Genetic analysis and identification of DNA markers linked to a novel Phytophthora sojae resistance gene in the Japanese soybean cultivar Waseshiroge

The Glycine max (L.) Merr. cultivar Waseshiroge is highly resistant to several races of Phytophthora sojae in Japan. In order to determine which Rps gene might be present in Waseshiroge, 15 differential cultivars were challenged with 12 P. sojae isolates. None had a reaction pattern identical to that of Waseshiroge, indicating that Waseshiroge may contain a novel Rps gene. In order to characterize the inheritance of Waseshiroge resistance to P. sojae isolates, 98 F₂ progeny and 94 F_7:8 lines were produced from crosses between the susceptible cultivar Tanbakuro and Waseshiroge. Chi-square tests indicated that segregation fit a 3:1 ratio for resistance and susceptibility in two F₂ sub-populations of 42 and 56 seedlings. This and a 46.27:1.46:46.27 (or 63:2:63) ratio for resistance: segregation: susceptibility among the 94 F_7:8 lines indicated that resistance was controlled by a single dominant gene. DNA analyses were carried out on Tanbakuro, Waseshiroge and the 94 F_7:8 lines, and a linkage map was constructed with 17 SSR markers and nine new primer pairs that amplify marker loci linked to Rps1 on soybean chromosome 3 (linkage group N). The closest markers, Satt009 and T000304487l, map to locations 0.9 and 1.6 cM on each side of the estimated position of the Rps gene, respectively. The results showed that the Rps gene in Waseshiroge is either allelic to Rps1, or resides at a tightly linked locus in a gene cluster. A three-way-contingency table analysis indicated that marker-assisted selection with the two flanking markers could be used in the development of new resistant cultivars.     

A practical method for almond cultivar identification and parental analysis using simple sequence repeat markers

Early and accurate identification of almond [Prunus dulcis (Mill.) D.A. Webb] cultivars is critical to commercial growers and nurseries. Previously published simple sequence repeat loci were examined for their ability to distinguish commonly grown almond cultivars. Twelve highly polymorphic loci were selected for their ability to uniquely identify a set of 18 almond cultivars commonly grown in California, many of which are closely related. These markers also allow an accurate assessment of parent/progeny relationships among cultivars. This system can reliably identify at an early stage of development all major California almond cultivars in current production.     

Development, characterization and utilization of microsatellite markers in pigeonpea

Pigeonpea is a major legume of the semi‐arid tropics that has been neglected in terms of molecular breeding. The objectives of this study were to develop microsatellite markers and evaluate their potential for use in pigeonpea genetics and breeding. Two hundred and eight microsatellite loci were isolated by screening a non‐enriched partial genomic library. Primers were designed for 39 microsatellite loci, 20 of which amplified polymerase chain reaction products of the expected size. Nineteen of the primer pairs were polymorphic amongst 15 cultivated and nine wild pigeonpea accessions providing evidence for cross‐species transferability within the genus Cajanus. A total of 98 alleles were detected at the 19 polymorphic loci with an average of 4.9 alleles per locus. The observed heterozygosity ranged from 0.17 to 0.80 with a mean of 0.42 per locus. Less allelic variation (31 alleles) was observed within the cultivated species than across the wild species (92 alleles). The diversity analysis readily distinguished all wild relatives from each other and from the cultivated germplasm. Development of more microsatellites is recommended for future genomic studies in pigeonpea.     

Targeted identification of association between cotton fiber quality traits and microsatellite markers

Primitive and exotic accessions of cotton are potential sources of favorable alleles for genetic improvement, enriching diversity in the genetically constricted gene pool of elite cultivars. Three exotic accessions of cotton (MDN101, MDN063 and MDN257), collected from different parts of Central America and converted to day-neutral flowering; and four elite cultivars (PD94042, DES56, PMHS200 and Acala Maxxa) representing the US cotton gene pool were used as parents to create experimental populations. The corresponding F₂ and F_2:3 progenies of these populations were grown in two successive years (i.e., some in 2011–2012, some in 2012–2013) and phenotypes were scored in both F₂ and F_2:3 progenies in all 3 years (2011–2012–2013). These populations were screened with 113 polymorphic microsatellite markers selected from “hotspots” for fiber quality quantitative trait loci in the cotton genome and single marker analyses were performed to identify significant associations of the markers with six fiber quality traits. A total of 134 nominal marker-trait associations were identified, among which 15 were significant after Bonferroni correction for multiple comparisons. In 67 of 134 nominal associations and 4 of 15 significant associations, the exotic parents contributed favorable alleles to multiple backgrounds and for multiple traits, in addition to the traits for which they were selected. These results indicate that utilization of exotic and wild accessions of cotton is useful in introducing favorable alleles into the cultivated cotton gene pool for genetic improvement.     

Development of NBS-related microsatellite (NRM) markers in hexaploid wheat

NBS (nucleotide binding site) genes, one type of the most important disease-resistance genes in the plant kingdom, are usually found clustered in genome. In this study, a total of 2288 full-length NBS protein-coding sequences were isolated from the wheat (Triticum aestivum L.) genome, and 903 TaNBSs of which were found expressed in wheat. Meanwhile, 2203 microsatellite loci were detected within 1061 scaffolds containing TaNBS. The distribution of these microsatellite loci across wheat homologous groups (HG) is 20% HG2, 16% HG7, 15% HG1, 15% HG6, 12% HG4, 12% HG5 and 10% HG3. We developed 1830 NBS-related microsatellite (NRM) markers for the microsatellite loci on TaNBS-scaffold sequences.Among them, 342 NRM markers were developed for HG2 with the largest number of microsatellite loci, and 69 out of these markers were anchored to the wheat genetic map using mapping population. Then, a total of 26 2AS-NRM markers, nine 2BL-NRM markers and nine 2DL-NRM markers were integrated into the genetic maps carrying Yr69, Pm51 and Pm43, respectively. Finally, candidate sequences, within the gene clusters where Yr5 and Sr21 located, were analyzed according to the genomic position information of TaNBS and NRM markers. These NRM markers have clear chromosome locations and are correlated with potential disease resistance sequences, which can be manipulated to mapping or adding linkage markers of disease-resistance genes or QTLs, especially for those in the NBS gene clusters.     

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

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

Discovery of single nucleotide polymorphism in <Emphasis Type="Italic">Capsicum</Emphasis> and SNP markers for cultivar identification

Molecular markers based on single nucleotide polymorphisms (SNPs) are abundant and evenly distributed in a whole genome enough to distinguish individuals in a population. In recent years, sets of SNP markers have been designed and applied for cultivar identification in various crop species. This paper is the first to report the development of a panel of SNP markers for variety identification in peppers. We used conserved ortholog set II (COSII) markers developed from conserved unigenes between tomato and Arabidopsis to identify SNPs in peppers. We tested 438 COSII primer sets amplified as single PCR products out of a total 600 COSII primer sets. Among the 438 COSII primers, 170 primer sets (38.8%) showed polymorphisms between Capsicum annuum ‘RNaky (RN)’and C. chinense ‘PI 159234 (234)’. In contrast, only 48 primer sets (11.0%) out of 438 primers sets were polymorphic between C. annuum ‘Perennial (PER), and ‘Dempsey (DEMP)’. The average frequency of SNPs plus InDels between C. annuum and C. chinense was 1/189 bp and between C. annuum spp. was 1/948 bp. Primer sets showing SNP between C. annuum PER and DEMP were re-designed to Allele Specific PCR (AS-PCR) primers and we finally selected a total of 40 SNP markers for cultivar identification. As the result, we were able to discriminate 97.5% of the 81 commercial hot cultivars and 100% of the 17 sweet pepper cultivars. We conclude the paper by discussing the use of the SNP marker set for cultivar identification and other applications.     

Isozyme polymorphism of Rosa spp. and cultivar identification

In order to study the polymorphism of the enzyme systems of rose cultivars, experimental conditions were determined to simultaneously extract three systems: esterase (EST), leucine aminopeptidase (LAP) and superoxide dismutase (SOD) which gave up to 9, 7 and 9 bands, respectively. The influence of environmental conditions on the polymorphism level was taken into account. Finally, the computation of the Jaccard distances from the distribution of the isozyme bands led to the improvement of the identification process within an Ancient Rose cultivar collection. This revised version was published online in July 2006 with corrections to the Cover Date.     

Stability of RAPD markers for determining cultivar specific DNA profiles in rye (Secale cereale L.)

Summary Cultivar specific DNA profiles in rye were revealed by polymerase chain reaction (PCR) using randomly amplified polymorphic DNA (RAPD) sequences. Ten base primers were used for the amplification of genomic DNA of rye cultivars by PCR. RAPD analysis was found to be reproducible among samples between PCR runs. When amplification profiles of different rye cultivars were compared using various primers, the overall profiles were cultivar specific. However, not all primers revealed polymorphisms. These primers appear to amplify conserved sequences in all rye cultivars. Intracultivar studies were conducted on two of the cultivars. In the cultivar Imperial, no polymorphisms were observed among ten plants analyzed with five primers. In the cultivar Balboa, polymorphisms were observed among fifty plants with four of the ten primers analyzed. Despite the small amount of intracultivar variability, RAPD analysis has the potential to be a rapid and reliable method of cultivar identification in this outcrossing species.     

Transferability of non-genic microsatellite and gene-based sunflower markers to safflower

Safflower (Carthamus tinctorius L.) DNA marker resources are currently very limited. The objective of this study was to determine the feasibility of transferring non-genic microsatellite (SSR) markers and gene-based markers, including intron fragment length polymorphism (IFLP) and resistance gene candidates (RGC)-based markers from sunflower (Helianthus annuus L.) to safflower, both species belonging to the Asteraceae family. Cross-species amplification of 119 non-genic SSRs, 48 IFLPs, and 19 RGC-based sunflower markers in 22 lines and germplasm accessions of safflower was evaluated. Additionally, 69 EST-SSR markers previously reported to amplify in safflower were tested. The results showed that 17.6% of the non-genic SSR, 56.2% of the IFLP, and 73.7% of the RGC-based markers were transferable to safflower. The percentage of transferable markers showing polymorphic loci was 66.6% for non-genic SSR, 70.6% for EST-SSR, 55.5% for IFLP, and 71.4% for RGC-based markers. The highest polymorphism levels were found for non-genic SSR. The average number of alleles per polymorphic locus and mean heterozygosity values were 2.9 and 0.46, respectively, for non-genic SSR, 2.2 and 0.35 for EST-SSR, 2.1 and 0.24 for IFLP, and 2.0 and 0.34 for RGC-based markers. The results of this study revealed a low rate of transferability for non-genic SSR sunflower markers and a better rate of transferability for IFLP and RGC-based markers. Transferable genic and non-genic sunflower markers can have utility for trait and comparative mapping studies in safflower.     

Assessment of EST- and genomic microsatellite markers for variety discrimination and genetic diversity studies in wheat

It is likely that in the near future sequence information from sequencing programmes and EST libraries will generate an abundance of genic microsatellite markers. This study is focused on the assessment of their likely impact and performance vis-à-vis their genomic counterparts. Microsatellites from two sources were used to assess the genetic diversity in 56 old and new varieties of bread wheat on the UK Recommended List. A set of 12 microsatellite markers generated from genomic libraries and 20 expressed sequence tag (EST)-derived microsatellites were used in the study, and the performance of both marker sets assessed. The EST-derived or genic microsatellites delivered fingerprints of superior quality, amplifying clear products with few stutter bands. Diversity levels as revealed bygenic microsatellites are similar to the few published results. The PIC values for the genic markers were generally lower than those calculated for the genomic microsatellites, though advantages of both marker classes for variety identification applications are discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Mapping of the Vrn-B1 gene in Triticum aestivum using microsatellite markers

An F₂ population segregating for the dominant gene Vrn‐B1 was developed from the cross of the substitution line ‘Diamant/'Miro‐novskaya 808 5A’ and the winter wheat cultivar ‘Bezostaya 1′. Microsatellite markers (Xgwm and Xbarc) with known map locations on chromosome 5B of common wheat were used for mapping the gene Vrn‐B1. Polymorphism between parental varieties was observed for 28 out of 34 microsatellite markers (82%). Applying the quantitative trait loci mapping approach, the target gene was mapped on the long arm of chromosome 5B, closely linked to Xgwm408. The map position of Vrn‐B1 suggests that the gene is homoeologous to other vernalization response genes located on the homoeologous group 5 chromosomes of wheat, rye and barley.     

Utilization of isozyme polymorphism for cultivar identification of 45 commercial peas (Pisum sativum L.)

Forty five Pisum sativum cultivars were analysed by isozyme electrophoresis with the objective to find protein markers for exact and reproducible discrimination of individual genotypes. The combination of six enzyme systems (acid phosphatase, amylase, esterase, leucine aminopeptidase, shikimate dehydrogenase and phosphoglucomutase) with two electrophoretic techniques (NATIVE-PAGE, isoelectric focusing) and use of seed and leaf tissue enabled to identify all 45 studied cultivars. Critical factors which may affect utilization of isozyme electrophoresis for commercial applications in pea breeding and seed production and testing are discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Assessing the genetic diversity of Portuguese maize germplasm using microsatellite markers

A collection of Portuguese maize accessions representing a valuable source of genes for introduction into modern cultivars is stored at the Portuguese Plant Germplasm Bank (Banco Português de Germoplasma Vegetal—BPGV). To assess genetic diversity among inbreds, microsatellite analysis was carried out for 54 inbred lines representing the diversity of Portuguese dent and flint maize germplasm. Fifty American and other European elite inbreds were also analysed for comparison. Fifteen microsatellite loci distributed throughout the maize genome were chosen based on their repeat unit and base composition. A total of 80 alleles were detected with an average allele number of 5.33 per locus. Polymorphism information content (PIC) values and observed genetic distances showed the existence of large variability among inbreds. Cluster analysis indicated that almost all of the inbreds could be distinguished from each other and Portuguese inbreds were present in all clusters formed. These associations were consistent with the known pedigree records of the inbreds, confirming a mixed origin of Portuguese materials. Comparative analysis of microsatellite diversity among groups was established according to important traits for both breeding and line identification. This revealed that, although most of the genetic diversity (>95%) was attributable to differences among inbreds of different groups, the existence of phenotypic differentiation in endosperm colour, kernel type and cob colour could be suggested for grouping. These findings support the joint use of molecular and morphological traits in management of the germplasm collection. In this study, SSR markers proved to be effective to characterise and identify maize inbred lines, and demonstrate associations among them. This revised version was published online in July 2006 with corrections to the Cover Date.     

Assessment of genetic variation of bread wheat varieties using microsatellite markers

The DNA fingerprinting profiles of 11 bread wheat cultivars, grown in Turkey, was obtained by using 19 highly polymorphic wheat microsatellites. Up to six randomly selected individuals from each of these cultivars were subjected to analysis. Estimated polymorphism information content (PIC) values among 65 individual genotypes for 19 loci were between 0.36 and 0.87 with an average value of 0.68. The numbers of observed alleles were between 2 and 9, with an average value of 5.42. These relationships were assessed among cultivars and among their individual genotypes using the mean pairwise average square distance (D ₁) (or the delta mu measure of distance (Ddm)) matrix and neighbor-joining method based on `Nei-72' distance matrix, respectively. Two spring cultivars, `Kaklic-88' and `Orso', were closely related to each other; the other 9 winter and facultative cultivars were dispersed. Pedigrees of cultivars represented very little shared information among each other, therefore, they could not been utilized in comparisons. This revised version was published online in July 2006 with corrections to the Cover Date.     

Complexity of indica-japonica varietal differentiation in Bangladesh rice landraces revealed by microsatellite markers

To understand the genetic diversity and indica-japonica differentiation in Bangladesh rice varieties, a total of 151 accessions of rice varieties mostly Bangladesh traditional varieties including Aus, Boro, broadcast Aman, transplant Aman and Rayada varietal groups were genotyped using 47 rice nuclear SSRs. As a result, three distinct groups were detected by cluster analysis, corresponding to indica, Aus and japonica rice. Among deepwater rice varieties analyzed some having particular morphological features that mainly corresponded to the japonica varietal group. Some small seeded and aromatic varieties from Bangladesh also corresponded to the japonica varietal group. This research for the first time establishes that the japonica varietal group is a prominent component of traditional varieties in Bangladesh, particularly in deepwater areas.     

Application of isozyme electrophoresis for purity testing and cultivar identification of F1 hybrids of Brassica oleracea

Summary Six isozyme genes were analyzed in seed samples of 65 commercial F₁ hybrids of four horticultural groups of Brassica oleracea (cabbage, Brussels sprouts, sprouting broccoli and cauliflower). Results obtained from electrophoretic assays led to the following conclusions: 1) the electrophoretic test of F₁ hybrid purity was possible in 59 (91%) of the hybrids analyzed, since their inbred parents were apparently fixed each for a different allele in at least on of the loci studied; 2) forty-eight (74%) of the hybrids were individually distinguished by their isozyme phenotype; 3) high levels of segregation in the inbred parents were inferred from the analysis of a sample of seeds of each hybrid.     

Assessment of genetic variability of olive varieties by microsatellite and AFLP markers

Fourteen developed microsatellite markers were characterized for their use in genotyping and diversity studies of olive varieties. After optimisation of microsatellite assay and allele sizing, ninety-six alleles were found in nineteen varieties, with an average of 6.8 alleles per locus. The characteristics of the microsatellite markers were used to identify markers that can be reliably applied for variety genotyping. Such features were the generation of complex banding patterns supported by underlying allele sequences, `short allele dominance', an unstable repeat structure and a low number of alleles. AFLP analysis was performed on the same set of olive varieties using eight primer pair combinations. The genetic relationships among nineteen olive varieties were compared on the basis of microsatellite and AFLP polymorphisms. Genetic distances between all pairwise combinations of the varieties were calculated using Jaccard's coefficient of similarity and dendrograms were constructed by the UPGMA method. The results of clustering analysis with both molecular systems showed the common genetic background of Tuscan varieties, and genetic divergence within Slovene olive germplasm. Slovenian varieties ‘Buga’, ‘Štorta’ and ‘Samo’ might represent regionally selected olives, while ‘Zelenjak’ and ‘Črnica’ are probably derived from the Central Italian region. The predominant local ‘Istrska belica’ was introduced to Slovenia independently from the other regional varieties and showed the lowest genetic similarity with the other regional varieties. This revised version was published online in August 2006 with corrections to the Cover Date.     

Comparative analysis of diversity based on morpho-agronomic traits and microsatellite markers in common bean

Morpho-agronomic traits and microsatellite markers were used to survey genetic diversity in 115 common bean genotypes that included 70 Indian landraces, 24 released varieties and 21 exotic accessions. Twelve morpho-agronomic traits, namely, days to 50% flowering, leaflet length, leaflet width, pod length, pod width, number of pods per plant, days to maturity, seed length, seed width, number of seeds per pod, 100 seed weight and seed yield per plant were studied. Field data of two consecutive years were subjected to multivariate analysis as proposed by Mahalanobis’s D²-statistics, Tochers method of clustering and combined analysis of variance. Seventeen microsatellite markers were also used to examine genetic diversity at molecular level that showed polymorphic information content (PIC) in the range of 0.00–0.684. Dendrograms based on Euclidean distances and UPGMA analysis showed the presence of majority of released varieties into single cluster, which pointed toward their low genetic base in comparison to indigenous landraces and exotic germplasm. Significant correlation existed between morphological genetic distance and microsatellite genetic distance tested by Mantel test (r = 0.876).