Development and Application of SCAR Markers for Discriminating Cytoplasmic Male Sterile Lines from Their Cognate Maintainer Lines in Indica Rice

The DNAfragments about 1 600 bp were amplified using random amplified polymorphism DNA （RAPD） primer OPAl2 with the templates of mitochondrial DNA of Zhenshan 97A and Zhenshan 97B, and were sequenced. The nucleotide sequences and lengths of the fragments from Zhenshan 97A and Zhenshan 97B showed no difference. The precise length of the fragment was 1 588 bp. Sequence characterized amplification region （SCAR） primers were then developed to discriminate the cytoplasmic male sterile （CMS） lines and their maintainer lines. A specific 1 588 bp fragment could be amplified with SCAR primers, CHI19F2/CHI19R2 and CHI20F3/CHI23R3, in the mitochondrial DNA of Zhenshan 97A, but not Zhenshan 97B. Furthermore, the specific fragment could be also amplified from the total DNA from green leaf tissues of Zhenshan 97A with SCAR primers, but not Zhenshan 97B. With the corresponding primers, the specific fragment could also be amplified from the total DNA of green leaves of other two CMS lines with wild abortive type cytoplasm （CMS-WA）, namely Zhenpin A and Tianfeng A, but not in their maintainer lines. Moreover, using total DNA as template, each of the four pairs of SCAR primers could also be used to amplify the 1 588 bp fragment in CMS-ID （Indonesia paddy type） line 11-32A but not in 11-32B, and the specific fragment was amplified from the DNA of both F1 and F2 seedlings of Shanyou 63. The results of detecting the genetic purity of a man-made mixture of the seeds of Zhenshan 97A using CHI20F3/CHI23R3 were completely consistent with the phenotypes. Taken together, these results indicated that the specific 1 588 bp-fragment amplified by CHI20F3/CHI23R3 was the unique amplification products of CMS mitochondrial DNA, and could be used to distinguish CMS-WA and CMS-ID lines from their corresponding maintainer lines at the seedling stage.

A Set of SCAR Markers Efficiently Differentiating Hybrid Rice

Molecular markers have been widely used in crop genetic improvement, seed test and genetic mapping. Of which, sequence characterized amplified region （SCAR） markers are particularly popular for its diversity, stable reproducibility, and suitability for analyzing large number of samples. In this study, 500 random amplified polymorphic DNA （RAPD） primers were tested, and a set of SCAR markers comprising 37 pairs of loci-specific primers were developed from the DNA fragments ranging from 300 to 1000 bp which correspond to the stable, distinctive RAPD banding patterns. Using these SCAR markers, 59 hybrid rice combinations were assessed and distinguished into 58 subgroups at the similarity coefficient of 0.97 in a genetic clustering tree based on the allele diversities of the SCAR markers. Furthermore, 13 hybrid rice combinations were reassayed with 40 randomly selected simple sequence repeat （SSR） markers to evaluate the effectiveness of these SCAR markers. SSR markers produced similar results to SCAR markers as the 13 hybrid rice combinations were completely separated at the similarity coefficient of 0.91 in the clustering tree established from SSR patterns. Taken together, SCAR markers prove to be effective tools for identifying and differentiating hybrid rice combinations.

Assessment of genetic variation in tomato (Solanum lycopersicum L.) inbred lines using SSR molecular markers

A study was conducted to determine the genetic diversity of 39 determinate and indeterminate tomato inbred lines collected from China, Japan, S. Korea, and USA. Using 35 SSR polymorphic markers, a total of 150 alleles were found with moderate levels of diversity, and a high number of unique alleles existing in these tomato lines. The mean number of alleles per locus was 4.3 and the average polymorphism information content （PIC） was 0.31. Unweighted Pair Group Method with Arithmetic Mean （UPGMA） clustering at genetic similarity value of 0.85 grouped the inbred lines into four groups, where one USA cultivar formed a separate and more distant cluster. The most similar inbred lines are from USA, both with determinate type, whereas the most different lines are from USA （Us-16） and Japan （Ja-2） with determinate and indeterminate growth habit, respectively. Clustering was consistent with the known information regarding geographical location and growth habit. The genetic distance information reported in this study might be used by breeders when planning future crosses among these inbred lines.

Development of a SCAR marker for male gametophyte of Gracilariopsis lemaneiformis based on AFLP technique

The red alga Gracilariopsis lemaneiformis(Bory) is an economically valuable macroalgae. As a means to identify the sex of immature Gracilariopsis lemaneiformis, the amplifi ed fragment length polymorphism(AFLP) technique was used to search for possible sex- or phase-related markers in male gametophytes, female gametophytes, and tetrasporophytes, respectively. Seven AFLP selective amplifi cation primers were used in this study. The primer combination E-TG/M-CCA detected a specifi c band linked to male gametophytes. The DNA fragment was recovered and a 402-bp fragment was sequenced. However, no DNA sequence match was found in public databases. Sequence characterized amplifi ed region(SCAR) primers were designed from the sequence to test the repeatability of the relationship to the sex, using 69 male gametophytes, 139 female gametophytes, and 47 tetrasporophytes. The test results demonstrate a good linkage and repeatability of the SCAR marker to sex. The SCAR primers developed in this study could reduce the time required for sex identifi cation of Gracilariopsis lemaneiformis by four to six months. This can reduce both the time investment and number of specimens required in breeding experiments.

Identification of necrophagous fly species from 12 different cities in China using ISSR and SCAR markers

Objective:To identify necrophagous fly speies from different regions in China using inter simple sequenc repeat(ISSR) and sequence-characterized amplified region(SCAR) melocular markers and to analyze their gene difference and genetic relationship.Methods:Five carrion fly species were collected from 12 cities and regions in China,including Musca domestica(M.domestica), Lucilia sericata(L.sericata),Chrysomya megacephala(C.megacephala),Helicophagella melanura(H.melanura),Boethcherisca peregrina,and they were studied using ISSR and SCAR markers.Results:Eight ISSR primers were used for amplification of 121 samples.679 clear and stable bands were identified,of which 516 bands were polymorphic.Several species-specific ISSR fragment were cloned and sequenced as an initial effort to derive the SCAR markers.Using M.domestica SCAR specific primers,SCAR-PCR amplification was performed for 8 M.domestca population sample DNA from different regions in China as well as L sericata,C.megacephala, H.melanura and Lucillia cupirina.The result showed only M.domestica produced specificalty 600 bp fragment,but L sericata,C.megacephala,H.melanura and Lucillia cupirina did not produce the same specific fragment.Clustering analysis showed clustering of most flies of M. domestica,C.megacephala and L sericata.M.domestica samples from different regions in China yielded different banding patterns.Conclusions:Application of ISSR-PCR and SCAR markers to identify necrophagous fly species from 12 cities and regions in China is first reported.ISSR-PCR and SCAR markers provide a quick reliable molecular marker technique for the identification of different species of necrophagous fly.

Genetic Diversity in the Semi-Arid Grown Cowpea(<i>Vigna unguiculata</i>L. Walp)Accessions Using Morphological and Molecular Characterization

Assessment of genetic diversity of the indigenous crop accessions is extremely important for breeders to identify potential parents in cross-breeding programs. Fourteen cowpea accessions collected from different parts of Sudan were used for characterization at morphological and molecular levels. The seeds of the accessions were sown in the field using a randomized complete block design with three replicates. Sixteen morphological descriptors (9 qualitative and 7 quantitative) and 20 Random Amplified Polymorphic DNA (RAPD) markers were used for characterization of the accessions. The results of morphological data revealed considerable variability within and between state’s accessions. Some morphological traits revealed similarity between accessions from different states. Among the 20 RAPD markers used, 18 were polymorphic. A total of 379 polymorphic patterns were generated;polymorphic information content (PIC) ranged from 0.63 to 0.98 with an average of 0.9. The number of fragment detected ranged from 2 for OPL-11 to 51 for OPY-2 with an average of 26.06/primer and 27.07/genotype. One to five (1 - 5) unique fragments of different sizes were detected for particular accessions, which may provide a valuable resource for breeding superior cowpea cultivars in Sudan and other semi-arid zones. Genetic similarity was ranged from 0.02 to 0.47 with an average of 0.25. Highest genetic similarity was between genotypes HSD-2966 and HSD-2967 and between genotypes HSD-5131 and HSD-5627 and the lowest was between HSD-5131 and HSD-5861 followed by that between HSD-2976 and HSD-29130 accessions. The study recommends the combination of morphological and molecular data for more efficient genetic diversity assessment and management.

Genetic Characterization of Indigenous Rice Varieties in Eastern Himalayan Region of Northeast India

The eastern Himalayan region of Northeast （NE） India is home to a large number of indigenous rice varieties, which are traditionally classified as Oryza sativa subspecies indica, japonica or intermediate types. The classification based on traditional Cheng’s index is often inconclusive due to phenotypic plasticity of morphological characters, which are influenced by environmental conditions. We used molecular markers specific for indica and japonica subspecies to assess the degree of genetic relatedness of indigenous rice varieties in NE India. The results revealed that majority of upland （jum） and glutinous rice varieties, traditionally considered as japonica, were genetically close to the subspecies indica. All varieties of boro ecotype were found to be indica type, and only a few varieties cultivated in lowland and upland areas were japonica type. Some of the lowland varieties of the sali ecotype were intermediate between indica and japonica, and they showed a closer genetic affinity to O. rufipogon.

Assessment of Genetic Diversity of NIFOR Oil Palm Main Breeding Parent Genotypes Using Microsatellite Markers

The genetic diversity among 15 NIFOR breeding parents was assessed using 10 microsatellite markers. A high genetic diversity was observed with a total of 64 alleles including 23 rare alleles or alleles at frequencies less than 0.05. The NIFOR tenera parents recorded the highest number of rare alleles. The average observed heterozygosity and mean gene diversity across all parental groups were 0.6889 and 0.7029, respectively. Higher genetic diversity was detected among the NIFOR dura and tenera parents compared to that of the Deli dura parents in absolute terms. Analysis of molecular variance (AMOVA) showed that 87% of the total variation (p < 0.001) observed was due to differences among parents. Rogers’ genetic distance ranged from 0.2988 to 0.8000 (mean = 0.5570). The dendrogram constructed on the basis of Rogers’ genetic distance clustered the parents in three groups. They generally clustered in heterotic manner rather than by geographic origins. The groupings obtained through PCoA confirmed the results obtained by cluster analysis. The results obtained are strong assets for NIFOR breeding programme.

凡纳对虾、细角对虾和斑节对虾的RAPD鉴定标记

采用随机引物扩增DNA多态性(RAPD)技术分析比较了凡纳对虾、细角对虾和斑节对虾的分子标记。研究结果发现有3条引物(OPX 12、OPX 14和OPX 17)可扩增出重复稳定的图谱;3种对虾各有特定的RAPD标记:凡纳对虾有OPX 140940、OPX 171080、OPX 120760,细角对虾有OPX 141350、OPX 170850、OPX 121490,斑节对虾有OPX 140690、OPX 172260、OPX 120810。这些标记可作为3种对虾的鉴定标记。

金针菇子实体颜色基因的分子标记

用群体分离分析法从200个随机引物中筛选出S375,其扩增片段与金针菇子实体白色基因连锁.对S375扩增片段两端测序,以该序列为依据合成一对特征序列扩增区域(SCAR)引物,它能对带有白色基因的菌株扩增出单一片段,多聚酶链反应(PCR)产物不需电泳、加EB后可在紫外灯下直接检测.

典型工业恶臭源恶臭排放特征研究

恶臭污染具有主观性和复杂性特点,结合使用仪器分析和嗅觉方法,可以从成分和感官两方面充分反映恶臭污染特征.本文参考USEPA TO14A和GB/T 14675-93方法,选择天津滨海新区内的6个不同类型的工业恶臭源,包括制药、喷漆、炼油、石化、树脂合成和橡胶,采集了各类源工艺流程中通过有组织方式排放的恶臭废气,测定了废气的感官臭气浓度并定量分析了其中的恶臭VOCs物质.使用臭气浓度、恶臭指数及统计学方法进行研究,结果发现,炼油源和制胶源的废气具有非常严重的感官刺激性.甲硫醇等硫化物是炼油源和制胶源的主要特征恶臭物质;苯乙烯和甲苯分别是合成树脂源和喷涂源的特征恶臭组分;对苯二甲酸(PTA)源和制药源属于混合型恶臭源.甲苯是喷漆源和制药源的标识组分;二硫化碳是制胶源的标识组分;间,对-二甲苯可以用来标识石化PTA污染源;炼油源的标识组分为三氯乙烯、氯乙烷和1,2-二溴乙烷;苯乙烯是合成树脂源的标识组分.

中国舞毒蛾六个地理种群的RAPD分析及SCAR标记构建

舞毒蛾Lymantria disparL.是世界性农林害虫,包含不同的亚种,其中亚洲舞毒蛾的雌蛾具有较强的飞行能力,已成为国际性的重要检疫性有害生物。然而,不同舞毒蛾亚种及种群间形态难辨,因此采用传统的手段鉴别舞毒蛾亚种种群是很困难的。本研究首先采用RAPD标记分析了中国舞毒蛾6个地理种群的遗传多态性。结果表明,所检测的舞毒蛾种群的遗传分化系数Gst为0.7571,由此推算出的平均有效迁移数(基因流参数)Nem为0.1604,说明不同舞毒蛾种群间的遗传分化程度较高,缺乏广泛的基因流动。本研究在RAPD遗传分析基础之上,筛选出了4个舞毒蛾种群的特异性遗传位点,然后对这些特异性位点进行了克隆测序、序列分析和位点特异性引物设计。结果表明,其中2个舞毒蛾种群的位点特异性引物可产生序列特征性扩增区域(SCAR)标记。经验证,这些标记可被用来鉴别特定的舞毒蛾地理种群,因此有助于对这些舞毒蛾地理种群的分布与扩散进行监测。

黑龙江省春小麦主要品质性状和慢锈抗病基因的分子标记检测

1B.1R易位系、高分子量麦谷蛋白亚基(HMW-GS)、多酚氧化酶(Polyphenol oxidase,PPO)活性、黄色素含量等因素都对小麦加工品质有重要影响,慢锈基因Lr34既抗叶锈,又抗条锈、白粉和秆锈病。为了明确这些基因在黑龙江小麦品种中的分布,并为优质抗病小麦分子标记辅助育种提供材料和方法,本研究利用高分子量谷蛋白亚基Dx5、By8、1B.1R易位系、PPO、PSY基因和Lr34位点的特异性分子标记对黑龙江省不同地区的169份小麦品种进行鉴定。结果表明,Dx5亚基的频率为43.2%,By8亚基的频率为30.8%,1B.1R易位系的频率为4.7%,Ppo-A1b的频率为33.1%,Psy-A1b的频率为33.7%,含Lr34位点的频率为24.9%;不含1B.1R易位系、同时又具有Dx5和By8、Ppo-A1b、Psy-A1b及Lr34位点的品种只有2份,占1.2%。

玉米对生性状两个显性基因SCAR分子标记

把与玉米对生叶突变体对生性状两个显性位点紧密连锁的RAPD标记S312 3 77与S36 0 60 2 成功地转化成SCAR标记CBJ1与CBJ2。对RAPD片段的纯化、T A克隆、SCARPCR引物的设计等SCAR标记转化进行了探讨 ,并对CBJ1与CBJ2进行了验证 ,为对生玉米的分子标记辅助选择与定位克隆奠定了基础。

与瓠瓜品系‘J083’白粉病抗性基因连锁的SCAR分子标记

以瓠瓜抗白粉病品系‘J083’和感病品系‘J73’及它们的F1代和F2代分离群体为试验材料,经接种鉴定和抗性遗传规律分析表明:瓠瓜品系‘J083’对白粉病的抗性受单隐性基因控制;从100对扩增片段长度多态性(AFLP)引物组合中获得稳定的多态性引物组合1对,即E-ATG/M-CTC;经回收、测序,特异片段全长为105 bp,并成功将其转化为序列特征性扩增区域(SCAR)标记;经连锁分析,该SCAR标记与白粉病抗性基因的连锁距离为9.6 cM,将其命名为GPDSATG/CTC75.此标记可用于瓠瓜抗白粉病品种的辅助选育.

玉米抗灰斑病基因的分子标记

玉米灰斑病是我国玉米生产中的重要病害,培育和种植抗病品种是防治此病的有效途径。1年内在3个灰斑病重发区选取64份我国常用玉米自交系进行了抗灰斑病鉴定,分别筛选出高抗、抗病和中抗自交系2份、15份和12份。采用集团混合分组分析法,分别以10个抗病和10个感病玉米自交系基因组DNA构建抗病基因池和感病基因池。从100对AFLP引物组合中筛选出54对在抗感基因池间呈多态性的引物,进一步在组建抗池和感池的20份自交系之间检测到8个多态性片段。通过片段的回收、克隆和测序,将其中的P51M38-100扩增片段转化为SCAR标记(SCAR-100)。利用SCAR-100标记分析64份玉米自交系的基因型,结合田间抗病和感病表型进行相关分析,卡方测验表明SCAR-100标记与抗灰斑病显著相关。采用Mo17×黄早四群体(190个F2单株)和X178×B73群体(181个F8家系),结合已构建的SSR和AFLP标记连锁图谱,均将SCAR-100标记定位于第3染色体上,分别位于SSR标记umc1399-bnlg1754和umc1320-bnlg1754之间。开发抗病基因的分子标记可为分子标记辅助育种提供基础。

香菇135菌株特异SCAR标记的分布和遗传特性

用香菇(Lentinula edodes)135菌株的特异SCAR(Sequence Characterized Amplified Region)引物135F/R(扩增601bp的条带)对135菌株的58个原生质体单核体和97个孢子单核体的基因组DNA进行扩增.检验此特异标记在单核体中的分布和遗传规律。原生质体单核体分为两种交配型A1B1和A2B2,此标记仅存在于后一种核中;另外,此标记在四种交配型的孢子单核体后代中随机分布,显示这个SCAR标记可以稳定遗传到后代,而且与交配型A、B因子之间没有连锁关系。

中国春Ph1基因的分子标记及冬小麦ph1b代换系的获得

利用大麦 (HordeumvulgareL .)第 5染色体上RFLP探针衍生的 19个序列标志位点PCR(STS_PCR)引物对“中国春”小麦 (TriticumaestivumL .) (CS)及其ph1b突变体基因组总DNA进行PCR扩增 ,筛选出Ph1基因的一个连锁标记 ,再用“中国春”第 5部分同源群缺体_四体系和CS×ph1b突变体F2 群体证明并定位于离Ph1基因近着丝点端 5 .7cM (centiMorgan)处。然后将该标记转换成特异的序列特征扩增区 (SCAR)标记。以“阿勃”5B缺体为桥梁亲本 ,冬小麦“京 411”为受体亲本 ,“中国春”ph1b突变体为供体亲本 ,进行三轮杂交和一轮自交 ,每一轮经减数分裂分析和SCAR标记的辅助选择 ,快速地筛选出了ph1b基因型 ,并选得一个冬小麦“京 411”的ph1b中间代换系。

一种新型的分子标记—SCARs验证王浆高产蜜蜂的RAPD分子标记—W316bp的研究

为了验证王浆高产蜜蜂的RAPD分子标记—W 316bp正确性 ,采用了新型分子标记—SCARs ,研究结果获得的SCARs分子标记与原来RAPD分子标记有一致的显性行为 。

Morphological and Genetical Variability among Rhizoctonia solani Isolates Causing Sheath Blight Disease of Rice

Eighteen isolates of Rhizoctonia solani collected from infected rice plants in four different locations of Bangladesh were studied by using morphological characters and molecular markers. Anastomosis study with a reference isolate confirmed that all the isolates belonged to R. solani. Significant variation was observed in sclerotial size, shape and distribution. Un-weighted pair group method with arithmetic mean dendrogram constructed based on the Gower＇s general similarity coefficient showed that these isolates were grouped into four clusters at the 0.68 similarity coefficent according to morphological characters. Cluster I was a major cluster consisting of 13 isolates, while clusters Ⅱ to Ⅳ consisted of 1 or 2 isolates. Analyses by variable number of tandem repeat and amplified fragment length polymorphism markers showed that the isolates were grouped into five and three clusters at a similarity coefficient of 0.64 and 0.69, respectively. Although most of the variability was found between isolates from different regions as expected, significant variation was observed within the isolates collected from similar agro-ecological regions. Our results suggest the presence of different races of R. solani within the same local geographic regions.