苦荞产区种质资源遗传多样性和遗传结构分析

【目的】了解苦荞产区种质资源遗传多样性和遗传结构,为苦荞资源的有效利用提供参考。【方法】采用相关性分析、主成分分析方法对苦荞8个植株性状进行分析;温室内培养苦荞资源,于3叶期,每个资源选取10株新鲜叶片,采用CTAB方法提取苦荞基因组DNA,结合SSR分子标记方法进行PCR扩增,然后对扩增产物进行电泳检测并照相保存,根据SSR检测位点构建[0,1] 矩阵,最后利用PowerMarker3.25和Structure2.3.4软件对83份苦荞种质资源进行遗传多样性和群体遗传结构分析。【结果】8个植株性状分布较分散,大部分植株性状间呈现显著相关,植株性状的前4个主成分累计贡献率达到85.22%,基本可以显示苦荞种质资源植株性状的相关性关系;不同植株性状间株高和主茎粗变异系数最大,遗传变异最丰富。不同省份的资源表现出不同的遗传多样性,西藏资源的表型遗传多样指数H′ 均值最高,为1.82,其次为四川,遗传多样性指数H′ 均值为1.78;不同省份资源植株性状的遗传多样性存在差异,四川生育期、株高、主茎分枝的遗传多样性指数H′ 最高,陕西叶宽的遗传多样性指数H′ 最高,云南千粒重的遗传多样性指数H′ 最高;植株性状的主成分分析表明相似产区的植株性状具有一定的相关性。13条核心引物共检测出208条清晰的条带,其中200条（96.15%）具有多态性,平均每条引物扩增出的条带数和多态性条带数分别为16个和15.4个;不同引物等位基因变化范围为4-58个,重要的基因频率变化范围为0.02-0.86,多样性指数变化范围为0.38-0.98,多态信息量（PIC）变化范围为0.35-0.98;不同地理来源苦荞种质资源的遗传多样性表明,北方产区亲缘关系较近,西南产区亲缘关系较近,说明不同地理来源的群体类别与产区存在一定的关系;来自陕西群体的等位基因数量最多、基因多样性指数和多态性信息量最高,分别为12.0769、0.8365和0.8265;基于模型的遗传结构分析将苦荞资源划分为3个类群,基于遗传距离的聚类显示苦荞种质资源穿插分布,资源的地理来源地分化不明显,但是同一产区的资源遗传距离较近,资源之间具有一定的产区分化。【结论】苦荞产区种质资源PIC较高,遗传多样性丰富,2大产区具有一定的资源交流和遗传物质交换。     

小麦wzy2-1基因的克隆及功能分析

脱水素是一类植物胚胎发育后期丰富蛋白(late embryogenesis abundant protein, LEA蛋白),属于LEA D-11家族,植物受非生物胁迫会大量表达。利用同源克隆技术,从郑引1号小麦中克隆了1个Kn型脱水素基因,命名为wzy2-1。该基因全长1740bp,编码579个氨基酸,含有9个保守的K片段,与大麦的Dhn5基因具有较高同源性。生物信息学预测该蛋白属于高度亲水性的无序蛋白。通过该蛋白对大肠杆菌的保护作用研究表明,WZY2-1蛋白能够提高大肠杆菌对低温、高温、高盐以及高渗胁迫的耐受性。荧光实时定量PCR分析表明,wzy2-1基因受低温、盐渍、干旱诱导表达,但不受外源ABA诱导,说明wzy2-1基因属于非ABA依赖型脱水素基因。     

水分胁迫对糜子物质运转和籽粒灌浆特性的影响

为探索不同生育期不同程度水分胁迫下糜子籽粒灌浆特性及产量形成特点,以榆糜2号为材料,采用盆栽试验,利用Richards方程对不同处理强、弱势粒的灌浆过程进行比较,进而分析研究了拔节期、孕穗期和开花成熟期中度(占田间最大持水量的40%~45%)、重度水分胁迫(占田间最大持水量的20%~25%)对糜子强、弱势粒灌浆特性与抽穗后物质积累和转运及产量形成的影响。结果表明:与对照(全生育期充足供水,占田间最大持水量的70%~75%)相比,重度水分胁迫下,籽粒的起始生长势R0降低,最大灌浆速率出现的时间Tmax延迟,灌浆后期时间缩短,灌浆中期和后期的贡献率下降,茎鞘向穗部的物质转运率降低,进而影响结实率和产量,其中孕穗期重度水分胁迫的穗粒数和穗粒质量比对照分别降低了44.62%和38.19%,是降幅最大的;中度水分胁迫加快了糜子灌浆进程,促进了糜子叶片和茎鞘向穗部的物质转运率,提高了结实率和籽粒充实度,提高了糜子产量,其中拔节期中度水分胁迫是增加幅度最大的,其穗粒数、结实率、千粒质量和穗粒质量比对照分别增加了24.17%、17.92%、7.19%、41.14%。因此,在适度水分胁迫下,协调并提高糜子籽粒的灌浆起始生长势和前期、中期的灌浆速率以及茎鞘向穗的物质转运率,对于保证和提高糜子产量有重要意义。     

苦荞遗传多样性分析与核心种质筛选

利用SSR分子标记技术,对来自西藏、陕西、四川、贵州等4省区的210份苦荞品种进行遗传多样性研究。结果表明,所选用的50对SSR引物中,有16对引物多态性良好,共扩增出178个条带,其中多态性条带达118个,占总数的66.3%;210份苦荞种质的遗传变幅为0.62~0.98,平均值为0.80,在遗传相似系数为0.88处可划分为5大类。聚类结果显示,来自同一地区的苦荞品种显示出聚为一类的趋势,表明苦荞的遗传信息受地理分布的影响较大;第V类所聚的41份分别来自西藏(27份)、陕西(8份)和贵州(6份)的品种表现出较为丰富的遗传多样性,与其他种质相比,这41份材料不仅遗传差异较大,而且遗传来源广泛,可作为苦荞核心种质筛选的基础。     

Effects of progressive drought on photosynthesis and partitioning of absorbed light in apple trees

To understand how drought stress affects CO2 assimilation and energy partitioning in apple(Malus domestica Borkh.), we investigated photosynthesis and photo-protective mechanisms when irrigation was withheld from potted Fuji trees. As the drought progressing, soil relative water content(SRWC) decreased from 87 to 24% in 15 d; this combined the decreasing in leaf relative water content(LRWC), net photosynthesis rate(P n) and stomatal conductance(G s). However, the concentrations of chlorophylls(Chl) remained unchanged while P n values were declining. Photochemistry reactions were slightly down-regulated only under severe drought. Rubisco activity was significantly decreased as drought conditions became more severe. The actual efficiency of photosystem II(ΦPSII) was diminished as drought became more intense. Consequently, xanthophyll-regulated dissipation of thermal energy was greatly enhanced. Simultaneously, the ratio of ΦPSII to the quantum yield of carbon metabolism, which is measured under non-photorespiratory conditions, increased in parallel with drought severity. Our results indicate that, under progressive drought stress, the reduction in photosynthesis in apple leaves can be attributed primarily to stomatal limitations and the inhibited capacity for CO2 fixation. Xanthophyll cycle-dependent thermal dissipation and the Mehler reaction are the most important pathways for dispersing excess energy from apple leaves during periods of drought stress.     

Molecular variability of Apple chlorotic leaf spot virus in Shaanxi,China

Apple chlorotic leaf spot virus (ACLSV) is one of the latent viruses that occur in apple orchards worldwide but usually without visible symptoms. In 2010–2012, a total of 550 apple leaf samples from 12 different major apple-producing areas in Shaanxi, China, were tested by serological assay for ACLSV; the results revealed an infection level of 51.5 %. Because of the known variability in the putative amino acid sequences of the coat protein (CP), and thus the potential for non-detection by serological assay, the molecular variability of isolates of ACLSV collected in Shaanxi was analyzed using PCR and compared with isolates from the rest of the world. Sequences of 504 nt corresponding to 87 % of the CP gene of 12 isolates were acquired by RT-PCR and deposited in GenBank with the accession numbers KF134387–KF134298. Comparisons of the partial CP gene sequences of these 12 isolates as well as isolates previously reported in the world revealed the pairwise identities ranging from 68.9–99.8 % and 73.8–100 % at the nucleotide and amino acid level, respectively. Phylogenetic analysis based on these nucleotide sequences showed that the 72 isolates deposited in GenBank fell into three groups (P205, B6 and Ta Tao 5 Group). Our 12 ACLSV isolates were separated into the P205 and B6 groups, respectively. Multiple alignment analysis of the amino acid sequences of CP revealed that there was a combination of six amino acids at positions 40, 59, 75, 86, 130 and 184 in isolates from each group that could be used to distinguish among the three groups. Two recombination events were identified from all isolates by recombination analysis, and three ACLSV isolates collected in this study participated in these two events. Our results show that molecular variation was present in isolates of ACLSV collected in Shaanxi province and this may reflect introductions of the virus associated with different sources of germplasm.     

蚜虫内共生菌基因组DNA提取方法的比较和优化

为探究蚜虫共生菌基因组DNA的提取方案,以桃蚜为试验材料,比较了目前较为常用的4种蚜虫基因组DNA提取方法,从DNA纯度、完整性、PCR扩增效率及稳定性等方面进行了比较和评价,并通过调整蛋白酶K用量和水浴温度及时间对STE法进行了优化。结果表明,4种方法提取的蚜虫共生菌基因组DNA均可用于共生菌的PCR扩增检测;CTAB法和SDS法提取的DNA纯度较高,条带较完整,稳定性相对较高,不易降解,而STE法和PCR缓冲液法操作简便,适于快速提取单头蚜虫共生菌的基因组DNA,但纯度相对较低;可根据试验条件和要求进行选择。STE法优化条件为:用30 μL STE缓冲液将蚜虫匀浆,加入1.5 μL 20 mg/mL蛋白酶K,于56 ℃水浴1.5 h;再加入0.1 μL 10 mg/L RNA酶,于37 ℃培养1 h,95 ℃下处理5 min,5 000 r/min离心3 min,将提取到的DNA于-20 ℃保存或直接用于PCR扩增。优化后的STE法可作为提取蚜虫共生菌基因组DNA经济而快捷有效的方法。     

钾肥与吲哚-3-乙酸共同作用对小麦-麦长管蚜-短翅蚜小蜂三级营养关系的影响

为明确钾肥和吲哚-3-乙酸(IAA)的相互作用对调控小麦-麦长管蚜-短翅蚜小蜂载体植物系统内3个营养级生长发育的影响,通过外源浇灌含223.5、447和894 mg/L KCl的Hoagland营养液和喷施0、50、150和250 mg/L IAA溶液的组合,研究了其对小麦叶片各生理指标、麦长管蚜体重差及体内代谢酶活力、短翅蚜小蜂体长及体内代谢酶活力的影响。结果表明,不同KCl和IAA组合对3个营养级的影响差异显著。447 mg/L KCl与150 mg/L IAA处理下的小麦叶面积最大,为9.57±0.16 cm²;223.5 mg/L KCl与50、250 mg/L IAA处理组的叶片含水量最高,达89.69%;叶绿素和类胡萝卜素含量最高的处理组是447 mg/L KCl与250 mg/L IAA;麦长管蚜体重增长最多及短翅蚜小蜂体长最长的处理组均为447 mg/L KCl与50 mg/L IAA;各处理组对3个营养级酶活水平的影响程度不同。KCl和IAA在多个指标中表现出交互效应,表明选用合适的组合不仅能饲养出优异的适宜繁殖寄生蜂的替代寄主,还可最大限度地培养优秀蜂源。     

陕西省小麦条锈菌群体遗传结构分析

 利用TP-M13-SSR自动荧光检测技术,对陕西省小麦条锈菌群体遗传结构进行了分析。研究结果显示,在物种水平上,陕西省小麦条锈菌群体Nei’s基因多样性指数（H）为0.18、Shannon 信息指数（I）为0.29,表明该省条锈菌群体遗传多样性较为丰富。同时,条锈菌群体遗传多样性在地区之间也存在明显的差异,在7个条锈菌群体中,宁强种群遗传多样性相对较高（H为0.18,I为0.28）。AMOVA分析显示,陕西省小麦条锈菌在地区间、种群间和群体内的遗传分化分别为：12.26%、10.88%和76.86%,表明陕西省小麦条锈菌群体存在一定的遗传分化,但遗传变异主要发生在群体内部。