Single nucleotide polymorphism genotyping of the barley waxy gene by polymerase chain reaction with confronting two-pair primers

A high‐throughput single nucleotide polymorphism (SNP) genotyping procedure was developed to select amylose‐free barley mutants whose waxy genes had a C‐ to T‐base substitution in exon 5, which converted Gln‐89 of the wild‐type gene into a termination codon. An F₂ population carrying an amylose‐free waxy gene was checked for segregation. Polymerase chain reaction with confronting two‐pair primers (PCR‐CTPP) produced allele‐specific PCR products that have different sizes and are inherited in a co‐dominant manner. Two alleles of the barley waxy gene with SNP were correctly identified in parental strains using the PCR‐CTPP procedure. Segregation of the SNP as detected by PCR‐CTPP in an F₂ population fitted the expected 1:2:1 ratio. The PCR‐CTPP procedure can provide a time saving and cost‐effective alternative to derived cleaved amplified polymorphic sequence in marker‐assisted selection.     

A polymorphic microsatellite in the 3'end of 'waxy' genes of wheat, Triticum aestivum

Potential polymorphism of an (AT)_N microsatellite at the 3’end of waxy genes in bread wheat was examined. Primers were designed from a published cDNA sequence of a wheat waxy gene. Polymerase chain reaction (PCR) amplification of genomic DNA from 135 mainly Australian cultivars revealed eight alleles on chromosome 7A. This polymorphic microsatellite is a potential codominant marker for the Wx-A1 locus in breeding programmes. A distinguishable fragment was also amplified from chromosome 7D. This fragment was absent where a plant was null for the waxy gene on chromosome 7D, being a dominant marker for the Wx-D1 locus. The primers were also useful for amplifying genomic DNA from barley, rye and triticale and can be used to detect potential polymorphism in these species.     

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.     

Amplified fragment length polymorphism-derived microsatellite sequence linked to the Pch1 and Ep-D1 loci in common wheat

Amplified fragment length polymorphism (AFLP) markers linked to the Aegilops ventricosa‐derived chromosome segment in ‘VPM1’ on which the eyespot resistance gene, Pch1, and the endopeptidase gene, Ep‐D1b, occur were identified. One marker was isolated from the gel, cloned and sequenced. Sequence analysis revealed a microsatellite repeat motif. Sequence‐specific primers were designed to amplify a product containing the repeat motif, and the microsatellite marker was tested for cosegregation with the Ep‐D1b allele. Distinct alleles were produced by the Pch1 sources, normal wheat and wheat containing the Lr19 translocation. A recombination frequency of 0.02 was calculated between the microsatellite marker and Ep‐D1.     

Inter and intra-species Inter Simple Sequence Repeat (ISSR) variations in the genus Cicer

Intra and inter-species ISSR variation and use of ISSR markers in determination of genetic relationship were investigated in an accession collection representing twoperennial and six annual Cicerspecies. Screening of Ciceraccessions with SSR primers revealed highly reproducible amplicon profiles with relatively high multiplex ratios. Many of the primers generated amplicon profiles with which not only the differences among species can readily be identified, but also polymorphisms within species could be detected more efficiently. PCR products at 150 gel positions detected using six SSR primers in Cicer accessions were treated as dominant DNA markers and utilized to compute the distances among accessions and species. Cluster analysis of accessions and species revealed groupings that corroborate our previous studies of relationships based on allozyme and AFLP analysis. Consistent with the AFLP analysis carried out in the same accession collection, ISSR-based groupings indicated that perennial C. incisumis genetically close to the annuals of the second crossability group (C. pinnatifidum,C. bijugum, C. judaicum) while C. reticulatum is the closest wild species to the cultivated chickpea. ISSR-based variation estimates were relatively higher when compared to previous estimates computed from RAPD and AFLP data. Technically, ISSR analysis combines the PCR-based targeting of microsatellite-associated polymorphisms with no prior sequence requirement and stringent PCR conditions. Similarly, when compared to AFLP analysis, it is less technically demanding allowing to survey polymorphic loci in the genome. Thus, ISSR-PCR technology is a reliable, fast, and cost-effective marker system that can be used to study genetic variation and genetic relationships in the genusCicer. This revised version was published online in July 2006 with corrections to the Cover Date.     

Highly variable ‘Arhar’ simple sequence repeat markers for molecular diversity and phylogenetic studies in pigeonpea [Cajanus cajan (L.) Millisp.]

Pigeonpea is an important tropical grain legume widely cultivated in South and South‐East Asia for versatile end usage as food, feed, fodder and fuel. Recent publication of draft genome sequence of pigeonpea (‘Arhar’) has allowed mining of large numbers of genomic simple sequence repeat (SSR) markers most of which are either not validated or show very little polymorphism in simple agarose gel–based assays. Here, we describe a special category of 370 validated highly variable ‘Arhar’ genomic SSR (HASSR) markers, which provide much higher level of polymorphism than a random set of SSR markers. These markers were validated for consistent amplification in a set of eight pigeonpea varieties and 152 of these (41%) showed allelic polymorphism on agarose gel electrophoresis. Twenty‐four highest polymorphic HASSR markers were used on a larger set of forty pigeonpea varieties and eight wild Cajanus species for the analysis of genetic diversity and phylogenetic relationship. Genomic HASSR markers described here are highly suitable for genetic diversity and phylogenic studies in pigeonpea.     

Molecular tagging of Asiatic soybean rust resistance in exotic genotype EC 241780 reveals complementation of two genes

Asian soybean rust (ASR) caused by Phakopsora pachyrhizi severely reduces seed yield in soybean. Molecular tagging of ASR resistance can help in the process of resistance breeding. In this study, an F₂ population of cross (susceptible cultivar ‘NRC 7’ × resistant exotic genotype EC 241780) was used for bulked segregant analysis (BSA) with 25 SSR (simple sequence repeat) primers linked with six Rpp genes. Among them, five polymorphic SSR markers, viz., Sct 187, SSR 1859, Satt 191 (Rpp1b like loci) and Satt 215, Sat_361 (Rpp2 loci) distinguished the ASR resistant and susceptible bulks and individuals. In combined marker analysis, the markers Satt 191 (Rpp1b like loci) and Satt 215 (Rpp2 loci) were linked with ASR severity score and were also confirmed in individual 110 F₂ segregants. Hence, these markers could be utilized in the marker assisted rust resistance breeding of Rpp1b like and Rpp2 genes. In silico candidate gene analysis for hypersensitive response revealed that Satt 191 linked region was rich in genes encoding apoptotic ATPase having leucine‐rich repeat (LRR) domain.     

Genetic linkage map of Chinese native variety faba bean (Vicia faba L.) based on simple sequence repeat markers

Simple sequence repeat (SSR) marker is a powerful tool for construction of genetic linkage map which can be applied for quantitative trait loci (QTL) and marker‐assisted selection (MAS). In this study, a genetic map of faba bean was constructed with SSR markers using a 129 F₂ individuals population derived from the cross of Chinese native variety 91825 (large seed) and K1563 (small seed). By screening 11 551 SSR primers between two parents, 149 primer pairs were detected polymorphic and used for F₂ population analysis. This SSR‐based genetic linkage map consisted of 15 linkage groups with 128 SSR. The map encompassed 1587 cM with an average genetic distance of 12.4 cM. The genetic map generated in this study will be beneficial for genetic studies of faba bean for identification of marker‐locus‐trait associations as well as comparative mapping among faba bean, pea and grasspea.     

Development and molecular cytogenetic identification of a new wheat–Leymus mollis Lm#6Ns disomic addition line

We developed a new disomic addition line M11028‐1‐1‐5 (2n = 44 = 21” + 1”) from a cross between wheat cv. ‘7182’ and octoploid Tritileymus M47 (2n = 8x = 56, AABBDDN sNs ). Cytological observations demonstrated that M11028‐1‐1‐5 contained 44 chromosomes and formed 22 bivalents during meiotic metaphase I. The genomic in situ hybridization (GISH) investigations showed this line contained 42 wheat chromosomes and a pair of L. mollis chromosomes. SSR, EST and PCR‐based landmark unique gene (PLUG) markers were screened to determine the homoeologous relationships of the introduced L. mollis chromosomes in wheat background. Nine markers, i.e. Xwmc256, Xgpw312, Swes123, CD452568, BF483643, BQ169205, TNAC1748, TNAC1751 and TNAC1752, all of which were located on the homoeologous group 6 chromosomes of common wheat, amplified bands unique to L. mollis in M11028‐1‐1‐5. Gliadin analysis also confirmed that the added chromosomes in M11028‐1‐1‐5 were correlated with the sixth group chromosome. This indicated that M11028‐1‐1‐5 contained a pair of introduced L. mollis chromosome belonging to homoeologous group 6, which we designated it as Lm#6 Ns disomic addition line. This is the first report of a common wheat–L. mollis disomic addition line.     

Molecular mapping of a new gene for resistance to frogeye leaf spot of soya bean in 'Peking'

Amplified fragment length polymorphism (AFLP) and microsatellite (simple sequence repeat, SSR) techniques were used to map the _RGSp_eking gene, which is resistant to most isolates of Cercospora sojina in the soya bean cultivar ‘Peking’. The mapping was conducted using a defined F₂ population derived from the cross of ‘Peking’(resistant) בLee’(susceptible). Of 64 EcoRI and MseI primer combinations, 30 produced polymorphisms between the two parents. The F₂ population, consisting of 116 individuals, was screened with the 30 AFLP primer pairs and three mapped SSR markers to detect markers possibly linked to Rcs_Peking. One AFLP marker amplified by primer pair E‐AAC/M‐CTA and one SSR marker Satt244 were identified to be linked to Res_Peking. The gene was located within a 2.1‐cM interval between markers AACCTA178 and Satt244, 1.1 cM from Satt244 and 1.0 cM from AACCTA178. Since the SSR markers Satt244 and Satt431 have been mapped to molecular linkage group (LG) J of soya bean, the Res_Peking resistance gene was putatively located on the LG J. This will provide soya bean breeders an opportunity to use these markers for marker‐assisted selection for frogeye leaf spot resistance in soya bean.     

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.     

基于RNA-seq牡丹SSR标记开发及通用性分析

从牡丹花色候选基因中开发一套SSR标记,为牡丹花色分子遗传学研究和分子标记辅助育种提供帮助。本研究基于高通量转录组测序技术(RNA-seq)获得了278条涉及调控牡丹花色形成的Unigenes序列,利用SSRIT在128条Unigenes中检测到215个SSR位点,出现频率为46.04%。其中优势重复基序为二核苷酸、三核苷酸和六核苷酸重复,分别占总SSR位点的18.60%、41.86%和36.28%,优势重复基元为TC/GA和AT/TA,分别占二核苷酸重复的30.00%和27.50%。在此基础上,从中选择100对SSR引物合成,以牡丹品种基因组DNA为模板,验证其有效性和多态性。结果表明,有效引物50对(占50%),多态性引物12对(占24%)。12对多态性引物在芍药属12个不同种质内进行通用性检测,转移率范围为83.33%~100%,平均转移率为96.53%,表明牡丹SSR标记在芍药属内具有较高的通用性。利用高通量RNA-seq开发牡丹候选基因SSR标记可为牡丹功能基因挖掘、品种分子身份证构建、遗传多样性分析和分子标记辅助选择育种等提供重要的遗传资源。     

Converting restriction fragment length polymorphism to single‐strand conformation polymorphism markers and its application in the fine mapping of a trichome gene in cotton

A well‐characterized and systematically organized collection of genetic markers is crucial in the study of any crop species. It is the basis of map‐based gene cloning and crop improvements through marker‐assisted selections. Single‐strand conformation polymorphism (SSCP) has been a robust way of discovering new polymorphisms in marker development without the requirement of sequencing. Here, we report the first approach of applying SSCP marker discovery methods in the genetic map construction and gene mapping of cotton species. A total of 80 restriction fragment length polymorphism (RFLP) markers were selected from a region on published cotton genetic maps around the T1 gene related to cotton trichome. Among the 80 RFLPs, 28 showed polymorphisms through SSCP, showing a polymorphic rate of approximately 35%, which is much higher than that of simple sequence repeat (SSR) markers in the same region (7.8%). By integrating these newly generated SSCP markers, a detailed genetic map was reconstructed around this region using an F₂ population derived from a cross between Gossypium arboreum and G. herboceum. The reconstructed region comprises 22 SSCP markers, eight SSR markers and the T1 gene, spanning 21.6 cM. The marker order of the new map agrees well with published reference RFLP maps. The above results suggest that SSCP method can be applied very efficiently and reliably to the marker development of cotton genomes. It will prove to be even more valuable and robust after the public release of cotton whole‐genome sequences.     

Identification of resistance gene analogue markers closely linked to wheat powdery mildew resistance gene Pm31

Specific oligonucleotide primers, designed for the sequences of known plant disease resistance genes, were used to amplify resistance gene analogues (RGAs) from wheat genomic DNA. This method was applied in a bulked segregant analysis to screen for the RGA markers linked to the powdery mildew resistance gene Pm31, introgressed into common wheat from wild emmer. Two RGA markers (RGA200 and RGA390) were found to be closely linked to Pm31 and completely co‐segregating with the marker allele of Xpsp3029 linked to Pm31, with a genetic distance of 0.6 cM. These two RGA markers were then integrated into the formerly established microsatellite map of Pm31 region. The result showed the effectiveness of the RGA approach for developing molecular markers linked to disease resistance genes and demonstrated the efficiency of denaturing polyacrylamide‐gel electrophoresis for detecting polymerase chain reaction polymorphism.     

Fine mapping of the genetic locus L1 conferring black pods using a chromosome segment substitution line population of soybean

The colour of plant organs is a useful trait in crop breeding. The pod colours of soybeans primarily include black, brown and tan types, which are controlled by two classical genetic loci, L1 and L2. Most wild soybeans have black pods, which reflect a possible role in adaptation to the natural environment. Here, an improved chromosome segment substitution line (CSSL) population SojaCSSLP3 was established to identify the L1 gene. The segment on the 19th chromosome represented by the SSR marker Satt313 was found to link with locus L1. The region was further delimited three times with increased SSR and InDel markers using a population derived from a heterozygous plant of CSSL124 from SojaCSSLP3. The L1 gene was finally located in a 184.43‐kb region between SSR_19p09 and Indel_19P7. Thirteen putative genes in this region were analysed with qRT‐PCR. The expression level of Glyma19 g27460, which is a member of the SANT superfamily with a MYB DNA‐binding domain, was significantly upregulated in black pods and was recognized to be the most likely candidate for the L1 gene.     

Molecular mapping of greenbug (Schizaphis graminum) resistance gene Rsg1 in barley

The greenbug, Schizaphis graminum (Rondani) is an extremely damaging aphid pest of barley (Hordeum vulgare L.) particularly in the southern Great Plains of the USA. The simply inherited, dominant resistance gene Rsg1 is in all greenbug‐resistant US barley cultivars. In this study, we conducted molecular mapping of Rsg1 using an F_2:3 population derived from a cross between the greenbug‐resistant Post 90*4/R015 and susceptible CI2260 inbred lines. Segregation of host responses to greenbug biotype E infestation confirmed that a single dominant gene is responsible for greenbug resistance in Post 90*4/R015. Simple sequence repeat (SSR) markers evenly distributed along the seven barley chromosomes were employed for the construction of a framework genetic map. Linkage analysis placed the Rsg1 locus in the long arm of chromosome 3H (3HL) flanked by SSR markers Bmag0877 and GBM1420 that were 35 cM apart. Polymorphic single‐nucleotide polymorphism (SNP) markers in 3HL were identified from an Illumina GoldenGate SNP assay and used for targeted mapping to locate Rsg1 to an 8.4‐cM interval. Comparative analysis identified syntenic genomic regions in Brachypodium distachyon chromosome 2, in which 37 putative genes were annotated including a NB‐LRR‐type resistance gene homologue that may be a potential candidate gene for the Rsg1 locus of barley. Results from this study offer a starting point for fine mapping and cloning of this aphid resistance gene in barley.     

AFLP and SCAR markers linked to the suppressor gene (Rf) of a dominant genetic male sterility in rapeseed (Brassica napus L.)

Rs1046AB is a line which is true breeding for a dominant genetic male sterility gene (Ms) but which is a mixture of male fertile and sterile individuals (a two-type line) because it is segregating for a dominant suppressor gene (Rf). This system provides a promising alternative to the CMS system for hybrid breeding in Brassica napus. In order to identify molecular markers linked to the rf gene, a near-isogenic line (NIL) population from the cross between a sterile individual (MsMsrfrf) and a fertile individual (MsMsRfrf) in Rs1046AB was subjected to amplified fragment length polymorphism (AFLP) analysis, with a combination of comparing near isogenic lines (NILs) and bulked segregant analysis (BSA). From 2,816 pairs of AFLP primers, six fragments showing polymorphism between the fertile and sterile bulks as well as the individuals of the bulks were identified. Linkage analysis indicated that the six AFLP markers are tightly linked to the Rf gene and all are distributed on the same side. The minimum genetic distance between the Rf gene and a marker was 0.7 cM. Since the AFLP markers are not suitable for large-scale application in MAS (marker-assisted selection), our objective was to develop a fast, cheap and reliable PCR-based assay. Consequently, three of the four closest AFLP markers were converted directly to sequence characterized amplified region (SCAR) markers. For the other marker a corresponding SCAR marker was successfully obtained after isolating the adjacent sequences by PCR Walking. The available SCAR markers of the Rf gene will greatly facilitate future breeding programs using dominant GMS to produce hybrid varieties.     

GenBank数据库中黄麻EST-SSR标记的开发及其通用性评价

本研究从GenBank公共数据库中下载黄麻表达序列标签838条,利用SSRPrimer软件对其进行SSR位点查找,利用Primer 3.0软件设计66对SSR引物,通过琼脂糖凝胶研究这些SSR引物的PCR扩增特点,以检测其多态性。结果表明,66对SSR引物在黄麻属6个不同类型材料的扩增中,42 (63.6%)对引物至少在2个材料之间存在多态性。(AT)n重复基元和(GC-)n丰富的三核苷酸重复基元多态性较高,可作为黄麻SSR标记引物设计的首选。黄麻EST-SSR标记开发效率较高,不仅可以丰富黄麻分子标记的数量,而且为剖析黄麻重要性状的遗传机制奠定基础,这对于黄麻的遗传基础研究具有重要应用价值。     

Identification of commercial chicory cultivars for hydroponic forcing and their phenetic relationships revealed by random amplified polymorphic DNAs and amplified fragment length polymorphisms

One‐hundred and twenty‐four amplified fragment length polymorphism (AFLP) and 49 random amplified polymorphic DNA (RAPD) markers have been used to distinguish between 20 and 23 commercial chicory cultivars, respectively. These were all Cichorium intybus var. foliosum F₁ hybrids, currently used in hydroponic forcing. Five‐hundred and twenty RAPD primers (OPERON) were tested, of which 156 resulted in reproducible patterns and 26 yielded polymorphisms. Two‐hundred and fifty‐six AFLP primer‐combinations were tested and six combinations were selected for identification purposes. Similarity indices were measured and clustering has been done using pairwise comparison. Both types of marker provide similar conclusions. Two major clusters are formed, representing late and early cultivars. All cultivars were identified using 10 informative RAPD primers or three AFLP primer combinations. A low degree of polymorphism was detected between some early cultivars, suggesting a narrow genetic base in their breeding strategy.