A CAPS marker set for mapping in linkage group III of pea (Pisum sativum L.)

A set of twelve CAPS markers was mapped for linkage group III of pea (Pisum sativum L.). New primers were designed to use a polymerase chain reaction to amplify fragments of sequenced pea genes containing at least one large intron. Amplification products were tested for polymorphism across three pea lines (Chi115, Flagman and WL1238) using eleven four-base restriction endonucleases. Nine STS markers for linkage group III from the literature were also tested for polymorphism, and five of these were used in this mapping study as anchor points. All polymorphic loci were located by genetic analysis of the F(2)population from the cross Chi115 x WL1238, and a map of linkage group III consisting of one morphological and twelve CAPS markers was created. The map covers the full length of the chromosome and is about 162 cM long. All the CAPS markers in a set were used to test for polymorphism among 10 additional pea DNA samples extracted from different marker lines and cultivars.     

Studying plant genome variation using molecular markers

The authors' studies on the organization and variation of plant genome with the use of molecular markers are briefly reviewed with special emphasis on random amplified polymorphic DNA (RAPD), inter simple sequence repeat (ISSR), sequence characterized amplified region (SCAR), and cleaved amplified polymorphic sequence (CAPS) markers detected with the use of polymerase chain reaction (PCR). These markers have been demonstrated to be promising for identifying cultivars and determining the purity of genetic strains of pea. Genetic relationships between strains, cultivars, and mutants of pea have been studied. The role of molecular markers in molecular genetic mapping and localizing the genes of commercially important characters of pea has been shown. The possibility of the use of molecular markers for studying somaclonal variation and detecting mutagenic factors in plants during long-term spaceflights is considered. The prospects of using DNA markers for understanding the organization and variability of higher plant genomes are discussed.     

Studying plant genome variation using molecular markers

The authors studies on the organization and variation of plant genome with the use of molecular markers are briefly reviewed with special emphasis on random amplified polymorphic DNA (RAPD), inter simple sequence repeat (ISSR), sequence characterized amplified region (SCAR), and cleaved amplified polymorphic sequence (CAPS) markers detected with the use of polymerase chain reaction (PCR). These markers have been demonstrated to be promising for identifying cultivars and determining the purity of genetic strains of pea. Genetic relationships between strains, cultivars, and mutants of pea have been studied. The role of molecular markers in molecular genetic mapping and localizing the genes of commercially important characters of pea has been shown. The possibility of the use of molecular markers for studying somaclonal variation and detecting mutagenic factors in plants during long-term spaceflights is considered. The prospects of using DNA markers for understanding the organization and variability of higher plant genomes are discussed.__________Translated from Genetika, Vol. 41, No. 4, 2005, pp. 480–492.Original Russian Text Copyright © 2005 by Gostimsky, Kokaeva, Konovalov.     

遗传标记及其在作物品种鉴定中的应用

本文评述了用于作物品种鉴定的形态标记（morphological markers）、细胞标记（cytological markers）、生化标记（biochemical markers）、分子标记（molecular markers）的优缺点。重点评述了分子标记在作物品种鉴定中的应用。文中除对蛋白质电泳指纹图谱——同工酶和贮藏蛋白（包括醇溶性蛋白、清蛋白、谷蛋白、球蛋白等）电泳产生的指纹图谱的应用外,较详细地介绍了近年来DNA指纹图谱技术;包括限制片段长度多态性（restriction fragment length polymorphism,简称RFLP）、随机扩增多态性DNA (random amplified po lymorphic DNA,简称RAPD)、小卫星DNA（minisatellite DNA）、微卫星DNA（microsatellite DNA）,简单重复序列间扩增（intersimple sequence repeats,简称ISSR）,扩增片段长度多态性（amplified fragment length polymorphism,简称AFLP）以及CAPS (cleaved amplified polymorphic sequences)和SNPS (single nucleotide polymorphisms)对作物品种鉴定和新品种登记,品种纯度和真实性的检验以及品种间亲缘关系的探讨和在分类研究中的贡献等。     

传标记及其在作物品种鉴定中的应用

本文评述了用于作物品种鉴定的形态标记（morphological markers）、细胞标记（cytological markers）、生化标记（biochemical markers）、分子标记（molecular markers）的优缺点。重点评述了分子标记在作物品种鉴定中的应用。文中除对蛋白质电泳指纹图谱－－同工酶和贮茂蛋白（包括醇溶性蛋白、清蛋白、谷蛋白、球蛋白等）电泳产生的指纹图谱的应用外,较详细地介绍了近年来ＤＮＡ指纹图谱技术;包括限制片段长度多态性（restriction fragment length polymorphism,简称ＲＦＬＰ)、随机扩增多态性ＤＮＡ（random amplified polymorphic DNA,简称ＲＡＰＤ）、小卫星ＤＮＡ（minisatellite DNA）、微卫生ＤＡＮmicrosatellite DNA）,简单重复序列间扩增（intersimple sequence repeats,简称ＩＳＳＲ）,扩增片段长度多态性（amplified fragment length polymor-phism,简称ＡＦＬＰ）以及ＣＡＰＳ（cleaved amplified polymorphic sequences）和ＳＮＰＳ（single mucleotide polymor-phisms）对作物品种鉴定和新品种登记,品种纯度和真实性的检验以及品种间亲缘关系的探讨和在分类研究中的贡献等。     

Rapid identification of 1-aminocyclopropane-1-carboxylate (ACC) synthase genotypes in cultivars of Japanese pear (Pyrus pyrifolia Nakai) using CAPS markers

In Japanese pear (Pyrus pyrifolia Nakai), fruit storage potential is closely related to the amount of ethylene produced. We have developed a rapid and accurate method for analyzing genes involved in high ethylene production during fruit ripening in Japanese pear. This involves cleaved-amplified polymorphic sequences (CAPS) of two 1-aminocyclopropane-1-carboxylate (ACC) synthase genes (PPACS1 and PPACS2). Two CAPS markers (A for PPACS1 and B for PPACS2), associated with the amount of ethylene produced, were identified. Marker A was associated with high ethylene producers and marker B with moderate ethylene producers. The absence of these two markers enabled the identification of low ethylene producers. Using these markers, we have identified ethylene genotypes for 40 Japanese pear cultivars and two Chinese pear (P. bretschneideri) cultivars that are commercially important and used in breeding programs. Furthermore, we performed linkage analysis of these two genes in the F(2) population, which revealed that the recombination frequency between the two markers was 20.8 +/- 3.6%. This information is critical to the selection of parents and in breeding strategies to improve storage ability of Japanese pears.     

Identification and mapping of polymorphic RAPD markers of pea (<Emphasis Type="Italic">Pisum sativum</Emphasis> L.) genome

Various pea cultivars, lines, and mutants were studied by the RAPD method. Polymorphic fragments characteristic of certain pea genotypes and which can be used for identifying genotypes were detected. Inheritance of some polymorphic RAPD fragments was studied. Mendelian inheritance of these fragments was shown. By analyzing the data obtained in studies of RAPD polymorphism, genetic distances between different pea cultivars, lines, and mutants were calculated and a genealogic dendogram showing a varying extent of differences between RAPD patterns was constructed. Ten new RAPD markers linked to various pea genes were detected. Genetic distances between RAPD markers and genes to which they are linked were calculated, and the respective disposition of RAPD markers on chromosomes was established.Translated from Genetika, Vol. 41, No. 3, 2005, pp. 341–348.Original Russian Text Copyright © 2005 by Koveza, Kokaeva, Konovalov, Gostimsky.     

CAPS markers improved by cluster-specific amplification for identification of octoploid strawberry (Fragaria × ananassa Duch.) cultivars, and their disomic inheritance

Cleavage amplified polymorphic sequence (CAPS) markers of strawberry (Fragaria × ananassa Duch.) can be useful for identifying mislabeled or patent-infringing cultivars in the marketplace. However, CAPS markers in octoploid strawberry tend to give unclear bands because multiple homologous sites are simultaneously amplified by the non-selective PCR. To overcome this problem, we used cluster-specific amplification based on the nucleotide sequences of PCR products and were able to improve the band clarity of 18 CAPS markers. By analyzing the marker segregation ratio, we demonstrated that 13 clarified markers were derived from single diploid loci that were transmitted to progeny in a manner consistent with Mendelian inheritance. We discuss the genomic structure of octoploid strawberry from the viewpoint of cluster and segregation analysis and suggest that it comprises independent genomes. We tested the utility of all of the markers we developed for cultivar identification and confirmed their ability to distinguish among 64 strawberry cultivars.     

Functional markers for <Emphasis Type="Italic">xa5</Emphasis>-mediated resistance in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis>, L.)

The recent cloning of several agronomically important genes has facilitated the development of functional markers. These markers reside within the target genes themselves and can be used with great reliability and efficiency to identify favorable alleles in a breeding program. Bacterial blight (BB) is a severe rice disease throughout the world that is controlled primarily through use of resistant cultivars. xa5 is a race-specific, recessive gene mediating resistance to BB. It is widely used in rice breeding programs throughout the tropics. Due to its recessive nature, phenotypic selection for xa5-mediated resistance is both slow and costly. Previously, marker assisted selection (MAS) for this resistance gene was not efficient because it involved markers that were only indirectly linked to xa5 and ran the risk of being separated from the trait by recombination. Recently, the cloning of the gene underlying this trait made it possible to develop functional markers. Here we present a set of CAPS markers for easy, quick and direct identification of cultivars or progeny carrying xa5-mediated resistance and provide evidence that these markers are 100% predictive of the presence of the xa5 allele. These markers are expected to enhance the reliability and cost-effectiveness of MAS for xa5-mediated resistance.     

CAPS markers using mitochondrial consensus primers for molecular identification of Panax species and Korean ginseng cultivars (Panax ginseng C. A. Meyer)

Cleaved amplified polymorphic sequence (CAPS) marker system using mitochondrial consensus primers was applied for molecular identification of Korean ginseng cultivars (Panax ginseng). Initially, a total of 34 primers were tested to six Korean ginseng cultivars and two foreign Panax species, P. quinquefolius and P. notoginseng. In the polymerase chain reaction (PCR) amplification results, four primers (mt7, mt11, mt13, and mt18) generated co-dominant polymorphic banding patterns discriminating the Korean ginseng cultivars from P. quinquefolius and P. notoginseng. In the CAPS analysis results, the majority of the cleaved PCR products also yielded additional latent polymorphisms between the Korean ginseng cultivars and two foreign Panax species. Specific latent CAPS polymorphisms for cultivar Gopoong and Chunpoong were detected from internal region amplified with mt9 primer by treating HinfI and Tsp509I endonucleases, respectively. Sequencing analysis revealed that the length of amplified region of Korean ginseng cultivars was 2,179 bp, and those of P. quinquefolius and P. notoginseng were 2,178 and 2,185 bp, respectively. Blast search revealed that the amplified region was a mitochondrial cytochrome oxidase subunit 2 (cox2) gene intron II region. Nineteen single nucleotide polymorphisms (SNP) including each specific SNP for Gopoong and Chunpoong, and three insertion and deletion (InDel) polymorphisms were detected by sequence alignment. The CAPS markers developed in this study, which are specific to Gopoong and Chunpoong, and between the Korean ginseng cultivars and two foreign Panax species, will serve as a practical and reliable tool for their identification, purity maintenance, and selection of candidate lines and cultivars.     

Development and Study of SCAR Markers in Pea (<Emphasis Type="Italic">Pisum sativum</Emphasis> L.)

In order to develop more specific markers that characterize particular regions of the pea genome, the data on nucleotide sequences of RAPD fragments were used for choosing more extended primers, which may be helpful in amplifying a fragment corresponding to the particular DNA region. Of the 14 STS markers obtained from 14 polymorphic RAPD fragments, 12 were polymorphic, i.e., they are SCAR markers that can be used in genetic analysis. The transition from complex RAPD spectra to amplification of a particular SCAR marker substantially facilitates analysis of large samples for the presence or absence of the examined fragment. Inheritance of the developed SCAR markers was studied in F₁ and F₂. SCAR markers were used to identify various pea lines, cultivars, and mutants. It was established that the study of amplification of STS markers in various pea genotypes at varying temperatures of annealing and the comparison with amplification of the original RAPD fragments in the same genotypes provide an approach for analysis of RAPD polymorphism origin.     

Interval mapping of QTLs controlling yield-related traits and seed protein content in Pisum sativum

A linkage map of garden pea was constructed on the basis of 114 plants (F2 generation) derived from a cross combination Wt10245 x Wt11238. The map, consisting of 204 morphological, isozyme, AFLP, ISSR, STS, CAPS and RAPD markers, was used for interval mapping of quantitative trait loci (QTLs) controlling seed number, pod number, 1000-seed weight, 1000-yield, and seed protein content. Characterization of each QTL included identification of QTL position with reference to the flanking markers, estimation of the part of variance explained by this QTL, and determination of its gene action. The yield-related traits were measured in F2 plants and in F4 recombinant inbred lines (RILs). The interval mapping revealed two to six QTLs per trait, demonstrating linkage to seven pea chromosomes. A total of 37 detected QTLs accounted for 9.1-55.9% of the trait's phenotypic variation and showed different types of gene action. As many as eight and ten QTLs influencing the analysed traits were mapped in linkage groups III and V, respectively, indicating an important role of these regions of the pea genome in the control of yield and seed protein content.     

Malting quality quantitative trait loci on a high-density map of Mikamo golden?×?Harrington cross in barley (Hordeum vulgare L.)

A high-density map consisting of 550 markers was constructed based on the segregation data of 95 doubled-haploid lines (DHLs) derived from the cross between a Japanese barley cultivar, Mikamo Golden and a North American barley cultivar, Harrington (MH-DHLs). Quality traits of malt extract (EX), total nitrogen (TN), soluble nitrogen (SN), Kolbach index (KI), diastatic power (DP), wort beta-glucan (WG) and viscosity (VS) were determined in three site/year crops. Quantitative trait loci (QTL) analyses were performed with these quality data sets, using the linkage map. Major QTL controlling EX, SN and KI were mapped on terminal region of 5H with Harrington as effective allele. Another QTL controlling EX was mapped on 2H with Mikamo Golden as effective allele. QTL controlling TN, DP, WG and VS were detected variably in terms of flanking markers and chromosomes depending on site/year. Cleaved amplified polymorphic sequences (CAPS) markers for EX based on the QTL detected on 2H and 5H were developed. Analysis of EX and genotypes of 33 malting barley cultivars from around the world as well as MH-DHLs revealed that the two CAPS marker on 2H and 5H affect EX by a significant difference, suggesting that the two CAPS markers were valuable for marker-assisted selection in malting barley breeding.     

植物功能基因组研究中出现的新型分子标记

随着功能基因组学的发展,表达序列标签(Expressed Sequence Tags, ESTs)已经成为开发以PCR为基础的新型分子标记的重要资源。本文综述了植物功能基因组研究中出现的EST-SSR、CAPS、SNP、SRAP和TRAP等新型分子标记的基本原理和特点。这些标记具有其显著的优势,如开发简便、信息量高和通用性好等,尤其是由于它们来源于基因编码区（ORFs),因此具有很高的物种间通用性,并且其多态性与基因功能变异相关联。目前,这些功能基因分子标记已广泛应用于遗传图谱构建、重要性状基因定位、比较作图、遗传多样性和品种鉴别、分子标记辅助选择育种等研究中。在文章最后简要介绍了作者所在实验室近年来所开展的功能基因分子标记工作,并说明了在使用这些功能基因分子标记时可能会出现的问题。     

Identification and mapping of polymorphic RAPD markers of pea (Pisum sativum L.) genome

Various pea cultivars, lines, and mutants were studied by the RAPD method. Polymorphic fragments characteristic of certain pea genotypes and which can be used for identifying genotypes were detected. Inheritance of some polymorphic RAPD fragments was studied. Mendelian inheritance of these fragments was shown. By analyzing the data obtained in studies of RAPD polymorphism, genetic distances between different pea cultivars, lines, and mutants were calculated and a genealogic dendogram showing a varying extent of differences between RAPD patterns was constructed. Ten new RAPD markers linked to various pea genes were detected. Genetic distances between RAPD markers and genes to which they are linked were calculated, and the respective disposition of RAPD markers on chromosomes was established.     

SNP-based markers for discriminating olive (Olea europaea L.) cultivars.

A set of 11 polymorphic markers (1 cleaved amplified polymorphic sequence (CAPS), 2 sequence-characterized amplified regions (SCARs), and 8 single-nucleotide polymorphism (SNP)-derived markers) was obtained for olive cultivar identification by comparing DNA sequences from different accessions. Marker development was more efficient, using sequences from the database rather than cloning arbitrary DNA fragments. Analyses of the sequences of 3 genes from 11 diverse cultivars revealed an SNP frequency of 1 per 190 base pairs in exons and 1 per 149 base pairs in introns. Most mutations were silent or had little perceptible effect on the polypeptide encoded. The higher incidence of transversions (55%) suggests that methylation is not the major driving force for DNA base changes. Evidence of linkage disequilibrium in 2 pairs of markers has been detected. The set of predominantly SNP-based markers was used to genotype 65 olive samples obtained from Europe and Australia, and was able clearly to discriminate 77% of the cultivars. Samples, putatively of the same cultivar but derived from different sources, were revealed as identical, demonstrating the utility of these markers as tools for resolving nomenclature issues. Genotyping data were used for constructing a dendrogram by UPGMA cluster analysis using the simple matching similarity coefficient. Relationships between cultivars are discussed in relation to the route of olive's spread.     

VCF2CAPS–A high-throughput CAPS marker design from VCF files and its test-use on a genotyping-by-sequencing (GBS) dataset

Next-generation sequencing (NGS) is a powerful tool for massive detection of DNA sequence variants such as single nucleotide polymorphisms (SNPs), multi-nucleotide polymorphisms (MNPs) and insertions/deletions (indels). For routine screening of numerous samples, these variants are often converted into cleaved amplified polymorphic sequence (CAPS) markers which are based on the presence versus absence of restriction sites within PCR products. Current computational tools for SNP to CAPS conversion are limited and usually infeasible to use for large datasets as those generated with NGS. Moreover, there is no available tool for massive conversion of MNPs and indels into CAPS markers. Here, we present VCF2CAPS–a new software for identification of restriction endonucleases that recognize SNP/MNP/indel-containing sequences from NGS experiments. Additionally, the program contains filtration utilities not available in other SNP to CAPS converters–selection of markers with a single polymorphic cut site within a user-specified sequence length, and selection of markers that differentiate up to three user-defined groups of individuals from the analyzed population. Performance of VCF2CAPS was tested on a thoroughly analyzed dataset from a genotyping-by-sequencing (GBS) experiment. A selection of CAPS markers picked by the program was subjected to experimental verification. CAPS markers, also referred to as PCR-RFLPs, belong to basic tools exploited in plant, animal and human genetics. Our new software–VCF2CAPS–fills the gap in the current inventory of genetic software by high-throughput CAPS marker design from next-generation sequencing (NGS) data. The program should be of interest to geneticists involved in molecular diagnostics. In this paper we show a successful exemplary application of VCF2CAPS and we believe that its usefulness is guaranteed by the growing availability of NGS services.

This is a PLOS Computational Biology Software paper.

     

Identification of Ppd-B1 alleles in common wheat cultivars by CAPS marker

Photoperiod response is a major determinant of the duration of growth stages in common wheat. In common wheat, many genes play a role in determining flowering time, but the Ppd genes located on the homoeologous group 2 play a major role. Of these Ppd-B1 is located on the short arm of 2B. In 107 common wheat cultivars grown in Poland and neighboring countries, the identification of Ppd-B1 alleles using in-del analysis by using a CAPS markers was investigated. 87 cultivars were shown to carry dominant Ppd-B1 alleles. This shows that Ppd-B1 alleles is have been widely used in common wheat breeding programme in these countries. Recessive ppd-B1 alleles were found only in 20 cultivars (12 Polish, 5 former Soviet Union, 2 German, 1 Swedish).     

烟粉虱Q与B隐种mtCOI基因的遗传变异及其对利用CAPS标记进行隐种鉴别的影响

【目的】为了评估VspI,StyI和StuI为基础的线粒体细胞色素氧化酶I基因（mtCOI）酶切扩增多态性序列（cleaved amplified polymorphic sequence, CAPS)标记方法鉴别烟粉虱Q与B隐种的有效性。【方法】本研究对国内外烟粉虱种群的mtCOI基因进行了测序并鉴定了其隐种;在对464个Q隐种、98个B隐种mtCOI序列分析的基础上,利用VspI,StyI和StuI对Q与B隐种分别进行了CAPS标记验证。检索并比对了GenBank中烟粉虱Q与B隐种mtCOI序列中VspI,StyI和Stu I酶切位点分布情况。【结果】对国内外烟粉虱种群研究发现,以Vsp I为基础的CAPS标记方法能够有效鉴别实验中的Q与B隐种;利用StuI或StyI的CAPS标记方法无法有效鉴别Q与B隐种。对GenBank中烟粉虱Q与B隐种mtCOI序列比对发现,利用VspI,StyI和StuI为基础的CAPS标记方法不能有效鉴别Q与B隐种。【结论】以VspI,StyI和StuI为基础的CAPS标记方法鉴别Q与B隐种方面均有一定的局限性。