The use of AFLP markers for cultivar identification in apricot

Amplified fragment length polymorphism (AFLP) molecular markers were employed for the fingerprinting of 118 accessions of apricot, including cultivated varieties and related apricot species. Five primer combinations were tested and 165 polymorphic bands produced which could uniquely differentiate all accessions under investigation. Primer combinations were rated according to the multiplex ratio, the polymorphic index content and the discrimination power parameters and superior combinations were identified. AFLP markers were used in cluster analysis carried out with the UPGMA and the neighbour‐joining methods and parsimony analysis. Four groups of similar accessions were identified: (i) those from the Mediterranean basin, (ii) from China, (iii) from continental Europe and (iv) mixed Europe‐North America accessions. In the principal component analysis the first three components accounted for 17% of the total variability existing among accessions. Because the most important regions where apricot is cultivated in the world were represented in the analysis, with a large number of varieties, it was possible to discuss the data in the light of current phylogenetic hypotheses on the origin and evolution of the species.     

Strawberry cultivar identification based on hypervariable SSR markers

We genotyped strawberry cultivars by two newly selected and two previously reported SSR markers. All four markers produced interpretable electropherograms from 75 accessions consisting of 72 Fragaria × ananassa cultivars or lines and three octoploid Fragaria species accessions. These SSR markers were highly polymorphic; in particular, one of the newly developed markers, FxaHGA02P13, was capable of distinguishing all of the accessions except for a mutant strain that was derived from another accession in the set. When two markers were combined, all 48 full-sib individuals could be distinguished. Fingerprinting patterns were reproducible between multiple samples, including the leaves, sepals, and fruit flesh of the same accession. Principal-coordinate analysis of the 75 accessions detected several groups, which reflect taxon and breeding site. Together with other available markers, these SSR markers will contribute to the management of strawberry genetic resources and the protection of breeders’ rights.     

Development and variability analysis of microsatellite markers in peach

A genomic DNA library enriched with AG/CT repeats has been developed from the peach cultivar ‘Merrill O'Henry’. The enrichment method was efficient, with 61% of the clones obtained carrying a microsatellite sequence and a yield of one polymorphic microsatellite every 2.17 sequenced clones. From 35 microsatellites detected, 24 were polymorphic in a set of 25 cultivars including 14 peaches and 11 nectarines. A total of 82 alleles were found with the polymorphic microsatellites, with an average of a 37% of observed heterozygosity. Microsatellites with a high number of repeats were generally those having the largest number of alleles. All cultivars except two (‘Spring Lady’ and ‘Queencrest’) could be individually distinguished with the markers used. Just three selected microsatellites were enough for the discrimination of 24 out of the 25 possible genotypes. Cluster analysis grouped all nectarines in a single cluster. Peaches, with 75 of the 82 alleles found, were more variable than nectarines, with only 64. Microsatellites appear to be powerful and suitable markers for application in peach genetics and breeding.     

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.     

Potential application of simple easy-to-use insertion-deletion (InDel) markers in citrus cultivar identification

We previously developed insertion-deletion (InDel) markers that distinguish three genotypes (two homozygous and one heterozygous) of diverse citrus cultivars. These InDel markers were codominant and could be clearly detected by using simple agarose gel electrophoresis. We sought to establish a method for cultivar identification using these 28 InDel markers to genotype 31 citrus cultivars. The results revealed that a minimum of 6 markers were required to identify individuals using the three-genotype classification method. Furthermore, we found that a simple method for distinguishing between two genotypes (homozygous and heterozygous) could be used to identify individuals using a minimum of 7 markers. Our findings provide a basis for the development of simple and rapid citrus cultivar identification methods.     

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.     

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.     

A practical method for apple cultivar identification and parent-offspring analysis using simple sequence repeat markers

Differentiation of cultivars with simple sequence repeat (SSR) markers is a very useful technique for the true-to-type characterization of cultivars and clarification of parent-offspring relationships. We developed an SSR marker set for cultivar identification comprising 15 markers that were screened from 46 previously published SSRs. This marker set could be used for apple varieties including Malus × domestica and/or other Malus species. These SSRs successfully characterized 95 apples, including the leading and major founding cultivars used worldwide for modern apple breeding. Therefore, this marker set could be applied to almost all apple cultivars. We also analyzed the parent-offspring relationships of 69 cultivars by considering allele transmissions. This analysis revealed the true parentage of the following seven cultivars: ‘Kizashi’, ‘Chinatsu’, ‘Honey Queen’, ‘Haruka’, ‘Seirin’, ‘Ozenokurenai’, and Morioka #48. This analysis also revealed a parentage discrepancy for ‘Hacnine’. From the parent-offspring analysis, two microsatellite mutation events at alleles inherited from pollen parents were observed.     

Development of microsatellite markers in sweet passion fruit,and identification of length and conformation polymorphisms within repeat sequences

The aim of this study was to construct and characterize an SSR‐enriched genomic library for Passiflora alata, a fruit species native to the Brazilian plateau and the eastern Amazon region. There is potential to improve this crop, as the fruit is attractive because of its pleasant aroma and flavour characteristics. Of 862 sequences, 391 (45%) were found to have SSRs. We identified 412 microsatellites: 69% were classified as perfect, 23% imperfect, 5% interrupted and 3% were compound microsatellites. The main types of repeat sequences were dinucleotide (62.5%) and trinucleotide (23%) repeats. It was possible to design 312 primer pairs, and 229 of them were synthesized and tested. The amplicons were electrophoresed using denaturing and non‐denaturing gels to screen for divergence between two phenotypically distinct parents of a mapping population of P. alata. Length and conformation polymorphisms within repeat sequences amounted to 35% and 28%, respectively. The importance of the development of SSR markers for sweet passion fruit and the effectiveness of the SSCP approach to increase the available number of polymorphic SSRs are discussed.     

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.     

Genic microsatellite markers for discrimination of spinach cultivars

A set of simple sequence repeat (SSR or microsatellite) markers was used to discriminate a collection of 33 Spinacia oleracea hybrid cultivars from seven different breeding stations all over the world. All SSR markers were genic microsatellite markers located in coding or non-coding regions of genes of known function. Cluster analysis based on 13 of the SSR markers showed that the spinach hybrids grouped into three clusters. The first two clusters consisted of European spinach types, which were well discriminated according to their origin from different breeding stations. The third cluster was a mixture of Asian as well as European types of spinach. Subclusters in this group did not reflect differences in morphology, earliness or company origin. The data show that genic microsatellites are a powerful tool for discrimination of spinach cultivars.     

红掌已知品种库的建立与应用

通过建立红掌已知品种数据库,了解国内市场上红掌品种的现状,为新品种选育提供指导,同时探讨已知品种数据库在新品种DUS测试中的应用。历时4年共收集了87个红掌品种,构建了红掌已知品种数据库。计算39个DUS测试性状的变异系数和遗传多样性指数,对数量性状进行相关性分析,比较了直接筛选和聚类分析方法在筛选近似品种上的应用。结果表明,红掌质量性状（假质量性状）的变异系数为14.00%~89.29%,平均变异系数为43.67%,平均遗传多样性指数为0.93;数量性状的变异系数为17.63%~32.34%,平均变异系数为26.59%,平均遗传多样性指数为1.02,表明红掌品种间多样性不够丰富,遗传基础较狭窄。相关性分析表明,所有数量性状间均显著正相关。根据调查问卷提供的性状直接从已知品种库筛选或以聚类方法均可有效地筛选或验证近似品种的有效性。可见红掌已知品种的遗传多样性指数偏低,不利于品种的进一步改良,已知品种库的建立可为DUS近似品种的筛选提供支撑。     

Development,characterization and mapping of microsatellite markers for lentil (Lens culinaris Medik.)

Lentil is the sixth most important pulse crop terms of production in the world, but the number of available and mapped SSR markers are limited. To develop SSR markers in lentil, four genomic libraries for (CA)n, (GA)n, (AAC)n and (ATG)n repeats were constructed. A total of 360 SSR primers were designed and validated using 15 Turkish lentil cultivars and genotypes. The most polymorphic repeat motifs were GA and CT, with a mean number of alleles per locus of 7.80 and 6.55, respectively. Seventy‐eight SSR primers amplified a total of 400 polymorphic alleles, whereas 71 SSR primers produced markers within the expected size range. For 78 polymorphic SSR primers, the average number of alleles per locus was 5.1 and PIC value ranged from 0.07 to 0.89, with an average of 0.58. A linkage map was constructed using 92 individual F₂ plants derived from a cross between Karacada? × Silvan, with 47 SSR markers. The SSR markers developed in this study could be used for germplasm classification and identification and mapping of QTL in lentil.     

Application of ISSRs for cultivar identification and assessment of genetic relationships in rose

The inter-simple sequence repeat (ISSR) technique was evaluated for its applicability to cultivar identification and assessment of genetic relationships in rose. Nine ISSR primers that revealed informative patterns were selected to fingerprint (by Resophor agarose gel electrophoresis) 33 cultivars, including unrelated cultivars, sports and offspring obtained by sexual propagation from the same initial variety. A total of 159 fragments were generated using these nine primers, 149 of which (93.7%) were polymorphic. All 33 rose cultivars, except for the known colour sports, were differentiated. A Jaccard's dissimilarity coefficient matrix of the cultivars showed a low dissimilarity level between them (mean dissimilarity: 0.45). An unweighted pair group method with the arithmetic averages dendrogram and principal coordinates analysis separated them into four main groups according to their horticultural classification and pedigree. The ISSR technique is therefore a potentially useful tool for cultivar identification and assessing genetic relationships in rose because it is simple, fast, cost-effective, reliable and highly discriminating.     

Development of chloroplast‐specific microsatellite markers for molecular characterization of alloplasmic lines and phylogenetic analysis in wheat

Wheat (Triticum aestivum L.) is strictly a self‐pollinated crop, where hybrid breeding requires well‐characterized cytoplasmic male sterile (CMS) lines. The CMS has mostly been developed by substituting nuclear genome of wheat into the cytoplasm from wild relatives. Molecular characterization of 90 genotypes including 82 CMS lines originating from five different species, namely Aegilops speltoides, Ae. kotschyi, Ae. variabilis, Triticum araraticum and T. timopheevii, and eight popular varieties was carried out. Consequently, a set of 25 microsatellite markers specific to chloroplast (cpSSRs) were designed and successfully validated for specificity of amplification. A total of 15 cpSSRs (60%) were found polymorphic, of which three cpSSRs (TaCM7, TaCM8 and TaCM11) in genic region and twelve cpSSRs were located in intergenic region. Phylogenetic analysis of genotypes using cpSSRs revealed two major groups well in accordance with respective origin. A set of cpSSRs and phylogeny of CMS belonging to different origins developed, which will be helpful for the improvement in CMS system in wheat. The genic cpSSRs can be used for the allele mining and evolutionary studies.