TILLING技术的形成和发展及其在麦类作物中的应用

TILLING（Targeting induced local lesions in genomes,定向诱导基因组局部突变技术）是一种高通量的等位变异创制和突变体快速鉴定技术,其实质是将传统的化学诱变方法和突变的高效筛选有效结合的反向遗传学研究方法。其技术原理是将传统的酶切技术与PCR技术相结合后采用红外双色荧光系统进行结果鉴定,从而筛选出相应的突变体。传统的TILLING技术主要用于筛选由人工诱导产生的突变体。Ecotilling技术由TILLING技术延伸而来,主要用于鉴定自然界中已经存在的突变体,其与传统的TILLING技术的区别主要为构建DNA池时略有差异。随着该项技术在拟南芥等模式植物中的成功应用,越来越多的人开始将其用于基因组较大的植物之中。本文对近年来TILLING技术在麦类作物中的应用进行了分析,并通过比较不同植物突变体库中的突变频率发现,经EMS处理的小麦等麦类作物突变体库中的突变频率显著高于其他植物,因此相信,TILLING技术将会作为一种常规手段在麦类作物尤其是普通小麦改良中得到越来越广泛的应用。     

利用EMS诱发玉米性状变异的效果

用含有不同浓度甲基磺酸乙酯（EMS）的石蜡油溶液处理5个玉米自交系的成熟花粉,处理后代采系谱法种植;在后代群体中筛选各种有利用价值的变异性状。本试验得出：经EMS处理的试材,M1代表现出来的叶色变异,并不能真实遗传。在诱变后代选择生育期突变体应从M2代开始,成株期质量性状未发生明显变异,但株高、穗位高、茎粗和叶型等数量性状出现了较高的突变频率,其中株高降低的突变明显。     

一个水稻低温移栽白条纹突变体wltt的鉴定和基因定位

【目的】叶色突变相关基因的鉴定与克隆为研究叶绿体发育、叶绿素合成和光合作用等分子机制提供理论基础。【方法】从常规粳稻镇糯19杂交后代中分离出一个低温移栽后叶色转成白条纹的自然变异突变体,命名为wltt (white stripe leaf after transplanting at low temperature)。成熟期测定野生型和wltt的主要农艺性状,分别在苗期、移栽后15 d和同时期直播条件下测定新生叶片的色素含量并观察叶绿体的超微结构;将wltt和野生型正反交进行遗传分析;用wltt与籼稻9311杂交产生的F_2作为定位群体进行基因定位;采用RT-qPCR分析叶绿体发育、叶绿素合成和光合作用相关基因在野生型和wltt中的表达水平。【结果】wltt突变体在苗期表现正常绿色,移栽15 d后心叶出现白条纹叶表型,至分蘖末期心叶叶色恢复;而不经移栽,突变体不会出现白条纹叶。人工模拟实验表明该表型是由低温条件下根损伤引起的。与野生型相比,wltt突变体移栽后的新生叶色素含量显著降低,光合速率下降;同时株高变矮,穗长、剑叶长和每穗粒数均显著降低。叶绿体的超微结构显示,突变体的叶肉细胞中,仅少数细胞含有正常的叶绿体,其余大部分叶肉细胞不含叶绿体。进一步研究发现,突变体中部分光合系统相关基因和叶绿体发育相关基因表达下调,叶绿素生物合成相关的14个基因表达也下调。遗传分析表明,该突变性状受一对隐性核基因控制。利用wltt突变体/9311的F_2群体,将该基因定位于水稻第2染色体着丝粒附近853kb区间内。目前,该区间内没有叶色相关基因的报道。【结论】WLTT是低温条件下移栽调控叶片转色的关键基因,在叶绿体发育过程中发挥重要作用。     

EMS诱变的普通小麦豫农201突变体库的构建与初步分析

为了构建小麦EMS突变体库,为小麦功能基因组学研究准备基础材料,利用化学诱变剂甲基磺酸乙酯(Ethyl methane sulfonate,EMS)溶液诱变处理豫农201种子,对获得的M2代植株进行农艺性状及其他生物学性状的表型筛选,结果共获得722份叶、茎、穗和其他性状发生变异的突变体,其中139份叶片性状突变、220份茎秆性状突变、38份穗部和籽粒性状突变、325份其他性状突变,突变频率分别为2.20%、3.49%、0.60%、5.15%.利用SDS-PAGE对突变体的高分子量麦谷蛋白亚基进行分析,共发现有21个缺失不同类型亚基的突变体.本研究初步构建的豫农201 EMS突变群体可应用于小麦功能基因组研究和小麦遗传改良.     

水稻白条纹叶突变体st11的遗传分析与基因定位

白条纹叶突变体st11是从粳稻品种Kitaake组培过程中获得的。该突变体在分蘖前叶色表现为正常,从分蘖期开始新生叶表现为白条纹直至成熟期。与野生型相比,该突变体的分蘖、株高、结实率和千粒重等农艺性状没有发生明显变化。遗传分析表明该突变体白条纹叶性状受一对隐性核基因控制。利用该突变体分别与水稻02428、Jodan杂交构建了两个F2群体用于基因定位。通过集群分离分析(bulked segregant analysis)发现该基因位于第1染色体端粒附近,并与分子标记RM151和RM10080连锁。进一步利用更多分子标记分析F2群体,我们将该基因定位于I10和I26两个标记之间大约270kb的区间内。     

春性甘蓝型油菜EMS突变体库的构建与筛选

创制突变体库是甘蓝型油菜品种选育和重要性状遗传解析的重要基础。春性甘蓝型油菜开花不需漫长的春化过程,生育期短,在创制突变体库方面具有独特优势。本研究采用甲基磺酸乙酯（EMS）诱变春性油菜862种子,以构建春性油菜EMS诱变突变体资源库。通过比较8种不同浓度EMS（0~1.4%,间隔0.2%）对发芽率的影响,最终确定使用0.8%的EMS作为最佳处理浓度,发芽率为37.2%。田间种植筛选与表型统计初步鉴定了M2代1823个单株的形态学性状,突变频率为3.91%,主要包括叶色、叶形、株高、株型、角果长短、种粒大小、种皮颜色、种子含油量以及其它性状变异等,突变类型十分丰富。该突变体库的构建为油菜功能基因组学研究和分子育种提供表型多样的种质资源。     

籼稻93-11辐射诱变突变体库的创建及其筛选

人工诱变突变体库是植物种质资源创新和基因组功能分析的有力工具。本研究以具有优异农艺性状且已完成基因组测序的籼稻常规品种93-11(O.sativa ssp.indica cv.93-11)为材料,创建了一个包含3617个家系的辐射诱变突变体库。通过在M2和M3代各生育期的筛选,从该突变体库中获得308个表型稳定的突变体。这些突变体的表型可以分成15类,包括株高、分蘖数、株型、穗型、小穗结构、育性、叶色、叶型、抽穗期、苯达松抗性等,突变频率为0.03%~2.74%。使用反向遗传学策略,鉴定出了2个雄性不育突变体和2个苯达松敏感突变体中的基因突变位点。其中,2个雄性不育突变体的突变表型是由于CYP704B2分别在第3和第4外显子发生碱基替换和片段缺失造成的,而2个苯达松敏感突变体的突变表型是由于CYP81A6中第1和第2外显子分别缺失了一个7bp和一个23bp的片段造成的。4个突变体家系中野生型和突变体植株的分离比均符合3∶1,属单基因隐性突变。这一籼稻突变体库将有助于籼稻基因组的功能分析,并且为水稻遗传改良提供了有价值的材料。     

叠氮化钠诱变普通小麦陕农33突变体库的构建和初步评估

为了用叠氮化钠(NaN3)构建一个小麦突变体库,分别用浓度为0、5、10、15、20 mmol·L-1的NaN3处理普通小麦陕农33的种子,检测了各处理发芽指标和田间M1群体的生理及形态特征。结果表明,10mmol·L-1的NaN3是诱变普通小麦较适宜的浓度。在以10mmol·L-1的NaN3诱变普通小麦得到的M2群体中发现了322份茎、叶、穗和其他性状变异的突变体,其中204份茎秆性状突变、65份叶片性状突变、24份穗部性状突变和115份其他性状突变,突变频率分别为5.18%、1.65%、0.61%、2.92%。采用RAPD分子标记技术估计该群体的突变密度为1/57.0kb。经M3代田间鉴定,共获得可遗传突变株系58个。本研究所构建的突变体库可为小麦功能基因组学研究和小麦育种提供材料。     

大麦EMS突变体库构建和突变体筛选

为研究大麦重要功能基因并给大麦遗传育种提供新的种质资源,通过甲基磺酰乙酯(EMS)诱变处理优质、高抗黄花叶病大麦品种苏啤6号,从2400个M_2代株系中筛选出152个突变株系,突变频率为6.33%。其中,幼苗习性突变株系36个,突变率为1.50%;叶部突变株系34个,突变率为1.42%;茎部突变株系33个,突变体率为1.38%;穗部突变株系22个,突变率为0.92%;成熟期和育性突变株系27个,突变率为1.13%。经过M_3代验证,获得可稳定遗传的各类突变株系43个。     

秋水仙素应用于大麦育种的初步研究

用0．04%的秋水仙素溶液处理分蘖盛期的二棱皮大麦（浙皮1号）的植株，在M1代即获得四倍体类型，同时还获得许多早熟、大穗、高植等性状变异的二倍体大麦植株，并从这些变异株中选得一优良株，经加速培育即在生产上示范推广．且很快通过了省级审定，定名为闻诱3号。     

甲基磺酸乙酯(EMS)对玉米自交系诱变效应的研究

用化学诱变剂甲基磺酸乙酯处理玉米自交系082的种子,研究了该诱变剂对玉米的诱变效应.结果表明:甲基磺酸乙酯对玉米自交系种子是一种有效的化学诱变剂,在M1代引起了较大的生理损伤和生物学效应,M2代质量性状未发生明显变异,但株高、穗位高、茎周长和穗上叶片长等数量性状出现了较高的突变频率,其中株高的负突变增加明显。     

Grazing management and tussock distribution in elephant grass

Soil occupation capacity via lateral expansion of tussocks in elephant grass (Pennisetum purpureum Schum.) may be associated with basal tillering. As grazing management alters the proportion of basal and aerial tillers in a tiller population, the hypothesis of this work was that grazing management affects tussock size and distribution with implications for plant population stability. The objective of this study was to evaluate the tiller population stability index, the proportion of basal and aerial tillers, tussock size, and the frequency of tussocks and bare ground in rotationally managed elephant grass cv. Napier. Treatments resulted from the combination of two post‐grazing heights (35 and 45 cm) and two pre‐grazing conditions (95% and maximum canopy light interception during regrowth – LI_0·95 and LI_Max) and were allocated to experimental units (850 m² paddocks) according to a 2 × 2 factorial arrangement in a randomized complete block design, with four replications. Measurements were taken from January 2011 to April 2012. The post‐grazing height treatments affected the tiller population stability index, but did not influence the pattern of tussock distribution. On the other hand, the different grazing frequencies (targets of LI pre‐grazing) altered the pattern of tussock distribution and the proportion of bare ground. In general, the tiller population stability index and frequency of tussocks were higher and the frequency of bare ground lower on swards managed with the LI_0·95 target relative to those managed with the LI_Max target, regardless of the post‐grazing height used, indicating a larger soil occupation capacity of plants under the more frequent defoliation regime. Such responses were associated with larger population of basal tillers and highlight the importance of tiller category and perennation pathway in defining patterns of plant growth and tussock distribution.     

新疆伊犁地区小麦条锈菌群体遗传结构分析

新疆伊犁地区是小麦条锈病的常发区和重发区，其年平均发生面积超过100×10⁴ hm²以上。为明确新疆伊犁地区小麦条锈菌群体的遗传结构，2021年，从新疆伊犁地区的新源县、巩留县、察布查尔县和伊宁县共采集到320份小麦条锈菌标样，利用16对小麦条锈菌SSR引物进行了群体遗传研究。结果表明，新疆伊犁地区条锈菌群体遗传多样性水平较高，群体间存在一定的遗传分化。群体间遗传变异仅占总变异的12%，群体内遗传变异占88%，不同群体间存在频繁的菌源交流，其中察布查尔县与新源县群体菌源交流最为频繁（N_m=4.717）。     

三个小麦-华山新麦草二体附加系分子细胞遗传学的初步鉴定

华山新麦草是我国华山地区特有小麦近缘物种，具有抗逆、早熟、优质等特点，对小麦的白粉病、条锈病、赤霉病也具有良好的抗性，是小麦重要的三级基因库。为进一步更好地发挥华山新麦草基因资源的作用，本研究利用细胞学方法、原位杂交技术、分子标记技术、农艺性状评估和抗病性鉴定对普通小麦-华山新麦草衍生后代材料进行鉴定，并获得了 20H11、17H21、18H28 三个小麦-华山新麦草衍生系。结果表明，20H11、17H21 和 18H28 的染色体数都是 44 条，均携带2 条属于华山新麦草的 Ns 染色体和 42 条普通小麦染色体。利用华山新麦草的特异分子标记进行检测，证明 20H11、17H21 和 18H28分别为 3Ns、4Ns和 2Ns二体附加系。通过农艺性状调查，3个试验异染色体系与亲本7182相比，株高和小穗粒数的差异不显著；分蘖数目与亲本7182有显著差异；20H11的穗长显著短于亲本7182；17H21的分蘖数显著少于亲本7182。经条锈病抗性鉴定，18H28 高抗条锈病小种 CYR32 和 CYR34。因此，这三个华山新麦草衍生系可作为中间材料用于小麦遗传育种研究。     

Regrowth patterns of elephant grass (Pennisetum purpureum Schum) subjected to strategies of intermittent stocking management

The morphogenetic responses of individual tillers and the composition of tiller populations throughout regrowth in response to all combinations of two post‐grazing heights (35 and 45 cm) and two pre‐grazing conditions (95% and maximum canopy light interception during regrowth – LI_95% and LI_Max) were studied in swards of elephant grass (Pennisetum purpureum Schum. cv. Napier) from November 2011 to April 2012. Swards were subjected to the same treatments from January 2011 until measurements began, to enable them to adapt to the defoliation regimes. Treatments were allocated to experimental units (850 m² paddocks) according to a 2 × 2 factorial arrangement in a randomized complete block design, with four replications. The total number of tillers (basal + aerial) was similar for both LI pre‐grazing targets. However, there was a change in the preferential growth pathway used by plants characterized by modification in the proportion of tiller classes in plant population. Swards managed with the LI_Max target had a smaller population density of basal tillers and used the recruitment of aerial tillers as the main growth pathway. Although effective for maintaining stable rates of leaf elongation and senescence, such a strategy resulted in greater tiller death. Additionally, stem elongation, a consequence of the competition for light under those circumstances, resulted in differences in sward herbage mass and morphological composition with potential implications for herbage feeding value.     

小黑麦杂交F_1代真假杂种的ISSR标记鉴定及遗传多样性分析

为了给ISSR标记技术应用于小黑麦杂种鉴定提供参考,利用ISSR标记对小黑麦品系P1和P_2有性杂交得到的F_1代群体进行分子鉴定,同时结合田间表型调查结果对F_1代群体进行遗传多样性分析。结果表明,66个杂交F_1代单株中,52个为真杂种,其中,5个单株无主茎、未结实,1个生长后期死亡。14条ISSR引物对46个正常结实真杂种扩增的多态性条带百分率为61.19%,其中,UBC822的多态性条带百分率为100%,UBC808和UBC815的多态性条带百分率均为75%。F_1代群体和父母本的遗传相似系数为0.62~0.94,说明杂交后代产生了丰富的遗传变异。聚类分析将杂交F_1代群体和父母本在阈值0.748处分为3大类群,第1类群包括1份材料(父本,P1),第2类群包括1份材料(母本,P_2),第3类包括46个杂交F_1代单株;杂交F_1代与父母本的遗传距离较远,先与母本聚为一类,之后与父本聚为一类。田间调查结果表明,7个性状中,除株高偏向于父本外,分蘖数、有效分蘖数、穗长、小穗数、穗粒数和穗粒重均偏向于母本,与父本的差距较大,这与ISSR分析结果基本相吻合。     

Simulating growth,development, and yield of tillering pearl millet: I. Leaf area profiles on main shoots and tillers

Pearl millet (Pennisteum americanum L.) is an essential crop in farming systems of the dry areas of the semi-arid tropics and its tillering habit is an important adaptive feature. This is the first paper in a series aiming at developing and validating a pearl millet simulation model that recognises tillers as functional entities, analogous to intercrops. The objective of this paper is to quantify the effects of total leaf number per axis (TLN), cultivar, plant density and axis number on parameters that are used to simulate potential leaf area per plant. Four cultivars with different phenology and tillering habit were grown under well-watered and well-fertilised conditions at two locations in India, covering a range of daylengths and plant densities. For selected plants, the area of fully expanded leaves was measured non-destructively. A bell-shaped function adequately described the relationship between individual leaf area and leaf position on an axis. Its shape was determined by the position (X₀) and area (Y₀) of the largest leaf and by the breadth and skewness of the leaf area profile curve. TLN affected all four parameters, although the association with Y₀ was weak. Cultivar only affected Y₀, suggesting that parameterising new cultivars is straightforward. The observed density effect confirmed that competition for light between axes started during stem elongation. The results highlighted the consistent differences between leaf area profiles of main shoots and tillers. For a high-tillering crop like pearl millet, modelling leaf area dynamics through individual leaves is justified, as this approach can potentially deal with cultivar and environmental effects on tillering.     

Grain weight response to foliar diseases control in wheat (Triticum aestivum L.)

Foliar diseases are the main biotic restriction reducing yield in wheat crops affecting both, grain number and/or grain weight, depending on developmental stage at which infection occurs (pre- or post-anthesis, respectively). Grain weight reductions due to foliar diseases were widely reported in the literature mostly associated with decreases on radiation interception during the grain filling period. However, different evidences in wheat showed variations on grain weight responses when fungicide was applied during the grain filling period, probably associated with the timing of fungicide application or with the amount of available resources per grain set when fungicides are applied. The present study was designed to determine the causes of grain weight reduction due to foliar diseases complex (including leaf rust, Septoria leaf blotch and tan spot) in wheat crops growing under contrasting agronomic and environmental conditions (i.e. different years, locations, cultivars and N supply). The experiments were carried out during 4 years under field conditions in different locations of Argentine and France. Five different commercial wheat cultivars were sown on early and late sowing dates; and two contrasting N availability and two fungicide treatments (protected and unprotected) were applied. Grain number was not affected by foliar diseases as their appeared after anthesis. Grain weight was strongly, poorly or not affected by foliar diseases and was not associated individually with both, the sink size and the source size. However, when the grain weight response due to fungicide application was plotted against the healthy area absorption per grain (HAA_G), a significant negative association (r² = 0.81; p < 0.0001) was found for the Argentine experiments. When the HAA_G was corrected by the grain weight potential (HAA_GW) all experiments conduced in Argentine and in France fit well to a common negative linear regression (r² = 0.74, p < 0.0001) for the relationship between grain weight variation and HAA_GW demonstrating that grain weight potential is an important feature to consider in diseases control programs. Foliar diseases forced the crop to use the accumulated reserved increasing the utilization rate of the water soluble carbohydrates (WSC_UR), depleting as a consequence the water soluble content at physiological maturity (WSC_PM) in all experiments. The association between WSC_UR and the healthy area absorption per grain corrected by grain weight of healthy crops (HAA_GW) suggest that foliar diseases in wheat cause source limitation, forcing to the crop to use the WSC reserve which could be insufficient to fill the grains previously formed.     

转TaSCL14基因小麦的遗传稳定性及农艺性状分析

为了获得农艺性状优良的转TaSCL14基因小麦品系,以普通小麦品种小偃39为受体获得的转TaSCL14基因小麦T1～T3代株(系)为材料,对其中TaSCL14基因的遗传稳定性和主要的农艺特性进行分析。结果表明,转TaSCL14基因小麦的T1和T2代植株的阳性率分别达到55.9%和85.6%,穗粒数、冬前分蘖与对照存在显著差异;T3代4个转基因株系3-4、3-7、3-8和3-11的阳性率达到90%以上,且TaSCL14基因在各株系中的表达水平都高于对照。与野生型相比,转基因小麦T3代部分株系的冬前分蘖、冬后分蘖、有效分蘖和小穗数均较野生型呈显著或极显著提高,但千粒重都较野生型有所降低,其中株系3-4和3-7降低幅度较大,分别达到显著和极显著水平。以上结果说明,TaSCL14基因的过表达能够影响转基因小麦的部分农艺特性,并且在株(系)间有差异。