苜蓿属4种不同类型材料亲缘关系研究

利用表型性状和ISSR分子标记技术对3份白花苜蓿、3份黄花苜蓿、5份紫花苜蓿和3份扁蓿豆共计14份苜蓿属材料进行研究,分析其遗传关系,进一步解析其之间的分类学关系。从表型性状来看,白花苜蓿区别于其他苜蓿的主要形态学特征是花色为乳白色,荚果1.0~1.5个螺旋,从聚类分析来看,3份扁蓿豆材料和11份苜蓿材料非常明显的聚成了2类,说明扁蓿豆与苜蓿还是有较大的区别,在遗传距离15的地方,白花苜蓿、紫花苜蓿和黄花苜蓿各单独聚为1类;从亲缘关系来看,扁蓿豆与黄花苜蓿的遗传距离最近,与紫花苜蓿的最远,白花苜蓿次之,相对于紫花苜蓿,白花苜蓿与黄花苜蓿的遗传距离较近;从ISSR分子标记水平来看,4类苜蓿种质资源在遗传距离0.80的地方分别聚为单独的4类,从遗传关系来看,白花苜蓿相对于黄花苜蓿与紫花苜蓿的遗传关系更近,但紫花苜蓿与白花苜蓿和黄花苜蓿的遗传关系几乎相等。     

利用微卫星标记鉴定扁蓿豆种质资源

对扁蓿豆种质资源进行遗传多样性和亲缘关系分析,为育种者选择亲本和种质资源的开发提供理论依据。采用微卫星分子标记对来自内蒙古野生扁蓿豆种质资源50份材料进行鉴定。用8份扁蓿豆基因组DNA从89对截形苜蓿SSR引物中鉴定筛选出扩增带单一、稳定清晰且多态性强的18对引物。用这18对引物,对扁蓿豆材料的DNA进行SSR扩增,以研究其遗传多态性。结果共检测到109个等位位点,平均每对引物扩增出6.1个等位位点。6对SSR引物BI4BO3,MTIC272,MAL369471,MTIC237,MTIC188和MTIC27对于检测扁蓿豆遗传变异最有效。供试材料间遗传距离介于0.023 6~0.807 5,平均遗传距离为0.177 8。聚类分析构建了亲缘关系树状图,大致分为9大类。     

扁蓿豆不同种质材料种子萌发期抗旱性比较

采用5个PEG渗透势梯度模拟干旱胁迫,以黄花苜蓿作对照,探讨了4份扁蓿豆种质材料种子萌发期的抗旱性。通过对种子相对发芽率、相对活力指数和半致死渗透胁迫强度3项指标的综合分析,扁蓿豆种质材料在种子萌发期的抗旱性高于黄花苜蓿,4份扁蓿豆种质材料抗旱性大小顺序为:材料4>材料1>材料3>材料2。其半致死渗透胁迫强度分别为:-1.30MPa、-1.03MPa、-0.85MPa、-0.78MPa。     

采用AFLP和SSR标记对扁蓿豆遗传多样性进行比较分析

为了筛选出优异的扁蓿豆育种新材料,本研究采用AFLP和SSR分子标记技术对来自于中国7个省市自治区的15份扁蓿豆种质资源进行遗传多样性的比较分析,结果表明:18对SSR引物扩增出109个多态位点,8个AFLP引物组合扩增出640条带,其中472条多态带.AFLP标记的平均Nei′s遗传多样性指数、Shannon多样性指数和遗传分化系数均高于SSR标记.15份扁蓿豆种质的遗传距离和遗传相似系数与地理类群很接近.AFLP和SSR数据的聚类分析显示:15份扁蓿豆种质分为4大类,但是聚类结果与地理类群不完全相符,主成分结果与聚类结果相似,Mantel 检测表明:AFLP和SSR数据有较高的显著相关性,AFLP和SSR标记能够有效地对扁蓿豆进行遗传多样性分析,其结果为扁蓿豆育种和资源保护具有指导意义.     

中国扁蓿豆种质资源遗传多样性的ISSR标记与生态因子相关性分析

采用ISSR分子标记技术对来自国内7个省市自治区的49个扁蓿豆居群的遗传多样性及生态因子的相关性进行分析,为扁蓿豆种质资源的保护利用提供理论依据和技术支撑。研究结果表明：采用筛选的15个条带清楚且稳定的引物,共获得127个扩增位点,其中,多态性位点110个,多态性位点的比例为85.61%,平均每个引物的多态扩增位点为7.33个;居群间基因多样性指数、Shannon指数、基因分化系数3个指标均大于地区间,内蒙古地区的遗传多样性较丰富,北京地区的遗传差异较小;聚类和主成分分析将49个扁蓿豆居群分为6大类,供试扁蓿豆种质资源呈现出较好的地域性分布规律;基因多样性指数、Shannon指数及多态位点百分率均与年降水量呈显著负相关。     

采用AFLP和SSR标记对扁蓿豆遗传多样性进行比较分析（英文）

为了筛选出优异的扁蓿豆育种新材料,本研究采用AFLP和SSR分子标记技术对来自于中国7个省市自治区的15份扁蓿豆种质资源进行遗传多样性的比较分析,结果表明:18对SSR引物扩增出109个多态位点,8个AFLP引物组合扩增出640条带,其中472条多态带。AFLP标记的平均Nei's遗传多样性指数、Shannon多样性指数和遗传分化系数均高于SSR标记。15份扁蓿豆种质的遗传距离和遗传相似系数与地理类群很接近。AFLP和SSR数据的聚类分析显示:15份扁蓿豆种质分为4大类,但是聚类结果与地理类群不完全相符,主成分结果与聚类结果相似,Mantel检测表明:AFLP和SSR数据有较高的显著相关性,AFLP和SSR标记能够有效地对扁蓿豆进行遗传多样性分析,其结果为扁蓿豆育种和资源保护具有指导意义。     

正交设计优化扁蓿豆ISSR反应体系的研究(英文)

以扁蓿豆(Medicago ruthenica)为试材,采用改良的CTAB法提取DNA,利用正交设计L16(45)探讨10×PCRBuffer(含Mg2+)、dNTPs、引物、TaqDNA聚合酶及模板DNA用量对扁蓿豆ISSR-PCR反应的影响,正交试验的结果采用直观分析和方差分析相结合。建立了扁蓿豆的ISSR-PCR优化反应体系,在25μL反应体系中,TaqDNA聚合酶1.5U,10×PCR Buffer(含Mg2+)2.0 mmol/L,模板DNA0.5 ng/μL,dNTPs 0.6 mmol/L,引物0.9μmol/L。同时探讨引物HZD09211的最适退火温度为52.3℃。2个不同引物对20份扁蓿豆材料DNA进行ISSR-PCR扩增,结果显示该体系具有较高的稳定性。     

不同生态型扁蓿豆种子性状的遗传多样性和相关性分析

对扃蓿豆(Medicago ruthenica (L.) Trautv.)不同生态区的20份野生材料种子性状的变异进行系统研究,分别测定千粒重、种子长、种子宽和种子长宽比共4个种子形态指标,方差分析表明:扁蓿豆种子性状变异幅度最大的为千粒重,变异系数为12.50％,所有性状均表现差异极显著.主成分分析表明:前2个主成分包含了原来4个性状指标的93.74％,足以能够代表所有性状变异的全部信息,特征值总和为3.749.聚类分析表明:在遗传距离为12时,可以把20份野生材料分为六大类.相关分析表明:种子长和种子宽、种子宽和千粒重均呈极显著正相关,而种子长和种子长宽比、种子长和千粒重均呈显著正相关.研究表明,扁蓿豆植物野生种质材料具有丰富的遗传多样性.     

扁蓿豆DNA甲基转移酶基因MrDRM2的克隆和生物信息学分析

由DNA甲基转移酶催化的DNA甲基化在调控基因表达及植物生长发育中发挥重要作用。本研究根据扁蓿豆(Medicago ruthenica L.)的转录组测序信息设计特异性引物,利用RT-PCR技术从中克隆到1个DNA甲基转移酶基因的c DNA片段,长1 767 bp,编码588个氨基酸残基。利用生物信息学分析推测这个基因属于DNA甲基转移酶中的结构域重排甲基转移酶(domains-rearranged methyltransferases, DRMs)亚家族。系统发育分析表明该基因与豆科其他植物的DRM2基因具有较高的同源性。利用实时荧光定量PCR分析其在扁蓿豆受到低温(4℃)、Na Cl、干旱胁迫及ABA处理下不同时间的表达量,发现其表达受非生物胁迫诱导。推测MrDRM2基因参与了扁蓿豆对非生物胁迫响应的调控。本研究结果可进一步丰富扁蓿豆DNA甲基转移酶基因信息,为深入理解扁蓿豆DNA甲基转移酶基因的功能及其在非生物胁迫应答中的表观遗传调控提供一定的基础。     

扁蓿豆开花习性及花荚脱落现象的初步研究

就扁蓿豆在呼和浩特地区栽培条件下的开花时间和花蕾、花、荚的脱落进行了研究.结果表明,在呼和浩特地区扁蓿豆每日内花朵集中开放的时间为10：00～14：00,开花时的最适宜温度为29～32℃,适宜湿度为54%～64%.扁蓿豆的花蕾、花及荚的脱落中,以落花为主,其次是落荚,花蕾的脱落最少.扁蓿豆结荚率为32%～43%,落花和落荚是限制其种子高产的重要因素.     

Organelle based molecular analyses of the genetic relatedness of cultivated alfalfa (Medicago sativa L) to Medicago edgeworthii Sirjaev, and Medicago ruthenica (L.) Ledebour

Medicago edgeworthii Sirjaev and M. ruthenica (L.) Ledebour are allogamous, diploid (2n = 2x = 16) perennials with flat pods.Medicago edgeworthii is indigenous to the Himalayas and alpine areas west to Afghanistan, and Medicago ruthenica is found in Siberia, Mongolia, and Manchuria on open hillsides and mixed grass steppes. Because both species have a remarkable ability to survive extreme cold and poor soils, the possibility of hybridizing them with alfalfa (M. sativa L.) is being investigated. The objective of this research was to conduct an organelle based molecular assessment of the genetic relatedness of cultivated alfalfa (2n = 4x = 32) to M. edgeworthii and M. ruthenica. A hypervariable, intergenic region of cpDNA was amplified, and mtDNA was amplified with two primer pairs developed from soybean (Glycine max L.) mtDNA sequences. Mean Nei and Li genetic distances (GDs) between alfalfa and M. edgeworthii and alfalfa and M. ruthenica were 0.56 and 0.48 (mtDNA), and 0.33 and 0.30 (cpDNA), respectively. Intra specific GDs were 0.37 (mtDNA) and 0.25 (cpDNA) for M. edgeworthii; 0.42 (mtDNA) and 0.15 (cpDNA) for M. ruthenica; and 0 = 0.50 (mtDNA) and 0 = 0.23 (cpDNA) for alfalfa. Cluster analyses grouped someM. edgeworthii and M. ruthenica entries with alfalfa entries. There is some chance that alfalfa and M. edgeworthii entries which clustered closely could be hybridized; chances of alfalfa × M. ruthenica hybridizations appear to be more problematic. This revised version was published online in July 2006 with corrections to the Cover Date.     

Effects of Temperature and Frost on Genotypes of Medicago truncatuh L. and Medicago aculeata L. from Contrasting Climatic Origins

The growth of M. truncatula and M. aculeata genotypes collected from sites of contrasting altitudes and winter temperatures was compared under different temperature regimes. Genotypes collected from mild winter environments produced more shoot dry matter, had higher leaf area and lower specific leaf weight than those from cold environments; however these effects were largely related to seed size for M. aculeata. Only genotypes from mild environments were responsive to temperature.
Frost tolerance of the genotypes was tested using a laboratory freezing test based on seedling survival. There was a relationship between frost tolerance and winter temperature at site of collection for M. aculeata , with the most frost tolerant genotypes coming from high altitudes. All genotypes of M. truncatula demonstrated low survival rates following frost damage. Genotypes from high altitudes represent a promising source for breeding for first tolerance with greater variation m M. aculeata than M. truncatula.     

几种多花黑麦草与紫花苜蓿品种的化感作用研究

本文对三个多花黑麦草与两个紫花苜蓿品种的茎、叶和根的水浸提液化感作用进行了研究分析。结果表明：三种花黑麦草与两种紫花苜蓿品种对受体赛迪7有一定的化感影响,对赛迪7的种子发芽都有抑制作用;多花黑麦草 Barextra和Barmega两个品种的根、茎、叶水浸提液对赛迪7的苗高、根长及生物量的积累都存在低促高抑的化感作用;供体不同部位化感作用不同;紫花苜蓿两个品种茎的水浸提液在三种浓度下均对赛迪7的根长有促进作用,对苗高有抑制作用。     

盐胁迫下金花菜和紫花苜蓿试管苗的转录组分析及其耐盐基因筛选

为筛选金花菜和紫花苜蓿的耐盐基因，本研究以盐胁迫下的金花菜(JHC)和紫花苜蓿(ZHMX)试管苗为试验材料进行转录组分析。结果表明：金花菜组和紫花苜蓿组差异基因数为26722，金花菜组vs紫花苜蓿组有15850个基因下调，有10872个基因上调。金花菜组和紫花苜蓿组差异基因KEGG富集显著的Pathways有核糖体、光合作用天线蛋白、糖酵解/糖异生、光合作用、苯丙烷生物合成等。金花菜组耐盐基因有碱性亮氨酸拉链43、NAC转录因子47、ABC转运A家族成员7、晚期胚胎发生丰富蛋白2、乙烯反应转录因子ERF110等基因；紫花苜蓿组耐盐基因有WRKY转录因子、蛋白TIFY 8、转录因子MYB13、核运输因子2A、低温盐响应蛋白等基因。本试验结果可为了解金花菜和紫花苜蓿的耐盐分子机制及选育金花菜和紫花苜蓿耐盐新品种提供理论依据。     

紫花苜蓿愈伤组织诱导和分化研究

以紫花苜蓿SANDITI无菌苗下胚轴、茎、叶3种不同外植体,在附加不同激素浓度的MS培养基上诱导愈伤组织。结果表明,SANDIT最佳外植体为下胚轴,最佳下胚轴愈伤组织诱导培养基为MS+2,4-D 2.0 mg/L+6-BA 0.5mg/L。15 d继代1次,或适当降低2,4-D浓度,提高6-BA或KT浓度,可以降低愈伤组织褐化率。愈伤组织在6-BA0.5 mg/L,NAA 0.1 mg/L,GA31.0 mg/L,YE 250 mg/L,CH 250 mg/L的MS培养基上,可获得较高的分化率。     

Transformation of Medicago sativa L. Using a Ti Plasmid Derived Vector

A cultivar of Medicago sativa was transformed with Agrobacterium tumefaciens strain At81 binary-vector carrying the plasmid pKan3A, with the neomycin phosphotransferase gene under the control of the mannopine biosynthesis promoter. Stem segments were infected with the bacterium and kanamycin resistant calli were obtained; plant regeneration via somatic embryogenesis and polyembryogenesis was achieved only in media devoid of the antibiotic. Genetic transformation was confirmed by the presence of the structural gene through DNA-DNA hybridization and the enzymatic assay showed its functional expression. Mesophyll protoplasts, leaf calli and S1 progeny of transformed plants grew in the presence of kanamycin (100 μgml^-1). Results are discussed in relation to the use of kanamycin-resistant plants in somatic hybridization in the genus Medicago.     

An improved breeding strategy for autotetraploid alfalfa (Medicago sativa L.)

Alfalfa cultivar development will be enhanced by breeding strategies whichutilize the full potential of autotetraploid population genetic structures. Thisstudy evaluates the effectiveness of an allelic selection scheme, which wasdeveloped to overcome limitations of inbreeding depression and to exploitgeneral and specific combining ability effects in autotetraploid populations.Allelic selection entails the minimization of non-additive genetic effects byselecting among full-sib families (F1) which are at uniform levels ofheterozygosity. Such F1 lines are developed by crossing individuals fromtwo unrelated random mating populations. Selected F1 lines wereintercrossed to form an improved population. Eight random matingpopulations of alfalfa were developed to study the effectiveness of allelicselection. Selection for increased dry matter yield resulted in alfalfapopulations with 38 percent greater yield than the parent populations. Twocycles of intercrossing, among selected F1 lines, did not dissipate the gainfrom selection. This result has important implications for synthetic cultivardevelopment in which a major limitation is the decline in productivity withadvancing generations of seed increase. A positively correlated response toselection for dry matter yield was observed for plant height and stemdiameter. The results of this research indicate that continued testing of theallelic selection scheme is warranted and could have a significant impact onthe breeding of autotetraploid alfalfa, particularly for synthetic cultivardevelopment.     

Characterization of factors affecting plant regeneration frequency of Medicago lupulina L.

Summary Different hormones, length of hormone pretreatment, and developmental status of plants were found to influence the shoot and root differentiation frequencies from immature inflorescence explants of Medicago lupulina L., a diploid autogamous species. Explants of immature inflorescence pretreated with phytohormones for a short period (10 min –6 h) were placed on hormone-free medium for shoot differentiation. Cytokinins, such as BA, were found to be essential for shoot differentiation, whereas treatment with 2,4-D could only induce root differentiation. Explants with hormone pretreatment for 30 min to 1 h produced the highest shoot regeneration rate. Plants at the beginning of flowering were found to have a better regeneration ability than those at fruiting or maturation. After a 10 mg/l BA pretreatment, respective shoot differentiation frequencies of 39%, 36% and 23% were observed for explants of self-progeny plants, regenerated plants, and plants propagated by cuttings, although the three categories of plants were originally derived from the same plants.Abbreviations BA 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - 2iP 2-iso-pentenyladenine     

紫花苜蓿生产性能构成因子研究

对3种不同秋眠类型9个不同秋眠级别的苜蓿品种进行生产性能构成因子以及构成因子间的相互关系的研究,结果表明,植株高度与节间长和再生速度达显著相关(P<0.05);生物量与植株高度和再生速度达显著相关(P<0.05);关于再生速度与植株高度、节间长和生物量的影响程度拟合方程分别如下:Y1=37.165+8.17x1;Y2=2.28+1.13x1;Y=3.03x1.再生速度与植株高度、节间长和地上生物量有着很高的相关性.植株高度是苜蓿产量一个很好的预测指标;决定苜蓿地上生物量的最终决定因子是再生速度,所以苜蓿的再生性是苜蓿高产育种首先考虑的因子.     

硒胁迫对大叶紫花苜蓿生理特性的影响

以新疆大叶紫花苜蓿为试验材料,采用土培试验方法,研究了硒(Se)胁迫对大叶紫花苜蓿不同组织可溶性蛋白、脯氨酸、超氧阴离子和丙二醛(MDA)含量的影响及大叶紫花苜蓿对Se的积累能力。结果表明:胁迫60d时,随Se胁迫浓度的增加,大叶紫花苜蓿不同组织中超氧阴离子、MDA、脯氨酸和可溶性蛋白质含量增加,同时,新疆大叶紫花苜蓿根的Se积累量大于叶和茎的Se积累量。综合研究表明,在900μM Se胁迫下,根据渗透调节物质的积累量,初步判断大叶紫花苜蓿有解毒和富集Se的能力,具有用于修复Se污染土壤的潜能。