Identification of sex in Carica papaya L. using RAPD markers

Brazil is currently the worlds largest producer of papaya (Carica papayaL.), producing fruits for both the domestic market and export. Only fruits from hermaphrodite plants are marketed because they have the necessary commercial characteristics, i.e. they are pear-shaped and have thicker flesh and a smaller internal cavity. Increased papaya yield has been limited mainly by the ratio of female to hermaphrodite (1: 2) plants normally occurring in orchards. This ratio causes great losses to papaya producers and the identification of the sex of seedlings during the nursery stage would be an important advance. In our study random amplified polymorphic DNA (RAPD) analysis was used to differentiate between the sexual forms of three commercial C. papaya cultivars belonging to the Solo group. RAPD assays using the BC210 primer were able to detect hermaphrodites in all of the cultivars tested. The BC210₄₃₈molecular marker was much better at papaya sex differentiation than other markers described in the literature. This revised version was published online in August 2006 with corrections to the Cover Date.     

Development and application of SCAR markers for sex identification in the dioecious species <Emphasis Type="Italic">Ginkgo biloba</Emphasis> L.

There is an urgent need for early sex identification to support field planting in Ginkgo biloba L., due to the different economic and medicinal values between male and female trees. An easy, rapid and reliable molecular method for sex type determination of G. biloba was reported in the paper. Random amplification of polymorphic DNA (RAPD) and sequence-characterized amplified region (SCAR) were used to search for specific molecular markers linked to the sex locus. A total of 48 primers were used for screening of specific RAPD markers in six male and three female samples. Only one primer, S10, showed different amplification band patterns associated with sex types. Then the sex-specific bands, S10-BandA and S10-BandB, were cloned and sequenced. Based on the sequences two pairs of SCAR primers, GBA and GBB, were designed. The GBA primers amplify a single 571 bp band in male samples but not in female samples, and DNA amplification using GBB primers could generate a 688 bp band only in the female individuals. Finally, the SCAR primers were used to test 16 sex-unknown samples. SCAR primers developed in this paper can be used as effective, convenient and reliable molecular markers for sex identification in G. biloba.     

RAPD and SCAR markers linked to the sex expression locus M in asparagus

Bulk segregant analysis (BSA), random amplified polymorphic DNA (RAPD) and sequence characterized amplified region (SCAR) methods were used to map molecular markers to the sex locus M of asparagus. Two parents, A19 (male, Mm) and MW25 (female, mm), and 63 progeny were used for the study. Two DNA bulks, one male and one female, were made by pooling equal amounts of DNA from 10 randomly selected progeny of each sex type. A total of 760 arbitrary decamer oligonucleotide primers were used for RAPD analysis. Primer OPC15 produced two RAPD markers, OPC15-98 and OPC15-30, both of which were linked to the M locus at a distance of 1.6 cM. Subsequently, amplified RAPD fragment OPC15-98 was cloned and sequenced. The sequence was then used to design flanking 24-mer oligonucleotide SCAR primers SCC15-1 and SCC15-2. Both of these SCAR primers amplified a single 980 bp fragment; the same size as the cloned RAPD fragment. However, the SCAR marker was dominant as was the original OPC15-98 band from which it was derived. These RAPD and SCAR markers could be used for scoring male and female progeny in the mapping population, but were not found to be applicable to other asparagus germplasm studied. This revised version was published online in July 2006 with corrections to the Cover Date.     

工业大麻雄性相关RAPD和SCAR标记的筛选与鉴定

利用42条RAPD（Random Amplified Polymorphic DNA）随机扩增引物分析工业大麻品种“火麻一号”组成的雄性或雌性DNA池（DNA pools）,结果显示,引物OPV-08扩增得到一条大小为869bp与工业大麻雄性相关的特异条带。根据测序结果,合成了两条SCAR（Sequence Characterized Amplified Region）标记引物,该SCAR标记不仅可以对工业大麻雌雄异株材料花期已知性别的雌雄植株进行准确鉴定,还可以对幼苗期未知性别的大麻雌雄植株进行鉴定;也可对雌雄同株材料可能出现的雄化进行早期鉴定。这不仅为工业大麻早期性别鉴定提供基础,且为减少雌雄同株材料的雄化提供支撑。     

Data to the sex determination in <Emphasis Type="Italic">Pistacia</Emphasis> species using molecular markers

Sex identification in Pistacia species during the long juvenile stage is an economically desirable objective. Due to the lack of morphological methods to identify sex at this stage, the application of molecular markers is expected to facilitate breeding programs. The aim of our study was to identify a marker closely linked to sex loci in Pistacia atlantica Desf subsp. mutica, P. khinjuk, and P. vera subsp. Sarakhs. Samples were collected from both male and female plants of each species, and their band patterns were analyzed according to the presence or absence of specific bands. Thirty random amplified polymorphic DNA (RAPD) primers and a pair of sequence characterized amplified region (SCAR) primers were tested as potential markers of sex in wild Pistacia species. Among the RAPD primers, only BC₁₂₀₀ was found to amplify a specific sex band present in female plants. Based on our analysis of all individual samples, a fragment of approximately 300 bp was amplified in female trees but absent in male ones. Although sex determination mechanisms in Pistacia are still unknown, they may be controlled by a single locus that acts as a trigger. The SCAR technique has proved to be a reliable technique in gender determination of pistachio genotypes at the seedling phenophase. This method could reduce both the time and costs associated with breeding programs.     

Development of male-specific markers and identification of sex reversal mutants in papaya

Papaya is a productive and nutritious fruit grown in tropical and sub-tropical regions worldwide. It is polygamous with three sex types: female, male and hermaphrodite. Sex determination in papaya is controlled by an XY sex chromosome system with two slightly different Y chromosomes, Y for males and Y^h for hermaphrodites. Comparative analysis of the hermaphrodite-specific region of Y^h chromosome (HSY) and male-specific region of Y chromosome (MSY) revealed 99.6% sequence identity, which explains why DNA markers that amplify for both males and hermaphrodites have easily been developed, but not for the male trait specifically. We examined the 0.4% sequence differences, and found 1887 indels and 21,088 SNPs between MSY and HSY. The vast majority of indels are single nucleotide or few base pairs. A large male-specific retrotransposon insertion of 8396 bp was used to develop two papaya male-specific markers, PMSM1 and PMSM2 that amplify 585 and 548 bp fragments, respectively. These two markers were tested in 11 gynodioecious and four dioecious varieties along with autosomal DNA marker 71E and male/hermaphrodite marker W11, and the results showed clear separation of male from hermaphrodite and female. PMSM1 and PMSM2 were also used to test the sex type of six sex male-to-hermaphrodite reversal mutants which are crucial materials for validating candidate genes for sex determination in papaya. Our result showed all six mutants were positive for the male-specific markers. These male-specific markers can be used to distinguish gynodioecious and dioecious cultivars in papaya seed market, and facilitate genetic and genomic research for papaya improvement.     

RAPD and SCAR markers linked to sex determination in Eucommia ulmoides Oliv.

Eucommia ulmoides Oliv. is strictly a dioecious perennial tree native to China. The pistillate plants are economically more useful than the staminate plants. The random amplified polymorphic DNA (RAPD) technique was used to screen markers of sex determination in this species. A 569 bp RAPD marker, marker linked to sex determination in E. ulmoides (MSDE), was found in all the pistillate but not in the staminate plants; its exclusiveness to pistillate plants was confirmed by Southern blotting. MSDE was sequenced and specific primers were synthesized to generate a 569bp pistillate-specific SCAR marker, SCARmr. SCARmr could be useful for screening E. ulmoides plants for gender even before they reach reproductive maturity, resulting in considerable saving of time and economic resources.     

Development of PCR-based markers linked to a restorer gene for cytoplasmic male sterility in radish (Raphanus sativus L.)

A random amplified polymorphic DNA (RAPD) marker named OPC06-1900 was previously found linked to a fertility restorer gene (Rfw) for cytoplasmic male sterility (CMS) in radish (Raphanus sativus L.). The RAPD marker was converted to a dominant sequence characterized amplified region (SCAR) marker SCC06-1894 by molecular cloning and nucleotide sequencing. A BLAST search revealed that the SCAR marker SCC06-1894 showed significant homology to the corresponding regions of Arabidopsis and Brassica sulfate transporter genes. The presence of the intron and exon of the DNA fragment SCC06-1894 was demonstrated by comparing RT-PCR and PCR products. Thus, allele-specific oligonucleotide primers were designed to amplify the SCAR marker SCC06-415. PCR test with F₂ plants and sequence analysis showed that SCC06-1894 and SCC06-415 were allelic, linked to Rfw/rfw gene at 8.0 cM. Nine oligonucleotide primers were designed based on a single radish nuclear restorer gene mRNA. A survey of these primer combinations by bulked segregant analysis (BSA) identified three polymorphisms. The three PCR-based markers were co-segregant in the coupling phase and distant from the Rfw gene by 1.4 cM. These specific markers distributed on both sides of the Rfw gene and will be helpful for breeding new rapseed (Brassica napus L.) restorer lines.     

Conversion of the RAPD OPC021150 marker of the Rfo restorer gene into a SCAR marker for rapid selection of oilseed rape

The Rfo fertility restorer gene for the Ogura cytoplasmic male sterility (CMS) applied for oilseed rape hybrid seed production can be monitored with the use of the RAPD OPC02₁₁₅₀ marker while molecular breeding. The aim of this work was to convert the RAPD marker into a more suitable SCAR marker. Total DNA was isolated from a doubled haploid line derived from the line BO20 (INRA, France). A fragment of 1150‐bp linked to the Rfo gene was PCR amplified with the use of the RAPD OPC02 primer, cloned and sequenced. A pair of primers was designed and PCR amplification was performed to develop a SCAR marker for the Rfo gene. The new marker was applied for analysis of 220 oilseed rape lines comprising doubled haploid and inbred restorer lines, restored hybrids as well as F₁ and F₂ recombinant generations involving restorer lines. Simultaneously, the RAPD OPC02 marker was used and it revealed that the markers are equivalent to each other. However, the developed new SCAR marker has made the analysis more practical, rapid and efficient.     

Occurrence and frequency of putatively Y chromosome linked DNA markers in Cannabis sativa L.

DNA from female and male hemp (Cannabis sativa L.) plants belonging to nine different varieties were screened with180 RAPD primers in a search for sex-associated DNA markers. About 1500bands were produced in total, nine primers were found yielding one or two DNA bands amplified in all nine male DNA bulks and absent in all female DNA bulks. These putatively male-associated markers were then scored in three different F1progenies, deriving from a cross between a common male parent and three different female plants. The sex of the progeny was accurately scored on the basis of the floral phenotype, and the presence of the nine male-associated markers was verified by RAPD analysis. In all three progenies examined, all the male plants showed the DNA markers previously identified by bulk segregant analysis (BSA) on the hemp varieties, while all the female plants lacked them. The fact that the association between these markers and the staminate phenotype is found when examining male plants of distantly related varieties, and that such linkage is never broken when different progenies are examined, strongly supports the hypothesis that the markers found are physically located on the Y chromosome, in a region excluded from recombination during meiosis. Another marker was shown to be present in the male parent, in all the male plants of each progeny, and in 50% of the female progenies, while it was absent in the female parent; the possible occurrence of markers deriving from multiple amplification sites of the genome is discussed. This revised version was published online in August 2006 with corrections to the Cover Date.     

Identification and characterization of RAPD and SCAR markers linked to anthracnose resistance gene in sorghum [Sorghum bicolor (L.) Moench]

Anthracnose, one of the destructive foliar diseases of sorghum growing in warm humid regions, is incited by the fungus Colletotrichum graminicola.The inheritance of anthracnose resistance was studied using the parental cultivars of Sorghum bicolor (L.) Moench, HC 136 (susceptible to anthracnose) and G 73 (anthracnose resistant). The F₁ and F₂ plants were inoculated with the local isolates of C. graminicola cultures. The F₂ plants showed a segregation ratio of 3 (susceptible): 1(resistant) indicating that the locus for resistance to anthracnose in sorghum accession G 73 segregates as a recessive trait in a cross to susceptible cultivar HC 136. RAPD (random amplified polymorphic DNA) marker OPJ 01₁₄₃₇ was identified as marker closely linked to anthracnose resistance gene in sorghum by bulked segregant analysis of HC 136 × G73 derived recombinant inbred lines (RILs) of sorghum. A total of 84 random decamer primers were used to screen polymorphism among the parental genotypes. Among these, only 24 primers were polymorphic. On bulked segregant analysis, primer OPJ 01 amplified a 1437 bp fragment only in resistant parent G 73 and resistant bulk. The marker OPJ 01₁₄₃₇ was cloned and sequenced. The sequence of RAPD marker OPJ 01₁₄₃₇ was used to generate specific markers called sequence characterized amplified regions (SCARs). A pair of SCAR markers SCJ 01-1 and SCJ 01-2 was developed using Mac Vector program. SCAR amplification of resistant and susceptible parents along with their respective bulks and RILs confirmed that SCAR marker SCJ 01 is at the same loci as that of RAPD marker OPJ 01₁₄₃₇ and hence, is linked to anthracnose resistance gene. Resistant parent G 73 and resistant bulk amplified single specific band on PCR amplification using SCAR primer pairs. The RAPD marker OPJ 01₁₄₃₇ was mapped at a distance of 3.26 cM apart from the locus governing anthracnose resistance on the sorghum genetic map by the segregation analysis of the RILs. Using BLAST program, it was found that the marker showed 100 per cent alignment with the contig{_}3966 located on the longer arm of chromosome 8 of sorghum genome. Therefore, these identified RAPD and SCAR markers can be used in the resistance-breeding program of sorghum anthracnose by marker-assisted selection.An erratum to this article can be found at     

An extended random primer amplified region (ERPAR) marker linked to a dominant male sterility gene in cabbage (Brassica oleracea var. capitata)

Similar to SCAR, an extended random primer amplified region (ERPAR) marker is a PCR amplified genomic DNA fragment at a single genetically defined locus. However, ERPAR uses specific primer pairs derived from RAPD primers by adding bases sequentially to their 3′-ends. As an example, an ERPAR marker was derived from a RAPD marker (OT11₉₀₀) linked to a dominant male sterility gene in cabbage (Brassica oleracea var. capitata). After two cycles of base adding and primer pair screening, a primer pair (5′-TTCCCCGCGACT-3′and 5′-TTCCCCGCGAGA-3′) amplified a single intense band with the same size as OT11₉₀₀. The identity of the new marker and OT11₉₀₀ was verified by segregation analysis. The new marker amplified by this extended primer pair was named as EPT11₉₀₀. The development of ERPAR exploits the importance of 3′-end bases of primers in PCR ERPAR shares advantages of SCAR, but eliminates the need for cloning and sequencing. It is a fast and universal way of converting RAPD markers into stable markers. This revised version was published online in July 2006 with corrections to the Cover Date.     

大葱雄性不育分子标记辅助选择的研究

大葱雄性不育在杂种优势利用中具有重要价值,传统方法选育不育系效率低,分子标记辅助育种可提高育种效率。本研究试图建立大葱雄性不育辅助育种的技术体系,加快大葱不育系、保持系的育种进程。利用RAPD技术分析了多态性片段S132800、S38960、S72300、S731100、S2002400与大葱育性的连锁关系,重组率分别为0、7．5％、0、4．2％、0。其中S132800、S2002400能在多数品种中区分N、S胞质,且重组率接近0,用于胞质鉴定具有很高的准确率,因而具有较高的利用价值。以S132800、S2002400为探针对不育系和保持系mtDNA的酶切片段进行了Southern杂交分析,结果表明它们在N、S胞质中存在多态性。预示着它们可能是与大葱CMS相关的片段。鉴于RAPD标记的应用有一定局限性,进一步对特异片段S132800、S2002400进行了克隆、测序和SCAR标记转化,其中S132800成功转化为SCAR标记,而S2002400转化后多态性消失。为进一步降低成本,简化了SCAR扩增产物的检测技术,初步建立了大葱不育系、保持系分子标记辅助选择的技术体系。研究表明,SCAR产物加入EB直接检测存在一定误差,而通过快速电泳可以快速、准确地鉴定单株的胞质类型。RAPD标记和SCAR标记在育种中应用有望提高大葱不育系、保持系的育种效率。     

Development and Validation of SCAR Markers Co-Segregating with an Agropyron Elongatum Derived Leaf Rust Resistance Gene Lr24 in Wheat

Summary An Agropyron elongatum-derived leaf rust resistance gene Lr24 located on chromosome 3DL of wheat was tagged with six random amplified polymorphic DNA (RAPD) markers which co-segregated with the gene. The markers were identified in homozygous resistant F₂ plants taken from a population segregating for leaf rust resistance generated from a cross between two near-isogenic lines (NILs) differing only for Lr24. Phenotyping was done by inoculating the plants with pathotype 77-5 of Puccinia triticina. To enable gene-specific selection, three RAPD markers (S1302₆₀₉, S1326₆₁₅ and OPAB-1₃₈₈) were successfully converted to polymorphic sequence characterized amplified region (SCAR) markers, amplifying only the critical DNA fragments co-segregating with Lr24. The SCAR markers were validated for specificity to the gene Lr24 in wheat NILs possessing Lr24 in 10 additional genetic backgrounds including the Thatcher NIL, but not to 43 Thatcher NILs possessing designated leaf rust resistance genes other than Lr24. This indicated the potential usefulness of these SCAR markers in marker assisted selection (MAS) and for pyramiding leaf rust resistance genes in wheat.     

Validation of male sex‐specific UBC‐8071200 ISSR marker and its conversion into sequence tagged sites marker in Jojoba: a high precision oil yielding dioecious shrub

There is a recent surge in the marker‐assisted selection of desired sex type among economically important dioecious crops. Simmondsia chinensis (Jojoba), a dioecious crop is of immense agricultural importance where only the female plants are preferred for commerce. DNA fingerprinting technology, ISSR analysis along with bulk segregant analysis (BSA), has been carried out on a diverse set of 17‐ to18‐year‐old mature male and female plants of Jojoba to validate a male sex‐specific ISSR marker, UBC‐807₁₂₀₀ on larger population that comprises 330 female and 255 male plants of Jojoba. This male sex‐specific DNA fragment of ~1200 bp was cloned and sequenced. The sequence was found to be 1120 bp in length and based on the sequence information, a pair of sequence tagged sites primers was developed that amplified a single ~800 bp band, consistently only in all the male populations while no amplification was seen in their female counterparts. The marker was named as Jojoba Male Sex Marker which was further validated on 330 female plants from 22 genotypes and 255 male plants from 17 genotypes.     

RAPD-SCAR在鱼类种质鉴定中的应用及前景

种质是利用和改良动植物、微生物的物质基础,更是实施各个育种途径的原材料,因此优良种质鉴定是一个很关键的问题。种质鉴定方法已由形态水平发展到蛋白质和DNA分子水平。RAPD技术具有敏感、快速、简便、产量高、重复性好及检测容易等突出优点,广泛应用于种质鉴定中,但也有不足之处。基于RAPD标记技术建立起来的SCAR（Sequence Characterized Amplified Region）标记克服了RAPD标记的不足,操作简捷,特异性和重复性较高,是一种有效的分子标记。RAPD-SCAR分子标记技术的利用使种质鉴定更为快速准确,提高了育种速度,缩短了育种周期,为相关的研究工作提供分子遗传学依据。     

Identification of a molecular marker linked to an Agropyron elongatum-derived gene Lr19 for leaf rust resistance in wheat

The leaf rust resistance gene Lr19, transferred from Agropyron elongatum into wheat (Triticum aestivum L.) imparts resistance to all pathotypes of leaf rust (Puccinia recondita f.sp. tritici) in South‐east Asia. A segregating F₂ population from a cross between the leaf rust resistant parent ‘HW 2046’ carrying Lr19 and a susceptible parent ‘Agra Local’ was screened in the phytotron against a virulent pathotype 77‐5 of leaf rust with the objective of identifying the molecular markers linked to Lr19. The gene was first tagged with a randomly amplified polymorphic DNA (RAPD) marker S73₇₂₈. The RAPD marker linked to the gene Lr19 which mapped at 6.4 ± 0.035 cM distance, was converted to a sequence characterized amplified region (SCAR) marker. The SCAR marker (SCS73₇₁₉) was specific to Lr19 and was not amplified in the near‐isogenic lines (NILs) carrying other equally effective alien genes Lr9, Lr28 and Lr32 enabling breeders to pyramid Lr19 with these genes.     

Evaluation of F2 and F3 plants introgressed with QTLs for clubroot resistance in cabbage developed by using SCAR markers

Clubroot disease caused by Plasmodiophora brassicae is one of the major diseases of Brassica crops, often devastating to the cultivation of cruciferous crops in temperate regions. In a previous study (Moriguchi et al. 1999) identified three major quantitative trait loci (QTLs) for clubroot resistance, each in a separate linkage group, in a population derived from a cross between a clubroot‐susceptible inbred cabbage line, Y2A and a resistant inbred kale line, K269. In this study, the original random amplified polymorphic DNA (RAPD) and restriction fragment length polymorphism (RFLP) markers were converted into sequence‐characterized amplified region (SCAR) markers to facilitate large‐scale marker‐assisted screening of clubroot resistance in cabbage breeding. Of 15 RAPD markers closely linked to the three QTLs, nine SCARs were developed as dominant markers after cloning and sequencing. In addition, two RAPD markers were converted into co‐dominant cleaved amplified polymorphic sequence (CAPS) markers, and one RFLP marker out of three tested was converted to a dominant SCAR marker. The effect of selection for resistance by the improved markers was evaluated in progeny plants in the F2 and F3. A total of 138 F2 plants were genotyped with nine SCARs and 121 well‐distributed makers consisting of 98 RAPD, 19 RFLP, two isozymes, and two morphological markers in order to estimate the level of resistance and the proportion of undesirable alleles from the kale in non‐target areas in each of the F2 populations. An F2 plant, YK118, had kale alleles at QTL1, QTL3 and QTL9. Three F2 plants, namely, YK107, YK25 and YK51 had kale alleles at only QTL1, QTL3 and QTL9, respectively. These F2 plants were selected for their low proportion of alleles derived from kale in non‐target regions. YK118, like the resistant kale parent, expressed very high resistance to three field isolates of Plasmodiophora brassicae, whereas the mean disease index in the F2 and F3 plants carrying only single QTLs was intermediate. The QTLs showed no differential response to the isolates. These plants with improved resistance will be useful as parental inbred lines for F1 hybrids.     

RAPD and SCAR markers for resistance to acochyta blight in lentil

Resistance to ascochyta blight of lentil (Lens culinaris Medikus),caused by the fungus Ascochyta lentis, is determined by a single recessive gene, ral ₂, in the lentil cultivar Indian head. Sixty F₂ individuals from a cross between Eston (susceptible) and Indian head (resistant) lentil were analyzed for the presence of random amplified polymorphic DNA (RAPD) markers linked to the ral ₂gene, using bulked segregant analysis (BSA). Out of 800 decanucleotide primers screened, two produced polymorphic markers that co-segregated with the resistance locus. These two RAPD markers, UBC227₁₂₉₀and OPD-10₈₇₀, flanked and were linked in repulsion phase to the gene ral ₂ at 12 cm and 16 cm, respectively. The RAPD fragments were converted to SCAR markers. The SCAR marker developed from UBC227₁₂₉₀ could not detect any polymorphism between the two parents or in the F₂. The SCAR marker developed from OPD-10₈₇₀ retained its polymorphism. The polymorphic RAPD marker UBC227₁₂₉₀ and the SCAR marker developed from OPD-10₈₇₀ can be used together in a marker assisted selection program for ascochyta blight resistance in lentil. This revised version was published online in July 2006 with corrections to the Cover Date.     

Sexual crossing between two genetically female plants and sex genetics of kakrol (Momordica dioica Roxb.)

Summary Application of 200 to 800 mg/l silver nitrate (AgNO₃) to the female kakrol or teasle gourd (Momordica dioica Roxb.) plant was effective for inducing hermaphrodite flowers. Pollen grain viability of the hermaphrodite flowers was as high as that of normal male flowers. Crossing among female genotypes of different fruit morphotypes, taking pollen from the induced hermaphrodite flowers, produced fruits and seeds. Sex of hybrids between female and normal male segregated into male and female in equal proportion, while the hybrids from female homo-sexual crosses produced only female plants. This indicated that sex of kakrol is controlled by a single factor; male being heterozygous and the female is homozygous recessive. The possibility to evaluate both parents for fruit characteristics should facilitate breeding.