RESUMO
The effects of 5 factors (template DNA, Mg(2+), dNTPs, Taq DNA polymerase, and primer) on the polymerase chain reaction (PCR) were investigated to optimize the start codon targeted polymor-phism (SCoT)-PCR system of Dactylis glomerata L., using an orthogo-nal design L16 (4(5)). A suitable SCoT-PCR system for D. glomerata was established; the 20 µL reaction volume contained 3.0 mM Mg(2+), 0.2 mM dNTPs, 1.0 U Taq DNA polymerase, 0.2 µM primer, 20 ng tem-plate DNA, and 2 µL 10X buffer. Each factor had a different effect on the amplification reaction, and the concentration of dNTPs had the larg-est effect on the SCoT-PCR system. We tested 10 orchardgrass samples to determine and verify the stability of the reaction system. The results showed that amplified bands from diverse materials were clear, stable, and rich in polymorphisms, indicating that the optimized system was very stable.
Assuntos
Códon de Iniciação , Dactylis/genética , Reação em Cadeia da Polimerase/métodos , Primers do DNA , DNA de Plantas/análise , DNA de Plantas/genéticaRESUMO
Dactylis glomerata L. is an important forage species in the Mediterranean region, and in other regions with a similar climate. Genetic material from 3 locations in north, central, and south Greece was studied, using morphological traits, SSR, and ISSR molecular markers. Morphological analysis revealed differences among the geographic locations studied for all morphological traits, except the number of reproductive tillers. Moreover, the highest phenotypic variation was observed on the accessions from south, while the lowest was observed on the accessions from the north. Although the results of the molecular marker analysis are indicative, a high level of genetic diversity at the species level was revealed by ISSRs (GST=0.291) and SSRs (FST=0.186). Analysis of molecular variance showed that a high level of genetic diversity existed for ISSRs and SSRs within populations (62 and 83%, respectively), rather than among populations (38 and 17%, respectively). Cluster analysis divided the 3 populations in 2 groups, with the population originating from the island of Crete forming 1 group, while the populations from north Greece (Taxiarchis) and central Greece (Pertouli) were clustered in a 2nd group. In general, the results indicate that SSRs are more informative compared to ISSRs about the genetic variation within a population, whereas the ISSRs were more informative about the genetic diversity among populations However, a similar trend in diversity (genotypic and phenotypic) was observed in the morphological traits and microsatellite-based (SSR/ISSR) markers at the locations studied.
Assuntos
Dactylis/genética , Variação Genética , Repetições de Microssatélites , Sementes/genética , Análise de Variância , Análise por Conglomerados , Dactylis/classificação , Genoma de Planta , Análise de Componente PrincipalRESUMO
Orchardgrass, or cocksfoot, is an important perennial forage grass worldwide. The comprehensive understanding of orchardgrass accessions will benefit germplasm collection and breeding progress, and it will enhance efforts to improve forage yield and quality. Therefore, 24 novel, simple, polymorphic, and reliable start codon-targeted (SCoT) markers were used to analyze the diversity and genetic relationships among 95 orchardgrass accessions. In total, 273 polymorphic bands were detected with an average of 11.4 bands per primer. The average polymorphic rate for the species was 83.4%, suggesting a high discriminating ability of the SCoT technique for orchardgrass. The molecular variance analysis revealed that 69.13 and 30.87% of variation resided within and among groups, respectively, demonstrating that the orchardgrass germplasms had a higher level of genetic diversity within groups than among geographical regions and distributions. The distinct geographical divergence of orchardgrass was revealed between North America and Oceania. The unweighted pair-group method with arithmetic mean dendrogram revealed a separation of 7 main clusters between 95 accessions according to the geographical origin. Furthermore, each cluster was divided into subgroups mainly according to the origin of its state. The genetic divergence of orchardgrass might be influenced by the ecogeographical conditions, climatic types, breeding systems and gene flow with variations in cultures, bird migration, and breeder selection. These results could facilitate orchardgrass germplasm collection, management, and breeding worldwide.
Assuntos
Códon de Iniciação , Dactylis/classificação , Dactylis/genética , Variação Genética , Evolução Molecular , Marcadores Genéticos , Genoma de Planta , Filogeografia , Polimorfismo GenéticoRESUMO
Six F1 populations derived from crosses among 4 orchardgrass (Dactylis glomerata L.) cultivars were studied by morphological and simple sequence repeat molecular markers to test for hybrid vigor and a correlation between genetic distance and heterosis. Heterosis was observed for days to length of culm, leaf traits, tiller numbers, etc. Significant differences between obverse and inverse crosses were found for eleven traits. A cytoplasmic effect existed for the agronomic traits considered in this study. The correlations between genetic distance and heterosis were investigated by analyzing the performance of 3 crosses. The results showed that genetic distance was significantly correlated with tiller number (r = 0.834) and negatively correlated with length of culm (r = -0.889). However, there was no significant correlation with heterosis for the other traits, including yield; the correlation coefficient were too small to allow prediction of orchardgrass heterosis from the parental genetics.
Assuntos
Dactylis/genética , Vigor Híbrido/genética , Hibridização Genética , Cruzamentos Genéticos , Dactylis/crescimento & desenvolvimento , Repetições de Microssatélites/genética , FenótipoRESUMO
The accurate identification of orchardgrass (Dactylis glomerata L.) cultivars is necessary to ensure purity for consumers, the effective utilization of cultivars, and to protect the intellectual property for breeders. Therefore, this study aimed to use SSR to construct DNA fingerprinting of orchardgrass cultivars. The genetic diversity of 32 orchardgrass cultivars originated from 21 countries, but grown in China, was assessed using a set of 29 SSR markers distributed across 9 linkage groups of the orchardgrass genome. A total of 229 bands were detected, with an average of 7.9 bands per marker. The average polymorphic rate for the species was 92.1%. The polymorphism information content ranged from 0.771 to 0.893. The genetic similarity ranged from 0.55 to 0.84, which confirmed a high level of genetic diversity among orchardgrass cultivars. The unweighted pair-group method, in combination with the arithmetic mean algorithm (UPGMA) dendrogram and principal coordinate analysis, showed a separation of 6 major clusters among 32 cultivars. The number of distinguishable cultivars ranged from 3 to 23, with an average of 12.1 per primer. Moreover, 11 bands that showed stable and repeatable SSR patterns were amplified by A01E14, A01K14, and D02K13. These bands were used to develop the DNA fingerprints for 32 orchardgrass cultivars. In the DNA fingerprints constructed, each cultivar had a unique fingerprinting pattern that was easily distinguished from the others. These results indicate that the SSR marker was polymorphic, and reliable for use in potential large-scale DNA fingerprinting of orchardgrass cultivars.
Assuntos
Dactylis/classificação , Dactylis/genética , Repetições de Microssatélites , Impressões Digitais de DNA , Ligação Genética , Variação Genética , Filogenia , Polimorfismo GenéticoRESUMO
Orchardgrass is a highly variable, perennial forage grass that is cultivated throughout temperate and subtropical regions of the world. Despite its economic importance, the genetic relationship and distance among and within cultivars are largely unknown but would be of great interest for breeding programs. We investigated the molecular variation and structure of cultivar populations, compared the level of genetic diversity among cultivars (Baoxing, Anba, Bote, and Kaimo), subspecies (Dactylis glomerata ssp Woronowii) and advanced breeding line (YA02-116) to determine whether there is still sufficient genetic diversity within presently used cultivars for future breeding progress in China. Twenty individuals were analyzed from each of six accessions using SSR markers; 114 easily scored bands were generated from 15 SSR primer pairs, with an average of 7.6 alleles per locus. The polymorphic rate was 100% among the 120 individuals, reflecting a high degree of genetic diversity. Among the six accessions, the highest genetic diversity was observed in Kaimo (H = 0.2518; I = 0.3916; P = 87.3%) and 02-116 had a lower level of genetic diversity (H = 0.1806; I = 0.2788; P = 58.73%) compared with other cultivars tested. An of molecular variance revealed a much larger genetic variation within accessions (65%) than between them (35%). This observation suggests that these cultivars have potential for providing rich genetic resource for further breeding program. Furthermore, the study also indicated that Chinese orchardgrass breeding has involved strong selection for adaptation to forage production, which may result in restricted genetic base of orchardgrass cultivar.