A microsatellite-based consensus linkage map for species of Eucalyptus and a novel set of 230 microsatellite markers for the genus

BACKGROUND: Eucalypts are the most widely planted hardwood trees in the world occupying globally more than 18 million hectares as an important source of carbon neutral renewable energy and raw material for pulp, paper and solid wood. Quantitative Trait Loci (QTLs) in Eucalyptus have been localized on pedigree-specific RAPD or AFLP maps seriously limiting the value of such QTL mapping efforts for molecular breeding. The availability of a genus-wide genetic map with transferable microsatellite markers has become a must for the effective advancement of genomic undertakings. This report describes the development of a novel set of 230 EMBRA microsatellites, the construction of the first comprehensive microsatellite-based consensus linkage map for Eucalyptus and the consolidation of existing linkage information for other microsatellites and candidate genes mapped in other species of the genus. RESULTS: The consensus map covers ~90% of the recombining genome of Eucalyptus, involves 234 mapped EMBRA loci on 11 linkage groups, an observed length of 1,568 cM and a mean distance between markers of 8.4 cM. A compilation of all microsatellite linkage information published in Eucalyptus allowed us to establish the homology among linkage groups between this consensus map and other maps published for E. globulus. Comparative mapping analyses also resulted in the linkage group assignment of other 41 microsatellites derived from other Eucalyptus species as well as candidate genes and QTLs for wood and flowering traits published in the literature. This report significantly increases the availability of microsatellite markers and mapping information for species of Eucalyptus and corroborates the high conservation of microsatellite flanking sequences and locus ordering between species of the genus. CONCLUSION: This work represents an important step forward for Eucalyptus comparative genomics, opening stimulating perspectives for evolutionary studies and molecular breeding applications. The generalized use of an increasingly larger set of interspecific transferable markers and consensus mapping information, will allow faster and more detailed investigations of QTL synteny among species, validation of expression-QTL across variable genetic backgrounds and positioning of a growing number of candidate genes co-localized with QTLs, to be tested in association mapping experiments

Variabilidade genética de variedades locais de arroz determinada por marcadores SSR

The objective of this study was to evaluate the genetic variability of rice (Oryza sativa) landraces collected in Brazilian small farms. Twelve simple sequence repeat (SSR) markers characterized 417 landraces collected in 1986, 1987 and 2003, in the state of Goiás, Brazil. The number of landraces with long and thin grain type increased in the evaluated period, probably due to market demand. Based on the molecular data, the genetic variability increased during this period and, as per to the factorial correspondence analysis, most of the accessions were grouped according to the year of collection. The incorporation of modern rice cultivars in landrace cultivation areas and the selection carried out by small farmers are the most probable factors responsible for increasing landrace genetic variability, during the evaluated period. Genotype exchange between farmers, selection practice and local environmental adaptation are able to generate novel adapted allele combinations, which can be used by breeding programs, to reinitiate the process.O objetivo deste trabalho foi avaliar a variabilidade genética de variedades locais de arroz (Oryza sativa), coletadas em pequenas propriedades rurais. Doze marcadores microssatélites (SSR) caracterizaram 417 variedades locais, coletadas em 1986, 1987 e 2003, no Estado de Goiás. O número de variedades locais com tipo de grão longo e fino aumentou no intervalo de tempo avaliado, provavelmente em razão da demanda de mercado. Conforme os dados moleculares, houve aumento na variabilidade genética e, por meio da análise fatorial de correspondência, observou-se que a maior parte dos acessos foi agrupada de acordo com o ano de coleta. A incorporação de cultivares modernas de arroz às áreas de cultivo das variedades locais e a seleção realizada pelos pequenos agricultores são, provavelmente, os fatores responsáveis pelo aumento observado na variabilidade genética, ao longo do período avaliado. O intercâmbio de genótipos entre fazendeiros, a prática da seleção e a adaptação ambiental ao local de cultivo são capazes de gerar novas combinações alélicas adaptadas que, por sua vez, podem ser usadas pelos programas de melhoramento, para reiniciar o processo

The characterization of a new set of EST-derived simple sequence repeat (SSR) markers as a resource for the genetic analysis of Phaseolus vulgaris

<p>Abstract</p> <p>Background</p> <p>Over recent years, a growing effort has been made to develop microsatellite markers for the genomic analysis of the common bean (<it>Phaseolus vulgaris</it>) to broaden the knowledge of the molecular genetic basis of this species. The availability of large sets of expressed sequence tags (ESTs) in public databases has given rise to an expedient approach for the identification of SSRs (Simple Sequence Repeats), specifically EST-derived SSRs. In the present work, a battery of new microsatellite markers was obtained from a search of the <it>Phaseolus vulgaris </it>EST database. The diversity, degree of transferability and polymorphism of these markers were tested.</p> <p>Results</p> <p>From 9,583 valid ESTs, 4,764 had microsatellite motifs, from which 377 were used to design primers, and 302 (80.11%) showed good amplification quality. To analyze transferability, a group of 167 SSRs were tested, and the results showed that they were 82% transferable across at least one species. The highest amplification rates were observed between the species from the <it>Phaseolus </it>(63.7%), <it>Vigna </it>(25.9%), <it>Glycine </it>(19.8%), <it>Medicago </it>(10.2%), <it>Dipterix </it>(6%) and <it>Arachis </it>(1.8%) genera. The average PIC (Polymorphism Information Content) varied from 0.53 for genomic SSRs to 0.47 for EST-SSRs, and the average number of alleles per locus was 4 and 3, respectively. Among the 315 newly tested SSRs in the BJ (BAT93 X Jalo EEP558) population, 24% (76) were polymorphic. The integration of these segregant loci into a framework map composed of 123 previously obtained SSR markers yielded a total of 199 segregant loci, of which 182 (91.5%) were mapped to 14 linkage groups, resulting in a map length of 1,157 cM.</p> <p>Conclusions</p> <p>A total of 302 newly developed EST-SSR markers, showing good amplification quality, are available for the genetic analysis of <it>Phaseolus vulgaris</it>. These markers showed satisfactory rates of transferability, especially between species that have great economic and genomic values. Their diversity was comparable to genomic SSRs, and they were incorporated in the common bean reference genetic map, which constitutes an important contribution to and advance in <it>Phaseolus vulgaris </it>genomic research.</p

Drought tolerance induced by the overexpression of the nuclear rbcL gene in rice

- The objective of this work was to determine whether the overexpression of the nuclear Rubisco large subunit (rbcL) improves the drought tolerance of the genetically modified (GM) BRSMG Curinga upland rice (Oryza sativa) cultivar. GM and non-genetically modified (NGM) plants of the same cultivar were compared under the two following water treatments: well watered (WW) and water deficit (WD). The performance of the agronomic traits of GM plants, including grain yield, was superior to that of NGM plants in both treatments. By quantitative polymerase chain reaction, GM plants show a significantly higher expression of the rbcL gene in both WW and WD, as well as a larger amount of abscisic acid. With the RNAseq analysis, almost three times more upregulated genes are identified in GM plants in stage 2 after water restriction, indicating a greater protection against water deficit. The higher expression of genes related to the protection of the cellular metabolism and a series of physiological alterations may be involved in the increase in the drought tolerance of GM rice plants overexpressing the rbcL gene

Tolerância à seca induzida pela superexpressão do gene nuclear rbcL em arroz

The objective of this work was to determine whether the overexpression of the nuclear Rubisco large subunit (rbcL) improves the drought tolerance of the genetically modified (GM) BRSMG Curinga upland rice (Oryza sativa) cultivar. GM and non-genetically modified (NGM) plants of the same cultivar were compared under the two following water treatments: well watered (WW) and water deficit (WD). The performance of the agronomic traits of GM plants, including grain yield, was superior to that of NGM plants in both treatments. By quantitative polymerase chain reaction, GM plants show a significantly higher expression of the rbcL gene in both WW and WD, as well as a larger amount of abscisic acid. With the RNAseq analysis, almost three times more upregulated genes are identified in GM plants in stage 2 after water restriction, indicating a greater protection against water deficit. The higher expression of genes related to the protection of the cellular metabolism and a series of physiological alterations may be involved in the increase in the drought tolerance of GM rice plants overexpressing the rbcL gene.O objetivo deste trabalho foi determinar se a superexpressão da grande subunidade nuclear da Rubisco (rbcL) aumenta a tolerância à seca da cultivar geneticamente modificada (GM) BRSMG Curinga de arroz (Oryza sativa) de terras altas. Plantas GMs e não geneticamente modificadas (NGMs) da mesma cultivar foram comparadas nos dois seguintes tratamentos de água: irrigação (WW) e déficit hídrico (WD). O desempenho dos caracteres agronômicos das plantas GMs, incluindo a produção de grãos, foi superior ao das NGMs em ambos os tratamentos. Por reação em cadeia da polimerase quantitativa, plantas GMs apresentam expressão significativamente maior do gene rbcL, tanto sob WW como sob WD, além de maior quantidade de ácido abscísico. Com a análise de RNAseq, quase três vezes mais genes regulados positivamente são identificados nas plantas GMs no estágio 2 após restrição hídrica, o que indica maior proteção contra o déficit hídrico. O aumento da expressão de genes relacionados à proteção do metabolismo celular e uma série de alterações fisiológicas podem estar envolvidos no aumento da tolerância à seca em plantas de arroz GMs que superexpressam o gene rbcL

Identification of stable quantitative trait loci for grain yield in rice

O objetivo deste trabalho foi identificar locos de caracteres quantitativos (QTLs) associados à produtividade em uma população segregante de arroz (GYP). Uma população de 245 linhagens puras recombinantes de arroz, do cruzamento 'Epagri 108' (Oryza sativa subsp. indica) x 'IRAT 122' (O. sativa subsp. japonica), foi avaliada em diferentes locais e anos e genotipada por marcadores de polimorfismo de nucleotídeo único (SNPs). Obteve‑se um mapa de 1.592,8 cM a partir de 9.831 SNPs, tendo-se identificado 25 QTLs. Os seguintes nove SNPs apresentaram estabilidade entre os diferentes ambientes: M1.37719614 e M6.9563117 para GYP; M4.29340056, M5.25588710, M7.29115624 e M12.4534450 para peso de 100 grãos (HGW); e M1.38398157, M4.28368337 e M7.25991230 para altura de plantas. Seis SNPs não estavam presentes nos blocos de ligação:  M6.9563117 e M4.1077080 para GYP; M5.25588710 e M6.8886398 para HGW; e M2.34471005 e M8.5955948 para altura de plantas. Os SNPs M6.9563117 e M5.25588710 foram considerados ambientalmente estáveis e não estiveram presentes em blocos de ligação, o que indica seu alto potencial para uso na seleção assistida por marcadores de produtividade de grãos, em programas brasileiros de melhoramento de arroz.The objective of this work was to identify the quantitative trait loci (QTLs) associated with grain yield in a rice segregant population (GYP). A population of 245 inbred recombinant rice lines from the 'Epagri 108' (Oryza sativa subsp. indica) x 'IRAT 122' (O. sativa subsp. japonica) cross was evaluated at different locations and years and genotyped by single nucletide polymorphism (SNP) markers. A map of 1,592.8 cM was obtained from 9,831 SNPs, identifying 25 QTLs. The following nine SNPs showed stability between the different environments: M1.37719614 and M6.9563117 for GYP; M4.29340056, M5.25588710, M7.29115624, and M12.4534450 for 100-grain weight (HGW); and M1.38398157, M4.28368337, and M7.25991230 for plant height (PH). Six SNPs were not present in the linkage blocks: M6.9563117 and M4.1077080 for GYP; M5.25588710 and M6.8886398 for HGW; and M2.34471005 and M8.5955948 for PH. The M6.9563117 and M5.25588710 SNPs were considered environmentally stable and were not present in the linkage blocks, showing their high potential for use in marker-assisted selection for grain yield in Brazilian rice breeding programs

Variabilidade genética de populações de seleção recorrente de arroz, influenciada por macho‑esterilidade ou recombinação manual

O objetivo deste trabalho foi determinar os efeitos da macho‑esterilidade ou da recombinação manual sobre a variabilidade genética de populações de seleção recorrente de arroz. Foram avaliadas as populações CNA‑IRAT 4, com gene de macho‑esterilidade, e CNA 12, recombinada manualmente. A variabilidade genética entre os ciclos de seleção foi estimada por 14 marcadores de sequências simples repetidas (SSR). Foram analisadas 926 plantas, incluindo dez genitores e 180 indivíduos de cada um dos ciclos avaliados (1, 2 e 5) da população CNA‑IRAT 4, e 16 genitores e 180 indivíduos de cada um dos ciclos (1 e 2) da CNA 12. A análise possibilitou a identificação de alelos não existentes nos genitores nas duas populações, em todos os ciclos, principalmente para a população CNA‑IRAT 4. Esses alelos foram resultantes da fecundação indesejada a partir de genótipos que não faziam parte das populações. Os parâmetros da estatística F de Wright (FIS e FIT) indicaram que a recombinação manual amplia a variabilidade genética da população CNA 12, enquanto a macho‑esterilidade reduz a de CNA‑IRAT 4.The objective of this work was to determine the effect of male sterility or manual recombination on genetic variability of rice recurrent selection populations. The populations CNA‑IRAT 4, with a gene for male sterility, and CNA 12, which was manually recombined, were evaluated. Genetic variability among selection cycles was estimated using14 simple sequence repeat (SSR) markers. A total of 926 plants were analyzed, including ten genitors and 180 individuals from each of the evaluated cycles (1, 2 and 5) of the population CNA‑IRAT 4, and 16 genitors and 180 individuals from each of the cycles (1 and 2) of CNA 12. The analysis allowed of the identification of alleles not present among the genitors for both populations, in all cycles, especially for the CNA‑IRAT 4 population. These alleles resulted from unwanted fertilization with genotypes that were not originally part of the populations. The parameters of Wright’s F‑statistic (FIS and FIT) indicated that the manual recombination expands the genetic variability of the CNA 12 population, whereas male sterility reduces the one of CNA‑IRAT 4

Efeitos fisiológicos e fenotípicos da superexpressão do gene OVP1 em arroz japonica

The objective of this work was to evaluate the physiological, phenotypic, and gene expression parameters in genetically modified (GM) rice plants that overexpress the Oryza sativa Vacuolar H+-Pyrophosphatase 1 (OVP1) gene, compared with non-genetically modified (NGM) rice. GM and NGM plants of the BRSMG Curinga cultivar were evaluated in two experiments, in a laboratory and greenhouse, in a randomized complete block design, with four replicates. Agronomic traits of interest were estimated, and transcriptome analysis and gene expression quantification were carried out. GM plants showed a 31 and 21% higher number of spikelets per panicle and total number of grains per panicle, respectively, in comparison with NGM plants. Physiological changes occurred during the grain-filling stage, in which GM plants presented a photosynthetic rate and carboxylation efficiency 61 and 89% higher than those of NGM plants, respectively. The overexpression of the OVP1 gene favors the upregulation of some photosynthesis genes and the increase in the number of spikelets and in the photosynthetic rate, but does not favor the increase in grain yield.O objetivo deste trabalho foi avaliar os parâmetros fisiológicos, fenotípicos e de expressão gênica em plantas de arroz geneticamente modificadas (GM) que superexpressam o gene Oryza sativa Vacuolar H+-Pyrophosphatase 1 (OVP1), em comparação ao arroz não geneticamente modificado (NGM). Plantas GM e NGM da cultivar BRSMG Curinga foram avaliadas em dois experimentos, em laboratório e casa de vegetação, no delineamento de blocos ao acaso, com quatro repetições. Foram estimados caracteres agronômicos de interesse, e realizadas análise de transcritoma e quantificação da expressão gênica. Plantas GM apresentaram número de espiguetas por panícula e número total de grãos por panícula 31 e 21% maiores, respectivamente, em comparação às plantas NGM. Ocorreram alterações fisiológicas durante a fase de enchimento de grãos, em que as plantas GM apresentaram taxa fotossintética e eficiência de carboxilação 61 e 89% mais altas do que as das plantas NGM, respectivamente. A superexpressão do gene OVP1 favorece a indução de alguns genes da fotossíntese e o aumento do número de espiguetas e da taxa fotossintética, mas não favorece o aumento da produtividade de grãos

Desenvolvimento de marcadores SNP para triagem de produtividade de grãos de cultivares brasileiras de arroz

The objective of this work was to identify and validate single-nucleotide polymorphism (SNP) markers related to grain yield in rice (Oryza sativa) core collection. The genome-wide association studies (GWAS) methodology was applied for genotyping of 541 rice accessions by 167,470 SNPs. The grain yield of these accessions was estimated through the joint analysis of nine field experiments carried out in six Brazilian states. Fifteen SNPs were significantly associated with grain yield, and out of the ten SNPs converted to TaqMan assays, four discriminated the most productive accessions. These markers were used for the screening of rice accessions with favorable alleles. The selected accessions were, then, evaluated in field experiments in target environments, in order to select the most productive ones. This screening reduces the number of accessions evaluated experimentally, making it possible to prioritize those with higher productive potential, which allows of the increase of the number of replicates and, consequently, of the experimental accuracy.O objetivo deste trabalho foi identificar e validar marcadores de polimorfismo de nucleotídeo único (SNP) relacionados à produtividade de grãos em coleção nuclear de arroz (Oryza sativa). A metodologia de estudos de associação genômica ampla (GWAS) foi aplicada à genotipagem de 541 genótipos por 167.470 SNPs. A produtividade de grãos desses acessos foi estimada por meio da análise conjunta de nove experimentos de campo, realizados em seis estados brasileiros. Quinze SNPs foram significativamente associados à produtividade de grãos e, dos dez SNPs que foram convertidos em ensaios TaqMan, quatro discriminaram os acessos com maior produtividade. Esses marcadores foram utilizados para identificar aceessos de arroz com os alelos favoráveis. Em seguida, os acessos selecionados foram avaliados em experimentos de campo, em ambientes-alvo, para identificar os mais produtivos. Essa triagem reduz o número de acessos avaliados experimentalmente, pois torna possível priorizar aqueles com maior potencial produtivo, o que permite aumentar o número de repetições e, consequentemente, a precisão experimental