12个地方鸡种遗传多态性的AFLP指纹分析

利用6对AFLP引物组合对中国12个地方鸡种进行了遗传检测，构建了各个品种的AFLP DNA指纹图谱，根据AFLP分析结果, 统计了每个引物组合在各个品种中检测到的多态性条带和特异性条带，计算了12个地方鸡种的遗传相似系数和遗传距离，并据此构建了UPGMA聚类关系图，分析了所研究鸡种的遗传关系。结果表明：6对AFLP引物组合在12个地方鸡种中共检测到279条多态性条带，平均每个引物组合产生46.5条多态性标记，同时在每个品种群体中还检测到了数量不等的特异性条带，其中寿光鸡和东乡黑鸡最多，为9条，旧院黑鸡和兴义矮脚鸡最少，为1条。12 个鸡种聚为3类，鸡种间的遗传相似系数及聚类结果与所保存的地方鸡种的地理分布、现实状况是相吻合的。从而表明AFLP指纹用于我国地方鸡种遗传多态性分析、品种的鉴定及品种间的亲缘关系分析是可行的。     

Rhizobia in tropical legumes—XIII. Biochemical basis of acid and alkali reactions

Three fast-growing rhizobia (Rhizobium meliloti isolated from Medicago saliva, R. trifolii from Trifolium subterraneum, and Rhizohium sp. from Leucaena leucocephala) and three slow-growing rhizobia R. japonicum from Glycine max, Rhizobium spp from Centrosema pubescens and Crotolaria anagyroides) were grown in defined media. The mean generation times of the fast-growing and slowgrowing strains were 3.8 h and 8.6 h respectively. Slow-growing organisms raised the initial pH of the defined medium while the fast-growing organisms lowered it. Rates of oxygen consumption tended to be higher in the slow-growing organisms.UMKL 19 (isolated from L. leucocephala) possessed all the normal reactions of fast-growing rhizobia but had a single sub-polar flagellum, similar to the three slow-growing strains studied.Certain combinations of amino acids and sugars (e.g. glutamine and galactose) induced an acidic reaction in the fast-growing organisms while the slow-growing ones changed the media to alkaline. Fast-growing organisms utilized more galactose for growth compared to slow-growing ones. Both types of organisms synthesized and released a wide range of amino acids into the medium.We suggest that pH changes produced by rhizobia growing on yeast-extract mannitol media are caused by the preferential utilization of sugars by fast-growing organisms and nitrogenous compounds by slow-growing ones.     

Intrinsic antibiotic resistance and competition in fast- and slow-growing soybean rhizobia on a hybrid of Asian and US cultivars

Summary Six fast-growing soybean rhizobia (Rhizobium fredii) and thirteen slow-growing soybean rhizobia (Bradyrhizobium japonicum) were examined for resistance to 10 antibiotics. Axenic studies were carried out to determine the competitiveness of dual-strain inocula consisting of fast- and slow-growing rhizobia isolated from subtropical-tropical soils for nodule occupancy on a hybrid of Asian and US soybean cultivars. Nodule occupancy was determined by intrinsic resistance to erythromycin and neomycin. The results showed wide variability in resistance to 10 antibiotics for fast- and slow-growing rhizobia. The intrinsic antibiotic resistance of fast- and slow-growing rhizobia was extremely high against nalidixic acid (400 g ml^–1) and penicillin (200 g ml^–1). The competitive ability of inoculant strains for nodule occupancy varied for different combination sets and with the plant growing media. Our results show that fast-growing rhizobia nodulate a hybrid of Asian and US soybean cultivars. Fast-growing soybean rhizobia did not completely exclude nodulation by the slow-growing strains, which formed 0–79% nodules, depending on the strain used in the inoculum.     

Some factors affecting the survival of root-nodule bacteria on desiccation

The average number of survivors of fast-growing medic rhizobia (3 strains), fast-growing Rhizobium leguminosarum types (6 strains) and slow-growing species (9 strains) following desiccation of sandy soil inoculated with 10⁶ bacteria·g^?1 soil was 727, 795 and 15,682 bacteria·g^?1 soil, respectively. Survival in desiccated sandy soil was not influenced by the degree of extracellular polysaccharide production in strains of R. trifolii, nor was it influenced by growth of R. meliloti and slow-growing species in media of low water activity before desiccation in sandy soil.A progressive increase in numbers of fast-growing bacteria surviving desiccation was observed in sandy soil amended with increasing concentrations of powdered montmorillonite, but not with mont-morillonite added as a suspension to the soil. The clay had either a detrimental effect or no effect on the survival of the slow-growing rhizobia. Maltose, sucrose and polyvinylpyrrolidonc provided a greater degree of protection to both fast- and slow-growing rhizobia than was obtained with montmorillonite. The effect of polyethylene glycol 6000 was similar to the effect of montmorillonite, as the polymer only protected the fast-growing rhizobia and not the slow-growing species.     

镰刀菌rDNA ITS区 RFLPs分析

本实验采用PCR-RFLP技术分析了镰刀菌菌种间的多态性。运用SDS碱裂解法抽提了采自于山西省不同地区的11株镰刀菌（Fusarium）基因组DNA，并用一对特异性引物对其rDNA ITS区段进行扩增，选用4种限制性内切酶（TaqⅠ AluⅠ HaeⅢ EcoRⅠ）酶切PCR扩增产物，共得到16条多态性片段，其中特异性条带8条，分子量大约在70-900bp之间。利用NTSYS-PC(2.1)软件包分析各菌株间的DNA限制性片段多态性，总共可以分为５大组，与传统分类一致，说明该方法可以用于镰刀菌菌株间的多态性分析。     

小麦AB基因组中一个重复序列的分离、定位与应用

利用102对微卫星引物对5份黑麦（Secale）、4份普通小麦（Triticum aestivum）和1份分枝小黑麦（Triticale）进行SSR分析,引物Xgwm614能在分枝小黑麦中扩增出一个387bp的特异DNA片段（记为FZ387,GenBank登录号为EF179137）,而黑麦未能扩增出。序列比对结果显示该片段与一粒小麦（T. monococcum）（AY485644）和栽培二粒小麦（T. turgidum）（AY494981）A基因组中Gypsy Ty3-LTR反转座子fatima的一部分分别有94%和95%同源性。根据序列同源性比对结果,在FZ387内部设计1对特异引物FaF和FaR。引物Xgwm614F和FaR能在含有A基因组的物种中扩增出约350bp的条带（记为A350）,而其不含A基因组的物种都未扩增出该条带。利用小麦二体和端体代换系材料对其进行定位,结果显示该片段分布在所有A染色体的长臂和断臂上。此外,引物FaF和Xgwm614R能在含有A、B或AB基因组的物种中扩增出约350bp的条带（记为AB350）,而不含AB基因组的材料未扩增出目标条带。利用这两对特异引物对小麦属近缘物种进行PCR扩增,发现只有中国春能够扩增出A350和AB350。序列比对结果和FZ387两侧SSR引物结合区的规律性变化表明该反转座子在进化上可能存在属间多样性和属内相似性。A350和AB350也可以分别作为分子标记检测A染色体和AB染色体。     

Rhizobia in tropical legumes—XIV. Ion uptake differences between fast- and slow-growing strains

We compared the uptake of nitrogen, potassium and phosphorus (as well as ¹⁴C-labelled mannitol, ³H-labelled glutamate, and ³²P-labelled phosphate) in three fast- and three slow-growing rhizobia. The fast-growing strains used were Rhizobium meliloti (isolated from Medicago sativa), R. trifolii (from Trifolium subterraneum), and Rhizobium spp from Leucaena leucocephala, while the slow-growing strains were R. japonicum (Glycine max), and two Rhizobium spp (from Centrosema pubescens and Crotolaria anagyroides). Slow-growing organisms preferentially utilized glutamate in the medium. Both fast- and slow-growing strains took up more NH⁺₄-N than NO^?₃-N on a per cell basis. In the presence of mannitol, fast-growing strains can cause either acid or alkaline reactions, an effect that is dependent only on the N-source (NH⁺₄ or NO^?₃). Uptake preferences of the fast-growing Leucaena isolate (UMKL 19) resembled those of the slow-growing rhizobia, further strengthening the argument that this organism (and others like it) may be intermediate between the normal fast- and slow-growing groups. Generally, the efficiency of uptake of N (either as NH⁺₄ or NO^?₃), P, and therefore K, was greater in the fast-growing organisms.     

山西省快生型大豆根瘤菌资源调查和鉴定

从山西省主要大豆产区的不同土壤和大豆品种中分离得到的38个快生型大豆根瘸菌株的鉴定表明,这些分离物的IAR除了氨苄青霉素外,均较慢生型为低。38个菌株被分为4个血清型,其中2个为新发现的,命名为2077和2120型。细胞成分N%含量为2.01-3.78,C%含量为50.52-55.53%,N/C值<10。所检测的7个菌株都有1-2个大质粒,且每个均有112 Md的大质粒。分离株的共生效应和结瘤竞争由于大豆品种不同而有显著差异。本研究表明,我国大豆起源地之一的山西省,快生型大豆根瘸菌的分布广泛,分离频率较高,菌株类型也多。     

Acid production,acid tolerance and growth rate of Lotus rhizobia in laboratory media

Twenty-seven strains of Lotus rhizobia were tested for acid tolerance in yeast-extract mannitol broth (pH 4.6) by multiplication from low initial cell densities. Acid production on unbuffered yeast-extract mannitol agar slopes incorporating bromothymol blue indicator was also determined. All slow-growing, alkali-producing strains were acid sensitive and four of the six fast-growing, acid-producing strains were acid tolerant as was one fast-growing, alkali-producing strain. The method of testing for acid tolerance proved suitable for fast-growing strains and results are discussed in relation to the ecological implications of acid and alkali production by rhizobia.     

甘薯抗茎线虫病基因的AFLP标记的开发

以甘薯抗茎线虫病品种徐781和感茎线虫病品种徐薯18的杂交F1分离群体的186株单株为材料,利用分离群体分组分析法（BSA法）和AFLP技术,在抗感池中共筛选了800对AFLP引物,结果表明其中245对引物具有多态性。用这245对引物检测两亲本以及建池单株,发现引物组合E2M23和E33M20分别在抗病单株中扩增出1条在感病单株中未出现的特异条带,长度分别约为500 bp和200 bp,认为这2个AFLP标记与甘薯抗茎线虫病基因连锁,分别命名为E2M23500和E33M20200。根据这2个AFLP标记对F1代186个单株的扩增结果,经Mapmaker 3.0软件分析,发现这2个分子标记与抗茎线虫病基因位于同一连锁群并紧密连锁,它们与抗茎线虫病基因间的遗传距离分别为6.94 cM和11.1 cM。用这2个分子标记对10个中国甘薯主栽品种进行检测,所得结果与常规方法鉴定结果完全一致,表明2个分子标记可用于甘薯抗茎线虫病分子标记辅助育种。     

Identification of sex-specific DNA markers in betel vine (Piper betle L.)

The Random Amplified Polymorphic DNA (RAPD) technique was used to amplify DNA segments, with the objective of finding markers linked to sex determination in male and female plants of Piper betle L. Two bulks of DNA were made drawing one each from male and female, by pooling an equal volume of DNA samples from each group of individual contributing to the bulk analysis. Fifty different random decamer primers were screened with the two bulks to identify markers associated with sex expression of which only four primers were found to be associated with sex expression. These four primers were then tested with individual plant DNA samples where sex-associated RAPD markers were identified. A ~1,400 and ~850?bp fragment from the primer OPA04 and OPN 02 respectively was found to be present in all the male individuals and absent in all the female plants. In another primer, a ~980?bp amplification product from the primer OPC 06 was present only in the female individuals. A common primer OPA 08 showed both male and female specific markers of 650 and 1,200?bp respectively. Thus, the three male- specific RAPD markers OPA04₁₄₀₀, OPA08₆₅₀ and OPN02₈₅₀ and two female-specific markers OPA08₁₂₀₀ and OPC06₉₈₀ can reliably differentiate the male and female plants of P. betle L. Ploidy comparison also showed the differences in male and female plants.     

基于BOX-PCR和REP-PCR技术青枯雷尔氏菌遗传多样性分析

青枯雷尔氏菌(Ralstonias olanace arum)能够对许多重要作物引起致命性萎蔫病害,广泛分布在热带、亚热带以及温带地区.研究青枯雷尔氏菌的遗传多样性对于了解青枯病的发生和流行具有十分重要的意义.本研究利用青枯雷尔氏菌特异性引物鉴定84株来自福建地区不同寄主的青枯雷尔氏菌,结果表明,这些菌株均在504 bp位置出现特异性条带.同时采用BOX插入因子PCR(BOX-PCR)和重复基因外回文序列PCR(REP-PCR)对这些菌株进行基因多样性研究,基于它们所扩增出的基因指纹图谱表明,BOX-PCR扩增出19条特异性条带,REP-PCR扩增出20条特异性条带.系统聚类结果表明,青枯雷尔氏菌的遗传分化与寄主作物和地理来源都存在相关性,其中,寄主植物是在遗传差异中起主导作用.进一步分析可知,地域性的差异主要由BOX-PCR提供,寄主间的差异主要由REP-PCR提供.不同地理来源的青枯雷尔氏菌在BOX-PCR中可扩增出各自特异性条带,在REP-PCR中同样具有与各个寄主相对应的特异性条带,利用这些特异性条带可以很容易区分不同寄主以及来源的青枯雷尔氏菌.BOX-PCR和REP-PCR多态性分析技术可为我国青枯雷尔氏菌基因多样性的研究提供另一条途径.     

Survival of fast- and slow-growing Rhizobium spp under conditions of relatively mild desiccation

When subjected to desiccation with Ca(NO₃)₂ at 27°C, strains of the fast growing Rhizobium meliloti and R. trifolii, survived better than slow-growing strains of R. japonicum and of the “cowpea miscellany”. At lower vapour pressures in a forced-draught oven, the pattern of survival changed and strains of slow-glowing Rhizobium withstood desiccation better than those of fast-growing species. The results are considered to be consistent with the interpretation that a lower internal water-retaining ability at any relative vapour pressure, renders strains of slow-growing Rhizobium more resistant to severe desiccation than strains of fast-growing species. It is suggested that under conditions of milder desiccation, this property is disadvantageous to the slow-growing Rhizobium because insufficient moisture is available to allow for the functioning of vital enzymes.     

Molecular characterization of salinity tolerance in wheat (Triticum aestivum L.)

Random amplified polymorphic DNA (RAPD) markers were used to estimate the genetical variability of three salt-resistant genotypes, SARC-1, SARC-5 and S-24, exposed to saline environment. High-yielding and salt-sensitive variety MH-97 was used as standard for comparison. The behavior of these genotypes under saline environment was analyzed by using the hydroponics screening methods at the seedling stage. One hundred and fifty primers were tested of which 52 primers revealed differences between SARC-1 and SARC-5, 54 revealed differences between SARC-1 and S-24 and 61 revealed differences between SARC-5 and S-24. Polymorphism differences between MH-97 and SARC-1, MH-97 and SARC-5 and MH-97 and S-24 were 53%, 64% and 42%, respectively. Four primer pairs amplified special fragments, which were located in all the three salt-resistant genotypes but none on the salt-sensitive genotype MH-97. Primer GLD-15 (5?-CCGTGGCATT-3?) generated a prominent fragment of length 1460 bp; primer GLF-18 (5?-ACCCGGAACC-3?) produced a fragment of length nearly 980 bp in the salt-resistant genotype; the primer pair GLE-5 (5?-TTCAAGCCCG-3?) located one polymorphic amplified band of 1290 bp and the primer GLH-9 (5?-ATCCAGGTCA-3?) performed as a weak polymorphic band of 640 bp, respectively.     

两个黄鸡品种Ghrelin基因的PCR-SSCP分析

摘要：本研究针对Genbank上检索的Ghrelin基因的序列设计了两对引物，用PCR-SSCP方法检测了140只京海黄鸡和30只苏禽黄鸡该基因的多态性，结果表明：引物1在两个鸡品种中均未检测到多态；引物2检测到多态：其中京海黄鸡有BB，BC，CC三种基因型，在苏禽黄鸡中只检测到BB，BC两种基因型。在两个鸡品种中B等位基因为优势基因，分布较高，其中在苏禽黄鸡中BB基因型频率达0.933，而京海黄鸡为0.657。对两个纯合基因型测序分析表明：位于Ghrelin基因546bp处发生了T→A的单碱基突变。     

小麦中国春背景下长穗偃麦草Ee染色体组特异AFLP及STS标记的建立

为建立长穗偃麦草Ee染色体组特异的分子标记,以普通小麦中国春、中国春-长穗偃麦草二体代换系、附加系为材料,用5对AFLP引物进行分析,共筛选出28个分布于1Ee-7Ee 7个染色体特异的AFLP标记,经PAGE凝胶电泳后回收、克隆和测序,并根据测序结果设计PCR特异引物,成功地将AFLP标记E-ATC/M-CGTA341bp, E-ATT/M-CTAG265bp, E-AAT/M-CCAG139bp和E-ATT/M-CTAG205bp转化为实验结果稳定、操作更简单的STS标记。利用获得的STS标记对5个长穗偃麦草居群和8个小麦品种进行了鉴定。结果表明其可以在长穗偃麦草居群中被检测到,但在其它小麦背景中检测不到。表明这些标记可以用于小麦-长穗偃麦草异源附加系和代换系中长穗偃麦草遗传物种的检测。     

5种笛鲷属鱼类的遗传多样性及分子标记

运用RAPD及SSR技术对笛鲷属的画眉笛鲷(Lutjanus vitta)、金带笛鲷 (L. fulvus)、金焰笛鲷 (L. fulviflamma)、千年笛鲷(L. sebae)和星点笛鲷(L. stellatus)等5种鱼的遗传多样性及其分子标记进行了研究。RAPD研究结果表明, 5种鱼的平均多态性位点比率(P)依次为89.30%、86.70%、92.11%、86.47%和86.00%; 种内两个体间平均遗传距离(D)分别为0.3431，0.2130，0.3121, 0.1825和0.1775；平均遗传多样性指数(Hi)分别为0.1634、0.1095、0.1353、 0.1022和0.1024。引物OPA8和OPP10的扩增产物中得到9个RAPD标记，分别为OPA8-413bp、OPA8-140bp、OPP10-418bp、OPA8-697bp、OPP10-526bp、OPA8-361bp、OPP10-449bp、OPA8-311bp和OPP10-599b，可鉴别5种鱼。SSR研究结果显示, 5种鱼的有效等位基因数依次为1.9610、3.3793、3.2957、1.7893和3.6591；群体的杂合度(H)分别为0.332、0.462、0.593、0.367和0.676；多态信息含量(PIC)分别为0.302、0.438、0.554、0.332和0.641。 在11个微卫星位点上得到6个微卫星标记，分别为13 Prs229-115 bp、4 Lca43-212 bp、4 Lca43-240 bp、13 Prs229-288 bp、19 Prs275-156bp和7Lca91-118 bp可用于鉴别5种鱼。     

Root-nodule bacteria from indigenous legumes in the north-west of Western Australia and their interaction with exotic legumes

Bacteria were isolated from root-nodules collected from indigenous legumes at 38 separate locations in the Gascoyne and Pilbara regions of Western Australia. Authentication of cultures resulted in 31 being ascribed status as root-nodule bacteria based upon their nodulation of at least one of eight indigenous legume species. The authenticated isolates originated from eight legume genera from 19 sites. Isolates were characterised on the basis of their growth and physiology; 20 isolates were fast-growing and 11 were slow-growing (visible growth within 3 and 7 d, respectively). Fast-growers were isolated from Acacia, Isotropis, Lotus and Swainsona, whilst slow-growers were from Muelleranthus, Rhynchosia and Tephrosia. Indigofera produced one fast-growing isolate and seven slow-growing isolates. Three indigenous legumes (Swainsona formosa, Swainsona maccullochiana and Swainsona pterostylis) nodulated with fast-growing isolates and four species (Acacia saligna, Indigofera brevidens, Kennedia coccinea and Kennedia prorepens) nodulated with both fast- and slow-growing isolates. Swainsona kingii did not form nodules with any isolates. Fast-growing isolates were predominantly acid-sensitive, alkaline- and salt-tolerant. All slow-growing isolates grew well at pH 9.0 whilst more than half grew at pH 5.0, but all were salt-sensitive. All isolates were able to grow at 37 °C. The fast-growing isolates utilised disaccharides, whereas the slow-growing isolates did not. Symbiotic interactions of the isolates were assessed on three annual, one biennial and nine perennial exotic legume species that have agricultural use, or potential use, in southern Australia. Argyrolobium uniflorum, Chamaecytisus proliferus, Macroptilium atropurpureum, Ononis natrix, Phaseolus vulgaris and Sutherlandia microphylla nodulated with one or more of the authenticated isolates. Hedysarum coronarium, Medicago sativa, Ornithopus sativus, Ornithopus compressus, Trifolium burchellianum, Trifolium polymorphum and Trifolium uniflorum did not form nodules. Investigation of the 31 authenticated isolates by polymerase chain reaction with three primers resulted in the RPO1 primer distinguishing 20 separate banding patterns, while ERIC and PucFor primers distinguished 26 separate banding patterns. Sequencing the 16S rRNA gene for four fast- and two slow-growing isolates produced the following phylogenetic associations; WSM1701 and WSM1715 (isolated from Lotus cruentus and S. pterostylis, respectively) displayed 99% homology with Sinorhizobium meliloti, WSM1707 and WSM1721 (isolated from Sinorhizobium leeana and Indigofera sp., respectively) displayed 99% homology with Sinorhizobium terangae, WSM1704 (isolated from Tephrosia gardneri) shared 99% sequence homology with Bradyrhizobium elkanii, and WSM1743 (isolated from Indigofera sp.) displayed 99% homology with Bradyrhizobium japonicum.     

适于玉米杂交种纯度鉴定的SSR核心引物的确定及纯度鉴定图谱绘制

为了确定一套适于玉米杂交种纯度鉴定的核心SSR引物，利用10个SSR引物对420份已知玉米杂交种进行DNA指纹分析。综合考虑多态性和杂合率两个指标，确定bnlg161和bnlg1450为玉米杂交种纯度鉴定的首选核心引物，利用这两个引物进行筛选，412个品种（占98%）能够找到具有杂合带型的鉴定引物；确定bnlg439、bnlg125、umc2105、umc1705、bnlg1792这5个引物为玉米杂交种纯度鉴定的备选核心引物；phi072、bnlg162和phi065因多态性或杂合率低，不推荐作为玉米纯度鉴定用引物。研究了纯度鉴定用特异引物的选择问题，并确定了部分品种的纯度鉴定用特异引物。基于以上研究结果，进一步筛选玉米杂交种的双亲互补型引物，建立已知玉米杂交种的纯度鉴定DNA指纹图谱。此外，对玉米杂交种纯度鉴定用核心引物及特异引物的选择标准进行了探讨。     

Analysis of Genetic Diversity and Relationships in Waxy Rice (Oryza sativa L.) using AFLP and ISSR Markers

Opaque endosperm is the main phenotypic indicator for waxy rice, but other phenotypic and genotypic variation among waxy rice accessions has largely been ignored. Previous studies showed that wide diversity in starch physiochemical properties exists in both indica and japonica waxy rices, especially for starch gelatinization temperature (GT) which could be divided into a high- and a low-GT group. In the present study, amplified fragment length polymorphism (AFLP) and inter-simple sequence repeat (ISSR) molecular markers were employed to examine genetic diversity and relationships of 56 waxy rice accessions. A total of 358 AFLP fragments were amplified with five primer combinations, showing a high level of polymorphism (78.3%). A total of 190 ISSR bands were generated with a single primer and a primer pair, showing a very high level of polymorphism (92.2%). The genetic distance matrices obtained from the two sets of markers were significantly correlated (r = 0.731, P = 0.004). The dendrogram generated with combined AFLP and ISSR markers could clearly differentiate the indica and japonica groups. Newly released varieties and breeding lines within each subspecies tended to be clustered together, whereas landraces were more distantly placed in the dendrogram. Only one AFLP band was found specific to the indica type, while no specific bands were found for starch GT. The implications for the conservation and breeding of waxy rice are discussed.