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.     

Characterization and fine mapping of <Emphasis Type="Italic">RppQ</Emphasis>, a resistance gene to southern corn rust in maize

Southern corn rust (SCR) is a fungal disease caused by Puccinia polysora Underw, which can infect maize and may result in substantial yield losses in maize production. The maize inbred line Qi319 carries the SCR resistance gene RppQ. In order to identify molecular markers linked to the RppQ gene, several techniques were utilized including random amplified polymorphic DNA (RAPD), simple sequence repeat (SSR), and amplified fragment length polymorphism (AFLP). In addition, sequence characterized amplified region (SCAR) techniques combined with bulked segregant analysis (BSA) were used. Seven RAPD markers, eight SSR markers, and sixty-three AFLP primer combinations amplified polymorphisms between two parents and two bulk populations. A large F₂ population was used for genetic analysis and for fine mapping of the RppQ gene region. One AFLP polymorphic band, M-CAA/E-AGC₃₂₄, was converted to a SCAR marker, MA7, which was mapped to a position 0.46 cM from RppQ. Finally, the RppQ gene was mapped between the SCAR marker MA7 and the AFLP marker M-CCG/E-AGA₁₅₇ with distances of 0.46 and 1.71 cM, respectively.     

Construction of a Genetic Map and Development of DNA Markers Linked to the Sex-Determining Locus in the Patagonian Pejerrey (Odontesthes hatcheri)

The process of sex differentiation in fishes is regulated by genetic and environmental factors. The sex of Patagonian pejerrey (Odontesthes hatcheri) appears to be under strong genotypic control (GSD) because the sex ratios are balanced (1:1) between 17°C and 23°C. However, sex ratios become female-biased at <15°C and male-biased at 25°C, which shows that this species also possesses some degree of temperature-dependent sex determination (TSD). Identification of the genetic sex of an individual will help elucidate the molecular basis of sex differentiation in this species. In this study, we used amplified fragment length polymorphism (AFLP) analysis to develop a genetic linkage map for both sexes and a sex-linked DNA marker for Patagonian pejerrey. The AFLP analysis of 23 male and 23 female progeny via 64 primer combinations produced a total of 153 bands. The genetic linkage map consisted of 79 markers in 20 linkage groups and 48 markers in 15 linkage groups for males and females, respectively. One AFLP marker tightly linked to the sex-determining locus was identified: the marker, ACG/CAA-217, amplified to the male-specific DNA fragment. Sequence analysis of this region revealed a single nucleotide polymorphism (SNP) between males and females, which was converted into a SNP marker. This marker provides genetic confirmation that the sex of Patagonian pejerrey is determined genetically and would be useful for the analysis of the molecular basis of GSD and TSD in this species.     

Sex-specific SCAR markers in the dioecious plant<Emphasis Type="Italic"> Rumex nivalis</Emphasis> (Polygonaceae) and implications for the evolution of sex chromosomes

We developed SCAR primers based on isolated and sequenced male-specific fragments as identified in an AFLP analysis of the dioecious plant Rumex nivalis. PCR amplification using these primers on females and males resulted in fragments exclusively present in males. Co-amplification of the nuclear rDNA internal transcribed spacer 2 together with the male-specific fragment was applied as an internal control for successful PCR reactions to avoid false-negative sex scoring. With a length of about 164 bp, the AFLP fragment was of a similar size as the tandemly arranged, repetitive sequences of 180 bp located on the Y chromosomes of Rumex acetosa. The genetic distances between the Y-chromosomal sequences of R. nivalis and R. acetosa, both members of the section Acetosa, were substantial. We found intra-individual divergence among cloned sequences of the male-specific fragment in R. nivalis. The patterns of interspecific and intra-individual sequence variation found are in accordance with proposed modes of the evolution of sex chromosomes. Y chromosomes possibly arose only once in the genus Rumex and consist mainly of heterochromatic DNA. Due to the almost complete absence of selection on them, Y chromosomes are likely to accumulate large numbers of mutations.Communicated by R. Hagemann     

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).     

CHARACTERIZATION OF GENETIC MARKERS LINKED TO SEX DETERMINATION IN THE HAPLOID‐DIPLOID RED ALGA GRACILARIA CHILENSIS1

Bulk segregant analysis, random amplified polymorphic DNA (RAPD), and sequence characterized amplified region (SCAR) methods were used to identify sex‐linked molecular markers in the haploid‐diploid rhodophyte Gracilaria chilensis C. J. Bird, McLachlan et E. C. Oliveira. One hundred and eighty 10 bp primers were tested on three bulks of DNA: haploid males, haploid females, and diploid tetrasporophytes. Three RAPD primers (OPD15, OPG16, and OPN20) produced male‐specific bands; and one RAPD primer (OPD12), a female‐specific band. The sequences of the cloned putative sex‐specific PCR fragments were used to design specific primers for the female marker SCAR‐D12‐386 and the male marker SCAR‐G16‐486. Both SCAR markers gave unequivocal band patterns that allowed sex and phase to be determined in G. chilensis. Thus, all the females presented only the female band, and all the males only the male band, while all the tetrasporophytes amplified both male and female bands. Despite this sex‐specific association, we were able to amplify SCAR‐D12‐386 and SCAR‐G16‐486 in both sexes at low melting temperature. The differences between male and female sequences were of 8%–9% nucleotide divergence for SCAR‐D12‐386 and SCAR‐G16‐486, respectively. SCAR‐D12‐386 and SCAR‐G16‐486 could represent degenerated or diverged sequences located in the nonrecombining region of incipient sex chromosomes or heteromorphic sex chromosomes with sequence differences at the DNA level such that PCR primers amplify only one allele and not the other in highly specific PCR conditions. Seven gametic progenies composed of 19 males, 19 females, and the seven parental tetrasporophytes were analyzed. In all of them, the two SCAR markers segregated perfectly with sexual phenotypes.     

半滑舌鳎雌性特异AFLP标记CseF783的克隆及其在遗传性别鉴定中的应用

半滑舌鳎性别控制和全雌育种等研究领域中迫切需要一种能够快速鉴定鱼类个体遗传性别的有效方法。文章采用AFLP技术, 利用选择性引物组合(E-ACT/M-CAA)从半滑舌鳎中筛选到一条雌性特异的AFLP标记。对该标记进行二次PCR扩增、琼脂糖凝胶回收、克隆、测序。分析表明, 序列全长为791 bp, 与GenBank中的序列无同源性。以该雌性特异AFLP标记DNA序列为模板, 设计了一对特异的PCR引物, 成功地将其转化为SCAR(Sequence characterized amplified regions)标记, 并在100尾已知性别的半滑舌鳎个体(雌雄各50尾)中进行验证, 结果表明, 该SCAR标记在所有雌性个体中均扩增得到一条长度为324 bp的DNA条带, 而在49尾雄性个体中均扩增不到该DNA条带(有1尾雄性个体例外), 证明该SCAR标记是雌性特异的, 并可用于半滑舌鳎个体遗传性别鉴定。随后, 利用该SCAR标记检测了3日龄半滑舌鳎幼苗, 结果表明, 雌性个体比例为41.7%。     

An AFLP marker that differentiates biotypes of the Asian rice gall midge (Orseolia oryzae, Wood-Mason) is sex-linked and also linked to avirulence

In an attempt to identify a specific marker for biotype 2 of the Asian rice gall midge (Orseolia oryzae, Wood-Mason), we used AFLP (amplified fragment length polymorphism) fingerprinting. We identified an AFLP marker that is specifically amplified in biotypes 1, 2 and 5 of the rice gall midge, but not in biotype 4. Biotypes 1, 2 and 5 are avirulent to hosts bearing the Gm2 resistance gene (found in rice variety Phalguna), whereas biotype 4 is virulent to Gm2. Based on the sequence of this AFLP marker, SCAR (sequence characterized amplified region) primers were designed and used in combination with previously developed SCAR primers to distinguish effectively all five biotypes in a multiplex PCR-based assay. The inheritance pattern of this marker in the progenies of inter-biotype crosses between biotypes 1, 2 and 4 shows that the marker can be amplified by PCR from all F₁ females, irrespective of the biotype status of their parents. However, the marker is present only in those male progenies whose mother was of a Gm2 avirulent biotype. The specific amplification of this marker in the avirulent biotypes and its pattern of inheritance show that avirulence with respect to carriers of the Gm2 gene in rice gall midge is sex-linked. Received: 16 August 1999 / Accepted: 27 December 1999     

A consensus map for <Emphasis Type="Italic">Cucurbita pepo</Emphasis>

Using random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), simple sequence repeats (SSR), and morphological traits, the first genetic maps for Cucurbita pepo (2n=2x=40) were constructed and compared. The two mapping populations consisted of 92 F₂ individuals each. One map was developed from a cross between an oil-seed pumpkin breeding line and a zucchini accession, into which genes for resistance to Zucchini Yellow Mosaic Virus (ZYMV) from a related species, C. moschata, had been introgressed. The other map was developed from a cross between an oil-seed pumpkin and a crookneck variety. A total of 332 and 323 markers were mapped in the two populations. Markers were distributed in each map over 21 linkage groups and covered an average of 2,200 cM of the C. pepo genome. The two maps had 62 loci in common, which enabled identification of 14 homologous linkage groups. Polyacrylamide gel analyses allowed detection of a high number of markers suitable for mapping, 10% of which were co-dominant RAPD loci. In the Pumpkin-Zucchini population, bulked segregant analysis (BSA) identified seven markers less than 7 cM distant from the locus n, affecting lignification of the seed coat. One of these markers, linked to the recessive hull-less allele (AW11-420), was also found in the Pumpkin-Crookneck population, 4 cM from n. In the Pumpkin-Zucchini population, 24 RAPD markers, previously introduced into C. pepo from C. moschata, were mapped in two linkage groups (13 and 11 markers in LGpz1 and LGpz2, respectively), together with two sequence characterized amplified region (SCAR) markers linked to genes for resistance to ZYMV.     

SCAR markers linked to the common bean rust resistance gene Ur-13

Rust in common bean (Phaseolus vulgaris L.) is caused by Uromyces appendiculatus Pers.:Pers. (Unger) which exhibits a high level of pathogenic diversity. Resistance to this disease is conditioned by a considerable number of genes. Pyramiding resistance genes is desirable and could be simplified by the use of molecular markers closely linked to the genes. The resistance gene Ur-13, present in the South African large seeded cultivar Kranskop, has been used extensively in the local breeding program. The purpose of this study was the development of a molecular marker linked to Ur-13. An F₂ population derived from a cross between Kranskop and a susceptible (South African) cultivar Bonus was used in combination with bulked segregant analysis utilizing the amplified fragment length polymorphism (AFLP) technique. Seven AFLP fragments linked significantly to the rust resistance and five were successfully converted to sequence characterized amplified region (SCAR) markers. The co-dominant SCAR markers derived from a 405 bp EAACMACC fragment, KB126, was located 1.6 cM from the gene. Two additional SCAR markers and one cleaved amplified polymorphic sequence marker were located further from the gene. The gene was mapped to linkage group B8 on the BAT 93/Jalo EEP 558 core map (chromosome 3).     

Isolation of sex-specific AFLP markers in Spotted Halibut (<Emphasis Type="Italic">Verasper variegatus</Emphasis>)

Spotted Halibut (Verasper variegatus) is a commercially important marine fish species. In the present study, to isolate sex-specific markers in Spotted Halibut, we screened the genomes of Spotted Halibut by AFLP technique with 64 different primer combinations. Two primer combinations, MseI-CAG/EcoRI-ACC and MseI-CAT/EcoRI-AGG, produced a female-specific fragment in all females (n = 88) and in no males (n = 60, except 3 individuals), respectively. Both fragments were excised from the gel, cloned and characterized. The first fragment (named VevaF533) was 533 bp long, while the length of the second one (named VevaF218) was 218 bp. The two sequences showed no similarity to each other, and to the known sequences existing in the GenBank database using BLASTn. Cross-species amplification showed that the marker VevaF218 is a species-specific marker which is present in Spotted Halibut females but absent in Barfin Flounder (Verasper moseri). So this marker could be used for discriminating unambiguously between Spotted Halibut females and Barfin Flounder. Examination of the patterns of inheritance of VevaF218 in an interspecific hybrid family (V. variegatus ♂×V. moseri ♀) showed a female-specific pattern of inheritance from mother to daughter, implying that the marker VevaF218 is located on the female sex chromosome. This study provides a reliable AFLP-based genetic sexing of Spotted Halibut that could be useful for genetic mapping of the sex chromosomes and identification of sex-linked genes.     

Mapping of AFLP markers linked to seed coat colour loci in<Emphasis Type="Italic"> Brassica juncea</Emphasis> (L.) Czern

Association mapping of the seed-coat colour with amplified fragment length polymorphism (AFLP) markers was carried out in 39 Brassica juncea lines. The lines had genetically diverse parentages and varied for seed-coat colour and other morphological characters. Eleven AFLP primer combinations were used to screen the 39 B. juncea lines, and a total of 335 polymorphic bands were detected. The bands were analysed for association with seed-coat colour using multiple regression analysis. This analysis revealed 15 markers associated with seed-coat colour, obtained with eight AFLP primer combinations. The marker E-ACA/M-CTG₃₅₀ explained 69% of the variation in seed-coat colour. This marker along with markers E-AAC/M-CTC₂₃₅ and E-AAC/M-CTA₂₅₀ explained 89% of the total variation. The 15 associated markers were validated for linkage with the seed-coat colour loci using a recombinant inbred line (RIL) mapping population. Bands were amplified with the eight AFLP primer combinations in 54 RIL progenies. Of the 15 associated markers, 11 mapped on two linkage groups. Eight markers were placed on linkage group 1 at a marker density of 6.0 cM, while the remaining three were mapped on linkage group 2 at a marker density of 3.6 cM. Marker E-ACA/M-CTG₃₅₀ co-segregated with Gene1 controlling seed-coat colour; it was specific for yellow seed-coat colour and mapped to linkage group 1. Marker E-AAC/M-CTC₂₃₅ (AFLP8), which had been studied previously, was present on linkage group 2; it was specific for brown seed-coat colour. Since AFLP markers are not adapted for large-scale applications in plant breeding, it is important to convert these to sequence-characterised amplified region (SCAR) markers. Marker E-AAC/M-CTC₂₃₅ (AFLP8) had been previously converted into a SCAR. Work is in progress to convert the second of the linked markers, E-ACA/M-CTG₃₅₀, to a SCAR. The two linked AFLP markers converted to SCARs will be useful for developing yellow-seeded B. juncea lines by means of marker-assisted selection.Communicated by H.F. Linskens     

Molecular identification of AFLP fragments associated with elytra color variation of the Asian ladybird beetle,Harmonia axyridis (Coleoptera: Coccinellidae)

The Asian ladybird beetle, Harmonia axyridis shows polymorphism in elytra color patterns. However, it is uncertain whether these color patterns are regulated by genetic factors. This investigation used amplified fragment length polymorphism (AFLP) analysis to determine any genetic causes of the variability of color patterns. Using four individuals of each group, AFLP analysis produced 37 polymorphic bands. Among several polymorphic bands, six AFLP markers were associated with elytra color patterns after further analysis using six additional individuals of each group. These polymorphic sites were sequenced but did not match DNA sequence data deposited in GenBank. Based on the color-associated AFLP markers, SCAR primers were designed for PCR amplification of genomic DNA. These primers (SCAR 12 and SCAR 44) were used to analyze color-associated loci and/or alleles of H. axyridis DNA. SCAR 12 primers designed from a Spectabilis type-specific fragment (AFLP 12) amplified a specific band of 530 bp in four Spectabilis individuals, but not in the insects with other color patterns.     

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。     

Isolation and Characterization of Male-Specific DNA Markers in the Rock Bream Oplegnathus fasciatus

Sex-specific DNA markers applicable were very useful for elucidating the sex-determination mechanism and sex control in fishes. In the present study, amplified fragment-length polymorphism (AFLP) approach with 144 primer combinations was employed to identify sex-specific markers in the rock bream. Four male-specific AFLP fragments were identified which were designated as Opl286, Opl237, Opl422, and Opl228. Further sequence analysis of the sex markers’ genomic region revealed subtle differences between the males and females. We identified four male-specific single-nucleotide polymorphisms (SNPs) and a deletion of 8 bp in marker Opl286, six male-specific SNPs in marker Opl237, three male-specific SNPs in marker Opl422, and eight male-specific SNPs and 1 bp inversions in marker Opl228. Specific primers were designed based on the nucleotide variation in the sequences to develop a simple polymerase chain reaction method for identifying the genetic sex of rock bream. As a result, three out of the four male-specific markers were converted into SNP markers. The male-specific AFLP markers and AFLP-derived SNP markers were tested in 100 individuals collected from three locations around the coast of Zhoushan, yielding reproducible sex identification. These male-specific DNA markers are a useful tool for the identification of the sex-determining locus in rock bream and for guiding artificial breeding programs.     

Cloning of Taiwan water buffalo male-specific DNA sequence for sexing

Random amplified polymorphic DNA (RAPD) fingerprinting was carried out to investigate the sex-specific DNA sequence for sexing in Taiwan water buffalos. One hundred and forty random primers were used for RAPD-PCR (polymerase chain reaction). One of these primers, OPC-16, produced a 321 bp fragment found only in tested males. This male-specific fragment was isolated and constructed into plasmids for nucleotide sequencing, a novel male-specific sequence was obtained. Two primers (BuSexOPC16-F and -R) were designed according to the cloned male-specific sequence to amplify the male-specific fragment using PCR for sexing. Sex-specific bands in the gel were represented in the males but none were found in the females when the Taiwan water buffalo genomic DNA samples were amplified with these two primers using PCR. The same results were also obtained from Taiwan yellow, Holstein, Angus, and Hereford cattle samples. This showed that the sex of these five breeds could be easily and effectively determined using the PCR technique.     

Conversion of AFLP markers to high-throughput markers in a complex polyploid,sugarcane

The application of DNA markers linked to traits of commercial value in sugarcane may increase the efficiency of sugarcane breeding. The majority of markers generated for quantitative trait locus mapping in sugarcane have been single sequence repeats or AFLPs (amplified fragment length polymorphisms). Since AFLP markers are not adapted for large-scale implementation in plant breeding, our objective was to assess the feasibility of converting AFLP markers to fast, cheap and reliable PCR-based assays in a complex polyploid, sugarcane. Three AFLP markers were selected on the basis of an association to resistance to the fungal pathogen Ustilago scitaminea, the causal agent of smut in sugarcane. We developed an approach which enabled the identification of polymorphisms in these AFLP markers. Towards this goal, we employed GenomeWalking and 454 sequencing to isolate sequences adjacent to the linked AFLP markers and identify SNP (single nucleotide polymorphisms) haplotypes present in the homo(eo)logous chromosomes of sugarcane. One AFLP marker was converted to a cleavage amplified polymorphic sequence marker, another to a SCAR (sequence characteristered amplified region) marker and the final AFLP marker to a SNP PCR-based assay. However, validation of each of the markers in 240 genotypes resulted in 99, 90 and 60% correspondence with the original AFLP marker. These experiments indicate that even in a complex polyploid such as sugarcane, polymorphisms identified by AFLP can be converted to high-throughput marker systems, but due to the complexity this would only be carried out for high-value markers. In some cases, the polymorphisms identified are not transferable to more sequence-specific PCR applications.     

Development of leucine-rich repeat polymorphism, amplified fragment length polymorphism, and sequence characterized amplified region markers to the Cronartium ribicola resistance gene Cr2 in western white pine ( Pinus monticola )

White pine blister rust (WPBR), caused by Cronartium ribicola, is a devastating disease in Pinus monticola and other five-needle pines. Pyramiding a major resistance gene (Cr2) with other resistance genes is an important component of integrated strategies to control WPBR in P. monticola. To facilitate this strategy, the objective of the present study was to identify leucine-rich repeat (LRR) polymorphisms, amplified fragment length polymorphisms (AFLPs), and sequence characterized amplified region (SCAR) markers linked to the western white pine Cr2 (BSA) gene for precise gene mapping. Bulked segregant analysis and haploid segregation analysis allowed the identification of 11 LRR polymorphisms and five AFLP markers in the Cr2 linkage. The closest LRR markers were 0.53 Kosambi cM from Cr2 at either end. After marker cloning and sequencing, AFLP marker EacccMccgat-365 and random polymorphic DNA marker U570–843 were converted successfully into SCAR markers. For a potential application in marker-assisted selection (MAS), these two SCAR markers were verified in two western white pine families. This study represents the first report of LRR-related DNA markers linked to C. ribicola resistance in five-needle pines. These findings may help further candidate gene identification for disease resistance in a conifer species.     

An RGA-Derived SCAR Marker Linked to PLRV Resistance from Solanum tuberosum ssp. andigena

A resistance gene analog (RGA)-derived sequence-characterized amplified region (SCAR) marker was successfully developed based on sequence homology with disease resistance genes of an AFLP molecular marker tightly linked to the Rl _adg gene of Solanum tuberosum ssp. andigena. The new marker was designated as ‘RGASC850’ (RGA-derived SCAR) based on the size of the amplified fragment. ‘RGASC850’ could be efficiently used for monitoring introgression of Rl _adg against backgrounds of improved gene pools with low likelihood of identifying false positives due to recombination. This SCAR proved to be highly predictive of Rl _adg -based resistance, as it did not amplify potato leafroll virus (PLRV) resistance sources other than andigena, and thus would be useful in developing cultivars with complementary sources of resistance to PLRV. In addition, a cleaved amplified polymorphic sequence (CAPS) marker based on ‘RGASC850’ was developed capable of distinguishing genotypes carrying Rl _adg . This CAPS marker would be useful for screening breeding populations derived from wide crosses, and confirming presence of the Rl _adg gene in those parents amplifying the ‘RGASC850’ marker.     

Development of strain-specific SCAR markers for authentication of Ganoderma lucidum

In this study we report the application of sequence-characterized amplified region (SCAR) markers in Ganoderma lucidum for strain identification, the first such study in this medicinal mushroom. One fragment unique to strain No. 9 was identified by inter-simple sequence repeats (ISSR), and then sequenced. Based on the specific fragment, one SCAR primer pair designated as GL₆₁₂F and GL₆₁₂R was designed to amplify a 612-bp DNA fragment within the sequenced region. Diagnostic PCR was performed using the primer pair. The results showed that this SCAR marker can clearly distinguish strain No. 9 from other related Ganoderma lucidum strains. Our data provided the foundation for a precise and rapid PCR-based strain-diagnostic system for Ganoderma lucidum.