Identification of random amplified polymorphic DNA (RAPD) markers highly linked to sex determination in the red alga Gracilaria gracilis

The random amplified polymorphic DNA (RAPD) technique was employed in the haplo-diploid dioecious species Gracilaria gracilis to identify sex-linked PCR markers. Sixty-nine decamer oligonucleotide primers were tested on two bulks of DNA, one from five haploid males and the other from five haploid females. One of these primers (OPD13) generated a 430-bp fragment specific to males and a 620-bp fragment specific to females. The diploid individuals (tetrasporophytes) showed the co-occurrence of these two fragments. In order to verify the linkage between the sexual phenotypes and these markers, a progeny array of 59 haploid individuals (male and female) born on a diploid individual was analysed, in all of which the two markers produced by the OPD13 primer segregated perfectly with sex.     

A molecular marker that segregates with sorghum leaf blight resistance in one cross is maternally inherited in another

Leaf blight-resistant sorghum accession SC326-6 was crossed to the susceptible cultivar BTx623 to analyze the genetic basis for resistance. Field scoring of inoculated F₂ progeny revealed that resistance was transmitted as a dominant single-gene trait. By combining the random amplified polymorphic DNA (RAPD) technique with bulked-segregant analysis, it was possible to identify PCR amplification products that␣segregated with disease response. Primer OPD12 amplified a 323-bp band (D12R) that segregated with resistance. Creation of longer primers, or SCARs (sequence characterized amplified regions) for D12R resulted in the amplification of a single major band of the predicted size from all the resistant F₂ progeny and the resistant parent SC326-6, but not from BTx623 or 24 of 29 susceptible F₂ progeny. The SCAR primers also amplified a single band with DNA from IS3620C, the female parent in a cross with BTx623 that has been used to produce a recombinant inbred population for RFLP mapping. An equivalent band was amplified from all 137 recombinant inbred progeny, indicating that organelle DNA is the amplification target in this cross. Received: 31 July 1998 / Accepted: 23 November 1998     

Identification of random amplified polymorphic DNA (RAPD) marker for differentiating male from female and sporophytic thalli of <Emphasis Type="Italic">Gracilaria changii</Emphasis> (Rhodophyta)

There have been limited reports on molecular sex markers for macroalgae. We report the use of random amplified polymorphic DNA analysis (RAPD) to identify molecular sex markers for Gracilaria changii (Xia et Abbott) Abbott, Zhang et Xia. Two DNA extraction methods were used: a modified CTAB and phenol-chloroform combination method and the DNeasy Plant Mini Kit. The CTAB and phenol-chloroform method gave the best yield of DNA in quality and quantity and is suitable for larger-sized specimens like G. changii. Sixty-nine RAPD primers were screened to search for sex-linked DNA markers for G. changii, and only one sex-linked marker (716 bp) was identified using OPA 18. RAPD was also used to investigate the molecular characteristics of the three life-stages (male, female, tetrasporophyte) of G. changii. Seven (OPA7, OPA18, S14, S61, S64, S75 and S76) out of the 69 primers showed polymorphism and were selected for interpopulation analysis for DNA isolated from 23 samples collected from Morib and Sungai Pulai in Malaysia. The combination of data produced by the seven primers generated a dendrogram that grouped the specimens into different clades according to their sex and life-stage using the unweighted pair group and arithmetic averages (UPGMA) method. It showed that RAPD was able to differentiate tetrasporophytes, females, and males. Presented at the 6th Meeting of the Asian Pacific Society of Applied Phycology, Manila, Philippines.     

A SCAR MOLECULAR MARKER SPECIFICALLY RELATED TO THE FEMALE GAMETOPHYTES OF SACCHARINA (LAMINARIA) JAPONICA (PHAEOPHYTA)1

PCR amplification was employed to identify female or male gametophyte associated markers in Saccharina japonica (Aresch.) C. E. Lane, C. Mayes et G. W. Saunders (=Laminaria japonica Aresch.). One pair of the primers, P5, was screened from five pairs designed based on a specific sequence (GenBank accession no. AB069714 ) of Marchantia polymorpha Y chromosome, resulting in a differential band ～500 bp in size between female and male gametophytes of Rongfu strain of S. japonica. According to the SCAR (sequence‐characterized amplified regions) strategies, one pair of primers, P51, was designed on the basis of the sequence of this band that was only present in female gametophytes. A SCAR marker, designated FRML‐494 (494‐bp Female‐Related Marker of S. japonica, GenBank accession no. EU931619 ), was developed successfully by PCR amplification using the designed P51 primer pair. The SCAR marker was verified to be present only in female gametophytes of another variety 901 of this kelp that was a hybrid between S. japonica as paternal and S. longissima (Miyabe) C. E. Lane, C. Mayes, Druehl et G. W. Saunders (=Laminaria longissima Miyabe) as maternal, suggesting that the FRML‐494 marker was specifically related to female gametophytes of the genus. This marker is the first molecular tool reported for sex identification in kelps. This study was beneficial for identifying gametophyte gender during vegetative growth and for judging whether the monogenetic sporophytes came from exclusive male or female gametophytes, as well as for further research on sex determination at the molecular level in kelps.     

烟草SCAR标记技术的建立

目的:通过烟草随机扩增多态性DNA(RAPD)标记技术建立烟草特征序列扩增区域(SCAR)标记技术,用于烟草品种鉴定。方法:对12个烟草品种的复烤叶片DNA进行RAPD分析,得到2个RAPD特异片段S1和S2,通过切胶回收,连接pUCm-T载体克隆转化,片段测序,设计特异性引物S1-1/S1-2和S2-1/S2-2,对SCAR-PCR扩增退火温度进行优化。结果:2个RAPD标记成功地转化为稳定快捷的SCAR标记,可将红花大金元和NC102等2个品种从12个烟草品种中快捷准确地鉴别出来。结论:SCAR标记可作为准确稳定的DNA水平的烟草品种鉴定方法,可对种植、复烤和配方品种的烟叶或叶片进行鉴别。     

谭清苏铁性别连锁的RAPD和SCAR分子标记

利用RAPD(Random amplified polymorphicDNA)分子标记技术,寻找谭清苏铁(Cycas tanqingii)中与性别相关的分子标记,筛选了160个10bp的随机引物,产生了2500多个RAPD条带。只有引物S0465(CCCCGGTAAC)产生了一条大约500bp的雌性特异RAPD标记,该分子标记出现在所有的供试雌性植株中,而所有的供试雄性植株都不具有该标记。对该特异片段进行了克隆和序列测定,并根据序列分析结果将RAPD标记转化为重复性和特异性更好的特异特征序列扩增区域(SCAR)分子标记,并命名为STQC-S465-483。分子标记的建立可用于谭清苏铁幼苗性别的早期鉴定,为谭清苏铁就地保护和迁地保护提供技术支持。     

谭清苏铁性别连锁的RAPD和SCAR分子标记

利用RAPD(Random amplified polymorphic DNA)分子标记技术,寻找谭清苏铁（Cycas tanqingii）中与性别相关的分子标记,筛选了160个10bp的随机引物,产生了2500多个RAPD条带。只有引物S0465 (CCCCGGTAAC)产生了一条大约500bp的雌性特异RAPD标记,该分子标记出现在所有的供试雌性植株中,而所有的供试雄性植株都不具有该标记。对该特异片段进行了克隆和序列测定,并根据序列分析结果将RAPD标记转化为重复性和特异性更好的特异特征序列扩增区域（SCAR）分子标记,并命名为STQC-S465-483。分子标记的建立可用于谭清苏铁幼苗性别的早期鉴定,为谭清苏铁就地保护和迁地保护提供技术支持。     

卡瓦胡椒SCAR分子标记的研究

目的:采用6份卡瓦胡椒材料、21份栽培胡椒和野生胡椒材料1、份不同属的草胡椒材料共计28份试验材料,开发1对特异SCAR引物。方法:在对它们进行了RAPD研究的基础上,通过克隆、测序和引物设计进行了SCAR分子标记研究。结果:研究开发了1对卡瓦胡椒特异SCAR引物P4.1和P4.2,用这对特异引物对试验的28份材料进行PCR扩增,结果只有6份卡瓦胡椒材料扩增出了预期大小562bp的特异带,其它材料均无任何扩增。结论:这说明引物P4.1和P4.2为卡瓦胡椒特异SCAR引物,可用于卡瓦胡椒的分子鉴定,这对卡瓦胡椒种质资源的真伪鉴定有一定帮助。     

Identification of DNA markers linked to the male sex in dioecious hemp (Cannabis sativa L.)

 A 400-bp RAPD marker generated by a primer of random decamer sequence has been found associated with the male sex phenotype in 14 dioecious cultivars and accessions of hemp (Cannabis sativa L.). The primer OPA8 generates a set of bands, most of which polymorphic among all the individual plants tested, and 1 of which, named OPA8₄₀₀, present in all male plants and absent in female plants. A screening of 167 plants belonging to different genotypes for the association of the OPA8₄₀₀ marker with the sex phenotype revealed that only in 3 cases was the 400-bp band was present in plants phenotypically female; on the contrary, in male plants the band was never missing, while in monoecious plants it was never present. Despite this sex-specific association, the sequences corresponding to OPA8₄₀₀ were present in both staminate and carpellate plants, as revealed by Southern blotting and hybridization with the cloned RAPD band. The RAPD marker was sequenced, and specific primers were constructed. These primers generated, on the same genotypes used for RAPD analysis, a SCAR marker 390 bp in length and male-specific. This SCAR is suitable for a precise, early and rapid identification of male plants during breeding programs of dioecious and monoecious hemp. Received: 16 January 1998 / Accepted: 30 April 1998     

卡瓦胡椒SCAR标记的研究

目的：采用6份卡瓦胡椒材料、21份栽培胡椒和野生胡椒材料、1份不同属的草胡椒材料共计28份试验材料,开发1对特异SCAR引物。方法：在对它们进行了RAPD研究的基础上,通过克隆、测序和引物设计进行了SCAR分子标记研究。结果：本研究开发了1对特异SCAR引物P10．1和P10．2,用这对特异引物对本次试验的28份材料进行PCR扩增,结果显示,6份卡瓦胡椒材料扩增出了三条带,三条带离的较近,中间一条为预期494bp特异片段。其它胡椒属材料均扩增出一条494bp的特异带,而不同属的草胡椒无任何扩增。结论：这说明引物P10．1和P10．2适用于卡瓦胡椒的分子鉴定(三条带),也可用于胡椒属植物的分子鉴定(一条带),这对卡瓦胡椒种质资源的真伪鉴定及胡椒属植物的分子分类有一定帮助。     

Development of male-specific SCAR marker in date palm (Phoenix dactylifera L.)

Genetics of control mechanisms that underlies sex differentiation in date palm is not known. Sex of the plants becomes known only at the time of first flowering, which takes around 5 years. In comparison, molecular diagnosis (if available/feasible) promises quick and reliable identification of sex types very early when plantlets are growing in seedbeds. To develop such an assay, genomic DNA from 45 individual plants (25 female and 20 male) belonging to different varieties of date palm was subjected to PCR amplification using 100 random amplified polymorphic DNA (RAPD) and 104 intersimple sequence repeat (ISSR) primers. Initially, two bulk genomic DNA samples (each made by pooling DNA from ten male and female plants, separately) were used. A primer showing sex-specific band in bulked samples was further used for amplification of the genomic DNA of the individual samples of that bulk. Only one RAPD primer, OPA-02, amplified a fragment of ~1.0 kb in all the individual samples of male genotypes, whereas this fragment was absent in all the female genotypes. This male-specific fragment was cloned and sequenced (GenBank accession no. JN123357), and a sequence-characterized amplified region (SCAR) primer pair was designed that amplified a 406-bp fragment in both female and male genotypes and a unique fragment of 354 bp in only male genotypes. The SCAR marker was further validated using 25 female and ten male date palm plants belonging to different varieties collected from different locations.     

一种新的近交系实验动物遗传监测方法及小鼠性别相关RAPD标记的发现

我们用21个10 bp的随机短引物对来自昆明、成都、上海、北京四个地方的BALB/c小鼠以及C57BL小鼠、昆明种小白鼠进行了随机扩增多态DNA(RAPD)分析,发现13个引物的扩增产物在BALB/c小鼠和C 57 BL小鼠中有差异,8个引物的扩增产物在BALB/c小鼠和昆明种小白鼠之间不同.在四个地方的BALB/c小鼠中,成都、上海、北京的BALB/c小鼠其遗传背景均一,而来自昆明的BALB/c小鼠中,有2只的4个引物的扩增产物不同于其它的BALB/c小鼠,表明这两只BALB/c小鼠可能曾发生过某种程度的遗传改变或污染。实验结果显示RAPD方法是一种有效的近交系实验动物遗传监测手段。实验中一个有趣的结果是,在OPG 2、OPE 4、OPE 9的扩增产物中,发现了严格的性别依赖的PAPD标记。OPE 9扩增产物中,凡雄性个体都有一条0.88 kb的标记.OPG 2、OPE 4则在所有的雄性个体中多扩增出一条约1.2 kb的带。通过交叉PCR扩增和斑点杂交证明OPG 2、OPE 4 得到的雄性特异性RAPD标记虽分子大小一致,但不具同源性。这些性别相关RAPD标记的染色体定位和性质分析正在进一步进行中.     

Female‐only sex‐linked amplified fragment length polymorphism markers support ZW/ZZ sex determination in the giant freshwater prawn Macrobrachium rosenbergii

Sex determination mechanisms in many crustacean species are complex and poorly documented. In the giant freshwater prawn, Macrobrachium rosenbergii, a ZW/ZZ sex determination system was previously proposed based on sex ratio data obtained by crosses of sex‐reversed females (neomales). To provide molecular evidence for the proposed system, novel sex‐linked molecular markers were isolated in this species. Amplified fragment length polymorphism (AFLP) using 64 primer combinations was employed to screen prawn genomes for DNA markers linked with sex loci. Approximately 8400 legible fragments were produced, 13 of which were uniquely identified in female prawns with no indication of corresponding male‐specific markers. These AFLP fragments were reamplified, cloned and sequenced, producing two reliable female‐specific sequence characterized amplified region (SCAR) markers. Additional individuals from two unrelated geographic populations were used to verify these findings, confirming female‐specific amplification of single bands. Detection of internal polymorphic sites was conducted by designing new primer pairs based on these internal fragments. The internal SCAR fragments also displayed specificity in females, indicating high levels of variation between female and male specimens. The distinctive feature of female‐linked SCAR markers can be applied for rapid detection of prawn gender. These sex‐specific SCAR markers and sex‐associated AFLP candidates unique to female specimens support a sex determination system consistent with female heterogamety (ZW) and male homogamety (ZZ).     

大麻性别的RAPD和SCAR分子标记

利用随机扩增多态性DNA(randomamplifiedpolymorphicDNA,RAPD)技术获得与大麻性别连锁的分子标记.将10株雄性大麻或10株雌性大麻的单个DNA样品等量混合分别组成雄性或雌性DNA池(DNApool),以提供具有相同遗传背景的雌、雄性DNA样品.每个随机引物分别用三个不同的循环程序进行PCR扩增.在30个随机引物中,用引物401扩增得到一条约2.5kb雄性多态性片段.对该片段进行了克隆和序列分析,并根据序列分析结果将上述RAPD分子标记转化为重复性和特异性更好的SCAR(sequencecharacterizedamplifiedregions)分子标记.     

Isolation of Female-Specific AFLP Markers and Molecular Identification of Genetic Sex in Half-Smooth Tongue Sole (<Emphasis Type="Italic">Cynoglossus semilaevis</Emphasis>)

The sex-specific molecular marker is a useful gene resource for studying sex- determining mechanisms and controlling fish sex. Artificially produced male and female half-smooth tongue sole (Cynoglossus semilaevis) were used to screen sex-specific amplified fragment length polymorphism (AFLPs) molecular markers. The phenotypic sex of 28 tongue soles was determined by histological sectioning of gonads. The AFLP analysis of 15 females and 13 males via 64 primer combinations produced a total of 4681 scorable bands, of which 42.11% and 43.39% of bands were polymorphic in females and males, respectively. Seven female-specific AFLP markers were identified and designated as CseF382, CseF575, CseF783, CseF464, CseF136, CseF618, and CseF305, respectively. One female-specific AFLP marker (CseF382) was amplified, recovered from the gels, cloned, and sequenced (accession no. DQ487760). This female-specific AFLP marker was converted into a single-locus polymerase-chain reaction (PCR) marker of a sequence-characterized amplified region (SCAR). A simple PCR method of using the specific primers was developed for identifying genetic sex of half-smooth tongue sole. PCR products demonstrated that the initial 15 females produced the female-specific band of about 350 bp, but the initial 13 male individuals failed to produce the band. We also investigated the applicability of the PCR primers in other tongue sole individuals. The same female-specific fragment of about 350 bp was found in the additional 59 female individuals, but not in the additional 58 male individuals. This AFLP-based molecular sexing technique may have great application potential in elucidation of sex determination mechanisms and sex control in half-smooth tongue sole.     

RAPD-based SCAR marker SCA 12 linked to recessive gene conferring resistance to anthracnose in sorghum [Sorghum bicolor (L.) Moench]

Anthracnose, caused by Colletotrichum graminicola, infects all aerial parts of sorghum, Sorghum bicolor (L.) Moench, plants and causes loss of as much as 70%. F₁ and F₂ plants inoculated with local isolates of C. graminicola indicated that resistance to anthracnose in sorghum accession G 73 segregated as a recessive trait in a cross with susceptible cultivar HC 136. To facilitate the use of marker-assisted selection in sorghum breeding programs, a PCR-based specific sequence characterized amplified region (SCAR) marker was developed. A total of 29 resistant and 20 susceptible recombinant inbred lines (RILs) derived from a HC 136 × G 73 cross was used for bulked segregant analysis to identify a RAPD marker closely linked to a gene for resistance to anthracnose. The polymorphism between the parents HC 136 and G 73 was evaluated using 84 random sequence decamer primers. Among these, only 24 primers generated polymorphism. On bulked segregant analysis, primer OPA 12 amplified a unique band of 383 bp only in the resistant parent G 73 and resistant bulk. Segregation analysis of individual RILs showed the marker OPA 12₃₈₃ was 6.03 cM from the locus governing resistance to anthracnose. The marker OPA 12₃₈₃ was cloned and sequenced. Based on the sequence of cloned RAPD product, a pair of SCAR markers SCA 12-1 and SCA 12-2 was designed using the MacVector program, which specifically amplified this RAPD fragment in resistant parent G 73, resistant bulk and respective RILs. Therefore, it was confirmed that SCAR marker SCA 12 is at the same locus as RAPD marker OPA 12₃₈₃ and hence, is linked to the gene for resistance to anthracnose.     

膜荚黄芪SCAR标记的建立

用RAPD方法对膜荚黄芪和蒙古黄芪进行指纹图谱的研究。采用BSA法从120个10碱基随机引物筛选出7个在膜荚黄芪基因池和蒙古黄芪基因池中表现多态性的引物。单株检测表明,引物OPD14具有膜荚黄芪特异性,在检测的膜荚黄芪个体中均能各自扩增出一条300 bp左右的特异带,而在蒙古黄芪的单株中则未见,将该膜荚黄芪特异性片断命名为OPD14_-300。获得的RAPD标记OPD14_-300经克隆、测序、重新设计一对特异性引物转化成更稳定的SCAR标记;该SCAR标记只在膜荚黄芪个体中出现,达到了在分子水平上快速、准确地鉴定膜荚黄芪和蒙古黄芪的目的。     

RAPD based DNA markers linked to anthracnose disease resistance in Sorghum bicolor ( L.) Moench

Anthracnose caused by Colletotrichum graminicola is one of the major diseases of sorghum. The locus for disease resistance in sorghum [Sorghum biocolor (L.) Moench] accession G73 was found to segregate as a simple recessive trait in a cross to susceptible cultivar HC136. In order to identify molecular markers linked to the locus for disease resistance, random amplified polymorphic DNA (RAPD) analysis was coupled with bulk segregant analysis. DNA from the parental cultivars and the bulks were, screened by PCR amplification with 114 RAPD primers. Three RAPD primers amplified a sequence that consegregated with the recessive resistance allele, while another three amplified a band linked to the susceptible allele. The six disease linked markers were screened with individual resistant and susceptible genotypes to observe degree of linkage of identified RAPD markers with the gene for resistance. Two primer sequences (OPI 16 and OPD 12) were found to be closely linked to the locus for disease resistance.     

Recombinant bovine interferon alpha causes only a temporary prolongation of the lifespan of the corpus luteum

Abstract

A method is described for developing a sheep‐ vs. goat‐specific DNA marker using sequence characterized amplified regions (SCARs) derived from a random amplified polymorphic DNA (RAPD) marker from sheep DNA samples. A sheep 645 bp DNA fragment that was absent in goat DNA was identified by analyzing pools of sheep and goat DNA with RAPD primers. This fragment was cloned and partially sequenced to design extended, strand‐specific 24‐mer oligonucleotide primers. Each primer contained the original 10 bases of the RAPD primer and the following 14 internal bases. The pair of primers resulted in the amplification of a single band of 645 bp when used to amplify sheep DNA, and in no amplification when used to amplify goat DNA. These SCAR primers successfully amplified the equivalent of DNA from one nucleated sheep cell in a sample of 5000 nucleated goat cells. This level of sensitivity is especially desirable for research involving the detection of interspecific chimerism.