Identification of simple sequence repeat markers tightly linked to plum pox virus resistance in apricot

Sharka disease, caused by the plum pox virus (PPV), is one of the major limiting factors for stone fruit production in Europe and America. Attempts to stop the disease through the eradication of infected trees have been unsuccessful. Introgression of PPV resistance for crop improvement is therefore the most important goal in Prunus breeding programs. Due to time- and labour-consuming protocols, phenotyping for sharka is still the major bottleneck in the breeding pipeline. In this context, screening of seedlings at early stages of development and marker-assisted selection (MAS) provide the best solution for enhancing breeding efficiency. In this study, we generated 42 simple sequence repeat (SSR) markers from the peach genome assembly v1.0 and an apricot bacterial artificial chromosome clone identified in the physical map of the PPV resistance locus previously defined in apricot. Using a linkage mapping approach, we found SSR markers tightly linked to PPV resistance trait in all our progenies. Three SSR markers, PGS1.21 PGS1.23 and PGS1.24, showed allelic variants associated with PPV resistance with no recombinants in the crosses analysed. These markers unambiguously discriminated resistant from susceptible accessions in different genetic backgrounds. The results presented here are the first successful application of their use in MAS for breeding resistance in Prunus species.     

Molecular sequence variations of the lipoxygenase-2 gene in soybean

Soybean lipoxygenase genes comprise a multi-gene family, with the seed lipoxygenase isozymes LOX1, LOX2, and LOX3 present in soybean seeds. Among these, the LOX2 isozyme is primarily responsible for the “beany” flavor of most soybean seeds. The variety, Jinpumkong 2, having null alleles (lx1, lx2, and lx3) lacks the three seed lipoxygenases; so, sequence variations between the lipoxygenase-2 genes of Pureunkong (Lx2) and Jinpumkong 2 (lx2) cultivars were examined. One indel, four single nucleotide polymorphisms (SNPs), a 175-bp fragment in the 5′-flanking sequence, and a missense mutation within the coding region were found in Jinpumkong 2. The distribution of the sequence variations was investigated among 90 recombinant inbred lines (RILs) derived from a cross of Pureunkong × Jinpumkong 2 and in 480 germplasm accessions with various origins and maturity groups. Evidence for a genetic bottleneck was observed: the 175-bp fragment was rare in Glycine max, but present in the majority of the G. soja accessions. Furthermore, the 175-bp fragment was not detected in the 5′ upstream region of the Lx2 gene on chromosome (Chr) 13 in Williams 82; instead, a similar 175-bp fragment was positioned in the homeologous region on Chr 15. The findings indicated that the novel fragment identified was originally present in the Lx2 region prior to the recent genome duplication in soybean, but became rare in the G. max gene pool. The missense mutation of the conserved histidine residue of the lx2 allele was developed into a single nucleotide-amplified polymorphism (SNAP) marker. The missense mutation showed a perfect correlation with the LOX2-lacking phenotype, so the SNAP marker is expected to facilitate breeding of soybean cultivars which lack the LOX2 isozyme.     

Mapping and validation of simple sequence repeat markers linked to a major gene controlling seed cadmium accumulation in soybean [Glycine max (L.) Merr]

Daily consumption of cadmium (Cd) contaminated foods poses a risk to human health. Cultivar selection is an important method to limit Cd uptake and accumulation, however, analyzing grain Cd concentration is costly and time-consuming. Developing markers for low Cd accumulation will facilitate marker assisted selection (MAS). Inheritance studies using a threshold value of 0.2 mg kg^?1 for low and high and an F_2:3 population showed that low Cd accumulation in soybean seed is under the control of a major gene (Cda1, proposed name) with the allele for low accumulation being dominant. A recombinant inbred line (RIL) population (F_6:8) derived from the cross AC Hime (high Cd accumulation) and Westag-97 (low Cd accumulation) was used to identify the DNA markers linked to Cda gene(s) or quantitative trait loci (QTLs) controlling low Cd accumulation. We screened 171 simple sequence repeat (SSR) primers that showed polymorphism between parents on the 166 RILs. Of these, 40 primers were newly developed from the soybean genomic DNA sequence. Seven SSR markers, SatK138, SatK139, SatK140 (0.5 cM), SatK147, SacK149, SaatK150 and SattK152 (0.3 cM), were linked to Cda1 in soybean seed. All the linked markers were mapped to the same linkage group (LG) K. The closest flanking SSR markers linked to Cda1 were validated using a parallel population (RILs) involving Leo × Westag-97. Linked markers were also validated with diverse soybean genotypes differing in their seed Cd concentration and showed that SSR markers SatK147, SacK149, and SattK152 clearly differentiated the high and low Cd accumulating genotypes tested. To treat Cd uptake as a quantitative trait, QTL analysis using a linkage map constructed with 161 markers identified a major QTL associated with low Cd concentration in the seeds. The QTL was also mapped to the same location as Cda1 on LG-K. This QTL accounted for 57.3% of the phenotypic variation. Potential candidate genes (genes with known or predicted function that could influence the seed Cd concentration) like protein kinase, putative Adagio-like protein, and plasma membrane H⁺-ATPase were found to be located in the locus of interest. Of the four SSR markers located in the region, SattK152 was localized in the plasma membrane H⁺-ATPase gene. SSR markers closely linked to Cda1 in seeds of soybean were identified and have potential to be used for MAS to develop low Cd accumulating cultivars in a breeding program.     

Two simple sequence repeat markers to select for soybean cyst nematode resistance coditioned by the rhg1 locus

The soybean cyst nematode (SCN) (Heterodera glycines Inchinoe) is the most economically significant soybean pest. The principal strategy to reduce or eliminate damage from this pest is the use of resistant cultivars. Identifying resistant segregants in a breeding program is a difficult and expensive process which is complicated by the oligogenic nature of the resistance and genetic variability in the pathogen. Fortunately, resistance at one SCN-resistance locus, rhg1, is generally accepted as a necessity for the development of resistant genotypes using any source of resistance and when challenged by any SCN race. Thus, the development of SCN resistant cultivars would be expedited if an effective and rapid system were available to identify breeding lines carrying a resistance allele at the rhg1 locus. In this study we report two simple sequence repeat (SSR) or microsatellite loci that cosegregate and map 0.4 cM from rhg1. Allelic variation at the first of these loci, BARC-Satt309, distinguished most, if not all, SCN-susceptible genotypes from those carrying resistance at rhg1 derived from the important SCN-resistance sources ’Peking’, PI 437654, and PI 90763. BARC-Satt309 was also effective in distinguishing SCN resistance sources PI 88788 and PI 209332 from many, but not all, susceptible genotypes. BARC-Satt309 cannot be used in marker-assisted selection in populations developed from typical southern US cultivars crossed with the important resistance sources PI 88788 or PI 209332 because these genotypes all carry the identical allele at the BARC-Satt309 locus. A second SSR locus, BARC-Sat_168, was developed from a bacterial artificial chromosome (BAC) clone that was identified using the primers to BARC-Satt309. BARC-Sat_168 distinguished PI 88788 and PI 209332 from southern US cultivars such as ’Lee’, ’Bragg’ and ’Essex’. Both BARC-Satt309 and BARC-Sat_168 were used to assay lines from SCN-susceptible×SCN-resistant crosses and proved to be highly effective in identifying lines carrying rhg1 resistance from those carrying the allele for SCN susceptibility at the rhg1 locus. Received: 5 November 1998 / Accepted: 3 February 1999     

Automated sizing of fluorescent-labeled simple sequence repeat (SSR) markers to assay genetic variation in soybean

 Simple Sequence Repeat (SSR) allele sizing provides a useful tool for genotype identification, pedigree analysis, and for estimating genetic distance between organisms. Soybean [Glycine max (L.) Merr.] cultivars are identified for Plant Variety Protection (PVP) purposes by standard pigmentation and morphological traits. However, many commercial soybeans arise from a limited number of elite lines and are often indistinguishable based on these traits. A system based on SSR markers would provide unique DNA profiles of cultivars. Fluorescent labeling of alleles combined with automated sizing with internal size standards in each gel lane was used as an alternative to standard [³²P] labeling to assess genetic variability in soybean. Allelic frequencies at 20 SSR loci were determined in 35 soybean genotypes that account for greater than 95% of the alleles in North American soybean cultivars based upon pedigree analysis. An average of 10.1 alleles per locus (range: 5–17), with a mean gene diversity of 0.80 (range: 0.50 to 0.87) were observed at the 20 SSR loci. The 20 loci successfully distinguished modern soybean cultivars that are identical for morphological and pigmentation traits, as well as 7 soybean genotypes reported to be indistinguishable using 17 RFLP probes. Pedigrees of 7 cultivars were studied to estimate stability of SSRs in soybean across generations. Of the 7 pedigrees 6 had one locus in the progeny with an allele(s) that was not present in either parent. These new alleles are most likely the result of mutation. The mutation rate of SSR alleles in soybean was similar to that reported in humans. To avoid difficulty associated with mutation, DNA fingerprint data should be determined from the bulk of 30-50 plants of a cultivar. Received: 24 March 1997 / Accepted: 4 April 1997     

Identification of species-diagnostic inter simple sequence repeat markers for ten Phyllanthus species

Phyllanthus has been widely used in traditional medicine as an antipyretic, a diuretic, and to treat liver diseases and viral infections. Correct genotype identification of medicinal plant material remains important for the botanical drug industry. Limitations of chemical and morphological approaches for authentication have generated the need for newer methods in quality control of botanicals. In the present study, attempts were made to identify species-diagnostic markers for ten Phyllanthus species using the inter simple sequence repeat-polymerase chain reaction (ISSR-PCR) fingerprinting method. PCR amplification using seven ISSR primers resulted in significant polymorphism among the populations from different species. P. angustifolius and P. urinaria showed monomorphic frequency of maximum (63.88%) and minimum (20.64%), respectively. Seventeen species-diagnostic markers were identified for seven species (P. acidus, P. emblica, P. fraternus, P. urinaria, P. rotundifolius, P. amarus, and P. angustifolius) while no marker was detected for P. reticulatus, P. nivosus, and P. virgulatus. A maximum of six species-diagnostic markers were identified for P. acidus and a minimum of only one of 755 bp was available for P. amarus. Among the seventeen markers, nine were present in all individuals of particular species. The species-specific differences in fragment numbers and sizes could be used as diagnostic markers to distinguish the Phyllanthus species quickly.     

Development of multiplex sets of simple sequence repeat DNA markers covering the soybean genome

Multiplexing involves the analysis of several markers in a single gel lane that is based on the allele size range of marker loci. Multiplex SSR marker analysis is conducted with primers that are labeled with one of three dyes. The development of an SSR multiplex system requires estimates of the allele size range of markers to strategize primer labeling and for grouping markers into multiplex sets. A method is presented that describes the development of multiplex sets of SSR markers in soybean (Glycine max (L.) Merr.) by the selective placement of primer sites and by the analysis of diverse germplasm. Primer sites were placed at specific distances from the SSR to adjust the allele size range of marker loci. The analysis of pooled DNA samples comprising diverse soybean genotypes provided robust estimates of the allele size range of marker loci that enabled the development of multiplex sets. Eleven multiplex sets comprising 74 SSR markers distributed across the 20 linkage groups of soybean were developed. Multiplex sets constructed from the analysis of diverse soybean germplasm should have a wide range of genotyping applications. The procedures used in this study were systematic and rapid and should be applicable for multiplex development in any species with SSR marker technology.     

Development of simple sequence repeat markers for the soybean rust fungus, Phakopsora pachyrhizi

Twenty-four simple sequence repeat markers were developed for Phakopsora pachyrhizi, a fungal pathogen of soybean (Glycine max) and other legumes. All 24 of the loci were evaluated on 28 isolates of P. pachyrhizi. Twenty-one loci were polymorphic, with allelic diversity ranging from two to eight alleles, and null alleles were observed for eight of the 24 loci. A preliminary screen with the closely related species, P. meibomiae, indicated that these primer pairs are specific to P. pachyrhizi.     

Identification and characterization of simple sequence repeat markers from Brassica napus expressed sequences

The availability of expressed sequence data derived from gene discovery programs enables mining for simple sequence repeats (SSR), providing useful genetic markers for crop improvement. These markers are inexpensive, require minimal labour to produce and can frequently be associated with functionally annotated genes. This study presents the development and characterization of 24 expressed sequence tags (EST)‐SSR markers from Brassica napus and their cross‐amplification across Brassica species. The markers show reliable amplification, genome specificity and considerable polymorphism, demonstrating the utility of EST‐SSRs for genetic analysis of wild Brassica populations and commercial Brassica germplasm.     

Identification and characterization of simple sequence repeat markers from a glandular Origanum vulgare expressed sequence tag

Oregano (Origanum vulgare) and marjoram (Origanum majorana) are two sensorial distinct spices within the genus Origanum (Lamiaceae). Simple sequence repeat (SSR) markers were developed from expressed sequence tags (ESTs) of essential oil glands of O. vulgare. Thirteen EST-SSR loci were evaluated using 20 individual plants of O. vulgare and 19 plants of Origanum majorana. The number of alleles per locus ranged from one to four. All loci developed from O. vulgare successfully cross-amplified in O. majorana.     

Simple sequence repeat markers linked to QTL for resistance to Watermelon mosaic virus in melon

A population of recombinant inbred lines (RIL) derived from a cross between the Watermelon mosaic virus (WMV) resistant genotype TGR-1551 and the susceptible Spanish cultivar ‘Bola de Oro’ has been evaluated for WMV resistance in spring, fall and growth chamber conditions. The quantitative trait loci (QTL) analyses detected one major QTL (wmv) on linkage group (LG) XI close to the microsatellite marker CMN04_35. This QTL controls the resistance to WMV in the three environmental conditions evaluated. Other minor QTLs affecting the severity of viral symptoms were identified, but they were not detected in all the assayed environments. The screening of the marker CMN04_35 in an F₂ progeny, derived from the same cross, confirmed the effect of this QTL on the expression of WMV resistance also in early generations, which evidences the usefulness of this marker for a marker assisted selection program.     

Identification of inter simple sequence repeat (ISSR) markers associated with seed size in wheat

The feasibility of identifying inter-simple sequence repeat markers associated with seed weight in hexaploid wheat was tested using 113 recombinant inbred lines developed by the single-seed descent method, from a cross between Rye selection111, an Indian genetic stock obtained through the introgression of genes for bold seed size from rye, and Chinese Spring having small seed size. Three markers were associated with low seed size with gene effects of 14.8%, 9.5%, and 6%, while four markers with contributions of 8%, 4.66%, 2.92% and 2.61% were found to be linked to high seed size, together contributing 31% of the phenotypic variance in seed size. Nulli-tetrasomic and di-telosomic analysis revealed the presence of three low seed size QTL-associated markers on three chromosomes, 6BL, 2DL, and 1DS respectively. This study clearly demonstrates that ISSRs are highly useful for finding markers associated with major and minor genes controlling agronomically important traits in wheat. Received: 24 February 2000 / Accepted: 31 March 2000     

Polymorphic simple sequence repeat markers in chloroplast genomes of Solanaceous plants

PCR-based markers were developed from mononucleotide simple-sequence repeats in the chloroplast genome of Nicotiana tabacum and applied to the analysis of genetic diversity. These markers were found to detect high levels of polymorphism at three taxonomic levels in Solanaceous plants. Of 36 chloroplast loci examined, 26 show some degree of polymorphism among potato accessions. Among a set of 30 tetraploid potato cultivars it is apparent that a single chloroplast haplotype is prevalent, presumably a result of the widespread use as a female parent of the imported US cultivar Rough Purple Chili in the latter half of the 19th century. Nonetheless, there is considerable chloroplast diversity in the cultivated potato, and it is clear that a large proportion of this variability has arisen through the use of wild or primitive cultivated species of potato in introgression programmes. This variability should be used in future breeding programmes. An examination of single accessions from 24 potato species, as well as representatives from tobacco and other members of the Solanaceae, reveals high levels of inter-specific chloroplast DNA variation. These data, and the ease of use and potential for multiplexing of these markers, suggest that cpSSRs will be of great utility in population genetics, germplasm management, evolutionary and phylogenetic studies as well as in, the analysis of material from introgression and somatic-fusion experiments. Interestingly, the polymorphism arising from one of the more-polymorphic chloroplast loci examined, does not originate solely from the SSR, and is due to variation in the copy number of two tandemly arrayed sequence elements. Received: 15 December 1998 / Accepted: 9 February 1999     

High-volume mapping of maize mutants with simple sequence repeat markers

Many genes in maize (Zea mays L.) are revealed by mutations that cause phenotypic variation from normal. These mutants are valuable resources of genetic information. From among the huge collection of maize mutants, it is ultimately necessary to establish which alleles are of the same genes and which are novel genes. Although any given mutant can be subjected to complementation tests or can be mapped by using conventional techniques, the number of mutants at this time makes these approaches prohibitive to encompass the whole collection. Here we describe procedures to efficiently map large numbers of mutants. Included are methods for generating polymorphic mapping progenies, for simply and rapidly preparing samples to use in polymerase chain reaction (PCR), for tissue pooling and application of simple sequence repeat (SSR), markers, and for stepwise determination of linkage followed by mapping to chromosomal region.     

Development of polymorphic simple sequence repeat markers for Pisolithus microcarpus

Six polymorphic simple sequence repeat (SSR) markers were developed for the ectomycorrhizal fungus Pisolithus microcarpus. A polymerase chain reaction (PCR)‐based technique was used in which random amplified polymorphic DNA (RAPD) fingerprints were probed with labelled SSR oligonucleotides by southern hybridization. The number of alleles per locus ranged from two to nine with expected heterozygosity values from 0.33 to 0.76. These loci will be potentially useful for genetic structure and gene flow studies of P. microcarpus populations. Cross‐species amplification with Pisolithus albus isolates at all loci was also observed.     

Cationic substrates of soybean lipoxygenase-1

Soybean lipoxygenase-1 (SBLO-1) catalyzes the oxygenation of 1,4-dienes to produce conjugated diene hydroperoxides. The best substrates are anions of fatty acids; for example, linoleate is converted to 13(S)-hydroperoxy-9(Z),11(E)-octadecadienoate. The manner in which SBLO-1 binds substrates is uncertain. In the present work, it was found that SBLO-1 will oxygenate linoleyltrimethylammonium ion (LTMA) to give primarily13(S)-hydroperoxy-9(Z),11(E)-octadecadienyltrimethylammonium ion. The rate of this process is about the same at pH 7 and pH 9 and is about 30% of the rate observed with linoleate at pH 9. At pH 7, SBLO-1 oxygenates linoleyldimethylamine (LDMA) to give primarily 13(S)-hydroperoxy-9(Z),11(E)-octadecadienyldimethylamine. The oxygenation of LDMA occurs at about the same rate as LTMA at pH 7, but more slowly at pH 9. The results demonstrate that SBLO-1 will readily oxygenate substrates in which the carboxylate of linoleate is replaced with a cationic group, and the products of these reactions have the same stereo- and regiochemistry as the products obtained from fatty acid substrates.     

Simple sequence repeat (SSR) markers linked to E1, E3, E4, and E7 maturity genes in soybean.

Soybean near isogenic lines (NILs), contrasting for maturity and photoperiod sensitivity loci, were genotyped with approximately 430 mapped simple sequence repeats (SSRs), also known as microsatellite markers. By analysis of allele distributions across the NILs, it was possible to confirm the map location of the Dt1 indeterminate growth locus, to refine the SSR mapping of the T tawny pubescence locus, to map E1 and E3 maturity loci with molecular markers, and to map the E4 and E7 maturity loci for the first time. Molecular markers flanking these loci are now available for marker-assisted breeding for these traits. Analysis of map locations identified a putative homologous relationship among four chromosomal regions; one in the middle of linkage group (LG) C2 carrying E1 and E7, one on LG I carrying E4, one at the top of LG C2, at which there is a reproductive period quantitative trait locus (QTL), and the fourth on LG B1. Other evidence suggests that homology also exists between the E1 + E7 region on LG C2 and a region on LG L linked to a pod maturity QTL. Homology relationships predict possible locations in the soybean genome of additional maturity loci, as well as which maturity loci may share a common evolutionary origin and similar mechanism(s) of action.     

水稻长穗颈基因eui紧密连锁SSR标记获得

02428h是从半矮秆材料02428体细胞培养后代中发现的隐性高秆突变体, 其株高性状由1对长穗颈基因eui和1对半矮秆基因sd-1共同控制。以02428h与半矮秆材料南京11杂交的F₂为作图群体, 利用Gramene公布的SSR标记和根据NCBI中的BAC序列自己新开发的SSR标记, 将eui基因定位在第5染色体上的RM3673和RM0012之间, 两侧遗传距离分别为0.3 cM和1.0 cM, 为该基因的分子标记辅助选择奠定了基础。     

Fifteen polymorphic simple sequence repeat markers from expressed sequence tags of Liriodendron tulipifera

We developed and evaluated simple sequence repeat (SSR) markers derived from expressed sequence tags (ESTs) of Liriodendron tulipifera. Characteristics of 15 EST‐SSR loci were investigated using 33 L. tulipifera individuals. The number of alleles per locus ranged from two to five. The expected and observed heterozygosities ranged from 0.216 to 0.751 and from 0.182 to 0.97, respectively. These loci were further tested for their cross‐species transferability to Liriodendron Chinense. Because of their high level of polymorphism and transferability, our 15 single‐locus EST‐SSR markers will be valuable tools for research on mating system, population genetics and systemic evolution of Liriodendron.