to simulate simultaneously the spread of three different genes. Four different regions were then surveyed from 1995 to 2001: one region comprised only farmers’ fields with frequent rape crops in the rotations; the three remaining regions comprised the French experimental GMO platforms with 2–3 GM and one non-GM rape variety as well as neighbour farmers’ fields. For all these fields, cropping history was recorded during the study and for at least three previous years. In the first region, rapeseed volunteers were assessed in crops as well as on road and field margins in 1 year. In the three remaining regions, volunteer densities and genotypes were assessed each year after harvest and rape harvest genotypes determined during 2 years. The comparison of simulated and observed data showed that the model usually satisfactorily predicts volunteer densities and genotype proportions as well as harvest genotype proportions. Several problem areas were identified: volunteers in road and field margins were usually badly predicted; the model frequently underestimated volunteer densities in spring crops; gene flow via pollen dispersal was underestimated when the distance from the pollen source increased. The model was then used to simulate the spread of herbicide-tolerant rape volunteers in a region grown with one or several GM herbicide-tolerant varieties and more particularly the stacking of several resistance genes in individuals. The simulations also showed the contamination of the harvest of non-GM rape varieties by GM seeds resulting from gene flow.     

Agronomic traits and gene containment capability of cleistogamous rice lines with the superwoman1-cleistogamy mutation

Pollen-mediated transgene flow is a major concern for the production of genetically modified (GM) rice. Cleistogamy is a useful tool for preventing this form of gene flow. We previously identified the cleistogamous rice mutant superwoman1-cleistogamy (spw1-cls) and determined its molecular genetic mechanism. In the present study, we cultivated spw1-cls over five years to examine effects of cleistogamy on agronomic traits. Simultaneously, we cultivated cleistogamous backcross lines created by continuous backcrossing with “Yumeaoba” (a japonica cultivar) as the recurrent parent and by application of a DNA marker. In these experimental cultivations, spw1-cls and its backcross lines showed almost equal or slightly lower, but acceptable, agronomic traits compared with each control line. We also conducted natural crossing tests in paddy fields to assess the gene containment capability of spw1-cls. In a series of field experiments, there was no natural crossing between spw1-cls (pollen donor) and pollen recipient lines, but the wild-type donor and recipient lines were crossed. Thus, the cleistogamy of the spw1-cls mutation is able to inhibit natural crossing effectively, without significant loss of commercial benefits, such as yield. We conclude that spw1-cls cleistogamy is a practical tool for gene containment in GM rice cultivation.     

Outcrossing frequencies from multiple high erucic acid oilseed rape fields to a central receptor field

Genetically modified oilseed rape is currently grown on about 23 % of the global oilseed rape acreage. In order to separate transgenic and non-transgenic oilseed rape production and to ensure co-existence of different agricultural cultivation schemes, as is specified by the European legislation, confinement measures have to be defined. Pollen-mediated gene flow is the most important means by which transgenes are dispersed between fields. In contrast to the majority of the previous investigations the objective of this study was to assess the extent of gene flow in the case of multiple pollen-donor fields. A high erucic acid rape genotype was used as biochemical marker for the quantification of outcrossing into a low erucic acid oilseed rape variety. Outcrossing data were obtained from two experimental locations. As expected, multiple pollen sources in a fragmented landscape can result in high gene transfer frequencies, thus requiring larger isolation distances than a field design with a single pollen donor source. The results of the study are transferable to homozygous transgenic oilseed rape varieties.     

Spontaneous outcrossing in tomato depends on cultivar and environment and varies between individual flowers

Knowledge about the degree of spontaneous outcrossing of diverse genotypes is essential for breeding programmes, maintenance breeding, and seed production. For tomato (Lycopersicon esculentum Mill.), very limited scientific evidence for genotypic differences is available and evidence from Europe is scarce. To close this knowledge gap, six cultivars were investigated in three Central European locations as part of the Organic Outdoor Tomato Project. To determine outcrossing rates, the monogenetic “cut‐leaf” trait, which is dominant over the “potato‐leaf” trait, was used as morphological marker. The observed range of outcrossing was 0.0%–5.2%. Outcrossing was significantly influenced by cultivar and environment. The outcrossing rate of individual flowers varied within cultivars ranging from 0% to 37%. The potential of newly opened flowers to accept foreign pollen varied largely with the cultivar. Genotypic differences could partly be linked to flower morphology traits. The potential for recombination between tomato genotypes is generally very low but can be a source for new variation in on‐farm management.     

Identification and mapping of a QTL for rachis internode length associated with cleistogamy in barley

General knowledge of the closed flowering trait, or cleistogamy, of barley is still limited. The relationship between cleistogamy and spike morphology characters was studied and linkage of cleistogamy genes with a highly significant quantitative trait locus (QTL) for rachis internode length on the long arm of chromosome 2H was detected. The mapping populations consisted of 129 doubled haploid lines of ‘Mikamo Golden’ × ‘Harrington’ and 150 F₂ plants of ‘Misato Golden’ × ‘Satsuki Nijo’. The phenotypic variance explained by this QTL accounted for 77.5% and 82.6% of the variance in rachis internode lengt, respectively, in these two populations. The peaks of the QTL coincided with the positions of the cleistogamy gene loci.     

Virtual modeling based on deep phenotyping provides complementary data to field experiments to predict plant emergence in oilseed rape genotypes

Breeding oilseed rape for oil and protein contents may have led to differences in seedling emergence in genotypes. New opportunities for deep automated phenotyping of germination and seedling growth are being developed on phenotyping platforms. Our aim was to demonstrate that using these data to parameterize a crop emergence model complements field experiments for the evaluation of differences among genotypes. Five genotypes, chosen in a diverse set of winter oilseed rape for their different germination speeds, were phenotyped for germination at different temperatures and water potentials as well as for radicle and hypocotyl growth. These data were used as parameters to run the SIMPLE crop emergence model over a period of 27 years (1985–2012), at two locations, one in France and one in Germany, and at four sowing dates. Field experiments were performed in 2012, 2013 and 2014, and the emergence of the five genotypes was measured at early and late sowing dates. First, model predictions were compared with observed field emergence in the French sowing trials in 2014. The model proved to be rather good at predicting the emergence of the genotypes. Then, for the simulation study, the model extended the observed differences between locations and sowing dates over a greater number of years. The model also identified the main reasons for non-emerging seedlings and their frequencies in the simulated sowings. Differences between the five genotypes were on average very small, but complex interactions appeared that led to bigger differences under certain sowing conditions. This study demonstrates that combining deep phenotyping with crop models in simulation studies paves the way for more precise and detailed evaluation of genotypes.     

Susceptibility of Faba Bean (Vicia faba L.) to Heat Stress During Floral Development and Anthesis

Experiments were conducted over 2 years to quantify the response of faba bean (Vicia faba L.) to heat stress. Potted winter faba bean plants (cv. Wizard) were exposed to temperature treatments (18/10; 22/14; 26/18; 30/22; 34/26 °C day/night) for 5 days during floral development and anthesis. Developmental stages of all flowers were scored prior to stress, plants were grown in exclusion from insect pollinators to prevent pollen movement between flowers, and yield was harvested at an individual pod scale, enabling effects of heat stress to be investigated at a high resolution. Susceptibility to stress differed between floral stages; flowers were most affected during initial green‐bud stages. Yield and pollen germination of flowers present before stress showed threshold relationships to stress, with lethal temperatures (t₅₀) ?28 °C and ~32 °C, while whole plant yield showed a linear negative relationship to stress with high plasticity in yield allocation, such that yield lost at lower nodes was partially compensated at higher nodal positions. Faba bean has many beneficial attributes for sustainable modern cropping systems but these results suggest that yield will be limited by projected climate change, necessitating the development of heat tolerant cultivars, or improved resilience by other mechanisms such as earlier flowering times.     

Floral Traits and Seed Production of Sorghum A-, B- and R-lines under Chilling Field Temperatures

In A-lines of sorghum ( Sorghum bicolor L. Moench), seed production under field conditions with manual pollination is generally lower than that in self-pollinated B-lines. This may be associated with floral differences. Six pairs of A/B-lines and four R-lines were evaluated during 2005 and 2006 at Montecillo, State of México (2240 m altitude). Rachis length, number of primary branches and fertile flowers per panicle, 100-seed weight, seed number, seed yield and seed set per panicle were evaluated. In the A-lines, the pistil characteristics were also measured and in the male-fertile lines, the size of anthers and the amount and size of viable pollen were recorded. Compared with the A- and B-lines, the R-lines exhibited significantly higher (P ≤ 0.05) numbers of flowers, seed set and seed yield per panicle and they also produced more pollen grains of larger size and with greater viability during their longer flowering period (FP). Between A- and B-lines, there were differences (P ≤ 0.05) in most of the yield traits, which also interacted with year. The proportion of viable pollen in B- and R-lines (75 %) was not considered to be a factor that might account for their low seed production. Chilling temperatures (3.5–8.4 °C) during the FP could have affected stigma receptivity in the three different line types and thus may have reduced seed set in the male-fertile lines.     

Chromosome location of genes controlling the content of dietary fibre and arabinoxylans in rye

Differences in fruit-set success reputed to exist between Heveabrasiliensis genetic clones were verified by an analysis of breeding recordsfor the clones PB 5/51, RRIM 600 and PR 107. Although the bestfruit-set success was obtained with PB 5/51 following artificial (hand)pollination, this clonal trait was not reflected in terms of greater numbersof pollen tubes developing it the styles of hand-pollinated PB 5/51 femaleflowers. It was observed, however, that more pollen tubes reached theovules in PB 5/51 than in RRIM 600 or PR 107. Significantly, PB 5/51female flowers required fewer pollen tubes to effect penetration of all threeof its ovules. As fruit formation in Hevea is dependent on all threeovules of the flower being successfully fertilised, PB 5/51 female flowershave hence a greater propensity for successful fruit-set. In all the threeclones studied, the frequency distribution of female flowers with 0, 1, 2 or3 of the ovules penetrated by pollen tubes did not conform to binomialexpectations. Flowers with no ovule penetrated and flowers with all threeovules penetrated were greatly over-represented. One explanation for thisnon-random distribution is the existence of `receptive' female flowers thatfavour successful fertilisation whereas `non-receptive' flowers tend to remainunfertilised even when hand-pollinated.     

Inheritance of cleistogamic flowering in durum wheat (Triticum durum)

Summary Generally durum wheat flowers are chasmogamous but very rarely, cleistogamy may be observed. Genetic studies based on six crosses and back crosses showed that chasmogamy was determined by a single dominant gene Cl, plants homozygous for the recessive allele, cl, were cleistogamous. The flowering behaviour phenotypes were unaffected by environmental conditions. Cleistogamy was attributed to poorly developed lodicules and stiff perianth.     

Use of diploid self incompatible rose genotypes as a tool for gene flow analyses in roses

A diploid rose genotype previously found to be self incompatible in greenhouse pollination experiments was analyzed in order to investigate the capacity to self fertilize under field conditions. A subset of 36 plants from open pollinations were analyzed by means of AFLP markers. All plants were found to be the result of outcrossing events, thereby demonstrating the utility of these kinds of rose genotypes as effective pollen sinks for future gene flow analyses under field conditions. Furthermore, it was demonstrated that isolation distances of 250 m from large rose plantations are insufficient to prevent gene flow between populations.     

应用基因型与播期互作效应分析水稻光温敏核不育系对光周期和温度的育性敏感性

应用AMMI模型、线性回归模型和系统聚类分析方法分析了9个水稻光温敏核不育系和2个对照不育系(培矮64,浙农大11S),在9个播期环境下花粉和种子育性的变化动态。从AMMI两维图可直观看到基因型与播期的交互模式,根据最大互作效应主成分轴IPCA1值和花粉育性平均值将不育系分成3个集团。第1集团是培矮64(1),浙大21S（10）和浙大22S（11）,具有较低花粉育性平均值和较高IPCA1值,表明它们对温度变化比较敏感,且有较大的负向互作效应。第2集团是浙农大11S（2）、浙大4S（3）、浙大7S（6）、浙大8S（7）和浙大9S（8）,其花粉育种平均值和互作值相对较低,变动在0.013-0.276间,暗示花粉育性对播期敏感度低。第3集团是浙大6S（5）和浙大10S（9）,花粉育性平均值高,互作效应大,该2个不育系尚有分离,且花粉育性对播期反映敏感度高。对光敏型和温敏型不育系而言,基因型IPCA值大小主要反映它们对光周期和温度敏感性强弱。花粉和种子育性在基因型、播期及其互作效应上都存在极显著差异。本文还提出利用育性相对稳定性的定量指标D_i界定光温敏核不育的育性稳定性,分析表明,Di值与育种实践结果较为接近。基于AMMI模型的基因型主效应和互作效应分析可以明确划分不育系的不育期、育性转换期和可育期,并将水稻光敏型温敏型不育系区分开,因而该模型可为不育系应用于种子生产提供信息和依据。     

Gene flow from transgenic oilseed Brassica juncea (L.) Czern. into weedy Sinapis arvensis L. (wild mustard)

Gene flow from acetolactate synthase‐resistant (HR) Brassica juncea oilseed canola to related weed, Sinapis arvensis (density 1 plant/m²) was assessed in a 100 m² field plot of HR B. juncea. Two HR F₁ hybrids (H1 and H2) were detected among 109 951 seedlings screened with imazethapyr (hybridization frequency – 1.8 × 10^?5). Hybridity was confirmed using flow cytometry, B. juncea‐specific amplified fragment length polymorphisms (AFLPs) markers, genomic in situ hybridization (GISH) and PCR‐based detection of B. juncea's HR gene. H1 and H2 had 2n = 27 and 2n = 45 chromosomes, corresponding 3x (SrAB) and 5x (SrAABB) genomic structures and reduced male fertility, 3.2 and 16.6% pollen viability, respectively. H1 was self‐incompatible, whereas H2 set seed when selfed (B. juncea trait). Selfed F₂, F₃ and F₄ plants showed HR trait persistence and vigorous growth and high (80–100%) pollen fertility in 22% and 39% of the F₂ and F₃ plants, respectively. No progeny were obtained from F_1, F₂ or F₃ hybrids × S. arvensis backcrosses, suggesting the likelihood of introgression of traits is low to negligible.     

花粉介导法获得油菜转基因植株研究

以甘蓝型冬油菜品种晋油7号为受体，载有GUS基因的质粒pBI121为供体，在7.5％的蔗糖等渗溶液中，通过花粉介导转化方法将GUS基因导入油菜花粉，随着花粉管的萌发进入胚囊，参与有丝分裂，将外源质粒转入受体材料。经田间植株性状比较、GUS组织化学定位检测、PCR扩增检测及PCR-Southern杂交检测，证明外源GUS基因已整合到油菜基因组中。     

Suitability of Canopy Temperature Depression in a Temperate Climate with Drought‐Stressed Winter Rye,Determined with Three Infrared Measurement Devices

Canopy temperature has been recognised as an indicator of crop water status and may thus be a useful secondary trait in selecting for yield under dry conditions. The aim of this study was to test the suitability of canopy temperature depression (CTD = T_air ? T_canopy) in a temperate climate with winter rye, by means of three infrared (IR) temperature measuring devices. In the years 2011 and 2012, 16 winter rye genotypes were examined under drought stress conditions in a rainout shelter and under well‐watered conditions. In each year, the CTD was determined several times during the growth period using two IR thermometers and an IR camera. By means of CTD, it was possible to detect drought stress and to differentiate between water regimes. The three measurement devices showed comparable results, despite greatly different costs. Under drought‐stress conditions, a significant positive correlation between grain yield and CTD was found on most measurement dates in 2011 and on some dates in 2012. When the CTD was pooled across water regimes, a significant positive correlation between grain yield and CTD was obtained on every measurement date. However, as genotypic differences for CTD were non‐existent, the correlations are less meaningful. The missing genotypic differences for CTD were rather caused by the limited genetic variability of the genotypes used in this study, than by climatic conditions. Due to this limitation, we were not able to make a concluding statement about the CTD in a temperature climate, although the results are quite promising and indicate that the CTD can potentially be used in a temperate climate.     

Comparative study of the efficiency of buffer zones and harvest discarding on gene flow containment in oilseed rape. A modelling approach

Oilseed rape (OSR) genes can escape fields in space via pollen and seeds and in time via volunteers resulting from seeds lost before or during oilseed rape harvests. Previous simulation studies and field observations showed that co-existence at the landscape level of contrasting OSR varieties such as genetically modified (GM) and non-GM varieties require costly measures that are difficult to implement, such as isolation distances between OSR fields and stringent volunteer control in all fields and road margins. In the present study, two local strategies, non-GM buffer zones around GM fields and discarding the harvest of boundary plants of non-GM fields, were tested in a simulation study using the GENESYS model. This model, which was modified here to improve the prediction of volunteer reproduction and pollen dispersal, quantifies the effects of cropping system on gene flow in landscapes over the years. The evaluation of the improved model version with independent field observations showed that cross-pollination of fields was accurately predicted up to 50 m and systematically underestimated above that distance, though fields were still ranked correctly. A corrective factor should therefore be applied to predictions in case of low regional OSR and GM proportions (which result into large distances between fields). To study the effects of the two local strategies, simulations were carried out in four contrasted case studies, based on different landscapes, regional OSR and GM proportions, cropping systems and OSR varieties. In these situations, harvest discarding was needless when volunteers were well controlled because the GM impurity in non-GM harvests was well below the EU labelling thresholds; it was useless when OSR contained male-sterile plants or when the landscape was infested by volunteers because large parts of the fields had to be discarded to respect impurity thresholds, which is financially unacceptable. Buffer zones were useful if the non-GM varieties comprised male-sterile plants or if the non-GM fields were small, but only if volunteers were well controlled and harvest impurity was already close to the labelling threshold. Buffer zones appeared considerably more efficient in reducing harvest impurity than harvest discarding because they not only increase the distance between GM and non-GM fields but also diminish the proportion of GM pollen in the total pollen cloud over landscapes. Harvest discarding could possibly be interesting for isolated OSR crops surrounded by bare ground or small crops, an effect that was not tested here. The present study showed that efforts should be dedicated to characterizing and modelling of gene flow at the landscape level, as well as the collection of datasets for the evaluation of these models in many landscapes and configurations. Local measures tested here are insufficient for ensuring harvest purity. Therefore, future work should focus on finding additional measures are necessary to manage OSR volunteers which constitute the major risk factor for OSR harvest purity.     

播期对川中丘陵区油菜倒伏性状的影响

油菜机械化生产中,植株倒伏是影响机械化收获的重要因素。为探讨不同播种期对川中丘陵区油菜倒伏性状的影响,以川油36和蓉油18为供试材料,分别设置9月22日、9月29日、10月6日、10月13日和10月20日5个播期处理,分析不同播期条件下油菜生育期气象因子、倒伏性状指标的变化情况。结果表明,随播种期推迟,油菜生育期内的降水量、积温、日均最高气温、日均最低气温和日照时长均逐渐下降,生育期逐渐缩短,成熟期植株株高、分枝部位、根颈粗、茎秆粗降低,倒伏系数增加。油菜抗倒伏能力在9月22-29日播期处理之间最强。因此,川中丘陵区油菜的适宜播期为9月下旬。     

Unilateral cross compatibility in Vaccinium elliottii × V. arboreum, an intersectional blueberry hybrid

Vaccinium elliottii Chapmn., a diploid blueberry in Vacciniumsection Cyanococcus, was crossed with V. arboreum Marsh, a diploid blueberry insection Batodendron. The goal was toproduce hybrids that could be used toincorporate traits from these species intotetraploid southern highbush cultivars. The crosses were made reciprocally usingtwo clones of each species. A recessiveseedling marker gene for anthocyanin-freecotyledons in the V. elliottii clonesused as female parents permitted thetesting of mentor pollination. For thementor pollinations, V. elliottiiplants that were homozygous for therecessive marker allele were pollinatedwith a small amount of viable V.elliottii pollen carrying the recessivemarker mixed with a large amount of V.arboreum pollen carrying the dominantmarker. Thousands of intersectional hybridseedlings were obtained when V.elliottii was the seed parent, both withand without mentor pollination, but nohybrids were obtained from 2735 flowerspollinated in the reciprocal crosses. Theseeds that gave rise to hybrid embryos weresmaller than normal V. elliottiiseeds, but germinated well. Hybridseedlings grew slowly at first, buteventually produced some vigorous plants inthe field, although many plants remainedmuch smaller than normal seedlings of thetwo parental species. Some of the hybridsproduced numerous flowers during the 6years they were observed in the field, buteven with many diploid, tetraploid, andhexaploid section Cyanococcus blueberryplants growing nearby that could haveprovided pollen for their flowers, thehybrids produced no viable seed.     

Characteristics of Pollen from Transgenic Lines of Apple Carrying the Exogenous CpTI Gene

It is fundamental for gene transformation and ecosystem hazard evaluation to study the pollen characteristics of transgenic plants. In this research,the characteristics of pollen from 7-or 8-year-old transgenic apple plants carrying an exogenous Cp TI gene were analyzed. The results showed that there was no significant difference in terms of size,morphology,or exine ornamentation between the pollen of the transgenic plants and the non-transgenic control. However,the transgenic plants had more abnormal pollen grains. Of the 13 transgenic lines tested,12 had a significantly lower amount of pollen and six exhibited a significantly lower germination rate when cultured in vitro. The pollen viability of three transgenic lines was determined,with two showing significantly lower viability than the control. The transgenic Gala apple pollen grains germinated normally via controlled pollination on Fuji apple stigmas. However,the pollen tubes extended relatively slowly during the middle and late development stages,and another 8 h were needed to reach the ovules compared with the control. The gibberellic acid concentration in transgenic Gala apple flowers was lower than in the non-transgenic control during all development stages tested. The abscisic acid concentration in the transgenic flowers was lower during the pink stage,and higher during the ball and fully open stages. Microscopic observation of the anther structure showed no difference. The tapetum of the pollen sac wall in transgenic plants decomposed late and affected pollen grain development,which could be one of the reasons for the lower number of pollen grains and poor viability in the transgenic plants.