Association between ACTA1 candidate gene and performance, organs and carcass traits in broilers

This study investigates the genetic association of the SNP present in the ACTA1 gene with performance traits, organs and carcass of broilers to help marker-assisted selection of a paternal broiler line (TT) from EMBRAPA Swine and Poultry, Brazil. Genetic and phenotypic data of 1,400 broilers for 68 traits related to body performance, organ weights, weight of carcass parts, and yields as a percentage of organs and carcass parts were used. The maximum likelihood method, considering 4 analytical models, was used to analyze the genetic association between the SNP and these important economic traits. The association analysis was performed using a mixed animal model including the random effect of the animal (polygenic), and the fixed effects of sex (2 levels), hatch (5 levels) and SNP (3 levels), besides the random error. The traits significantly associated (P < 0.05) with the SNP were analyzed, along with body weight at 42 days of age (BW42), by the restricted maximum likelihood method using the multi-trait animal model to estimate genetic parameters. The analysis included the residual and additive genetic random effects and the sex-hatch fixed effect. The additive effects of the SNP were associated with breast meat (BMY), liver yield (LIVY), body weight at 35 days of age (BW35); drumstick skin (DSW), drumstick (DW) and breast (BW) weights. The heritability estimates for these traits, in addition to BW42, ranged from 0.24 ± 0.06 to 0.45 ± 0.08 for LIVY and BW35, respectively. The genetic correlation ranged from 0.02 ± 0.18 for LIVY and BMY to 0.97 ± 0.01 for BW35 and BW42. Based on the results of this study, it can be concluded that ACTA1 gene is associated with performance traits BW35, LIV and BMY, DW, BW and DW adjusted for body weight at 42 days of age. Therefore, the ACTA1 gene is an important molecular marker that could be used together with others already described to increase the economically important traits in broilers.

A mixed model QTL analysis for sugarcane multiple-harvest-location trial data

Sugarcane-breeding programs take at least 12 years to develop new commercial cultivars. Molecular markers offer a possibility to study the genetic architecture of quantitative traits in sugarcane, and they may be used in marker-assisted selection to speed up artificial selection. Although the performance of sugarcane progenies in breeding programs are commonly evaluated across a range of locations and harvest years, many of the QTL detection methods ignore two- and three-way interactions between QTL, harvest, and location. In this work, a strategy for QTL detection in multi-harvest-location trial data, based on interval mapping and mixed models, is proposed and applied to map QTL effects on a segregating progeny from a biparental cross of pre-commercial Brazilian cultivars, evaluated at two locations and three consecutive harvest years for cane yield (tonnes per hectare), sugar yield (tonnes per hectare), fiber percent, and sucrose content. In the mixed model, we have included appropriate (co)variance structures for modeling heterogeneity and correlation of genetic effects and non-genetic residual effects. Forty-six QTLs were found: 13 QTLs for cane yield, 14 for sugar yield, 11 for fiber percent, and 8 for sucrose content. In addition, QTL by harvest, QTL by location, and QTL by harvest by location interaction effects were significant for all evaluated traits (30 QTLs showed some interaction, and 16 none). Our results contribute to a better understanding of the genetic architecture of complex traits related to biomass production and sucrose content in sugarcane.

Association of single nucleotide polymorphisms in the bovine leptin and leptin receptor genes with growth and ultrasound carcass traits in Nellore cattle

Given the important role of leptin in metabolism, we looked for a possible association of leptin and leptin receptor polymorphisms with carcass and growth traits in Nellore cattle. We examined associations of leptin and leptin receptor SNPs with ultrasound carcass (longissimus dorsi muscle area (ribeye area), backfat thickness and rump fat thickness and growth traits (weaning weight adjusted to 210 days of age, yearling weight adjusted to 550 days of age, weight gain of weaning to yearling and scrotal circumference adjusted to 550 days of age) of 2162 Bos primigenius indicus (Nellore) animals. Allele and genotypic frequencies were calculated for each marker. Allele substitution, additive and dominance effects of the polymorphisms were also evaluated. Some alleles of the molecular markers had low frequencies, lower than 1%, in the sample analyzed, although the same polymorphisms described for B. p. taurus cattle were found. Due to very low allelic frequencies, the E2JW, A59V and UASMS2 markers were not included in the analysis, because they were almost fixed. E2FB was found to be significantly associated with weight gain, ribeye area and backfat thickness. The promoter region markers, C963T and UASMS1, were also found to be significantly associated with ribeye area. T945M was significantly associated with weight gain. We conclude that the leptin and receptor gene markers would be useful for marker-assisted selection.

Genetic analysis of kernel oil content in tropical maize with design III and QTL mapping

Oil content and grain yield in maize are negatively correlated, and so far the development of high-oil high-yielding hybrids has not been accomplished. Then a fully understand of the inheritance of the kernel oil content is necessary to implement a breeding program to improve both traits simultaneously. Conventional and molecular marker analyses of the design III were carried out from a reference population developed from two tropical inbred lines divergent for kernel oil content. The results showed that additive variance was quite larger than the dominance variance, and the heritability coefficient was very high. Sixteen QTL were mapped, they were not evenly distributed along the chromosomes, and accounted for 30.91% of the genetic variance. The average level of dominance computed from both conventional and QTL analysis was partial dominance. The overall results indicated that the additive effects were more important than the dominance effects, the latter were not unidirectional and then heterosis could not be exploited in crosses. Most of the favorable alleles of the QTL were in the high-oil parental inbred, which could be transferred to other inbreds via marker-assisted backcross selection. Our results coupled with reported information indicated that the development of high-oil hybrids with acceptable yields could be accomplished by using marker-assisted selection involving oil content, grain yield and its components. Finally, to exploit the xenia effect to increase even more the oil content, these hybrids should be used in the Top Cross((TM)) procedure.

MUC1 gene polymorphism in three Nelore lines selected for growth and its association with growth and carcass traits

The objective of this study was to describe the VNTR polymorphism of the mucin 1 gene (MUC1) in three Nelore lines selected for yearling weight to determine whether allele and genotype frequencies of this polymorphism were affected by selection for growth. In addition, the effects of the polymorphism on growth and carcass traits were evaluated. Birth, weaning and yearling weights, rump height, Longissimus muscle area, backfat thickness, and rump fat thickness, were analyzed. A total of 295 Nelore heifers from the Beef Cattle Research Center, Instituto de Zootecnia de Sertozinho, were used, including 41 of the control line, 102 of the selection line and 152 of the traditional. The selection and traditional lines comprise animals selected for higher yearling weight, whereas control line animals are selected for yearling weight close to the average. Five alleles were identified, with allele 1 being the most frequent in the three lines, especially in the lines selected for higher means for yearling weight. Heterozygosity was significantly higher in the control line. Association analyses showed significant effects of allele 1 on birth weight and weaning weight while the allele 3 exert significant effects on yearling weight and back fat thickness. Despite these findings, application of this marker to marker-assisted selection requires more consistent results based on the genotyping of a larger number of animals in order to increase the accuracy of the statistical analyses.

Functional markers for gene mapping and genetic diversity studies in sugarcane

Abstract Background The database of sugarcane expressed sequence tags (EST) offers a great opportunity for developing molecular markers that are directly associated with important agronomic traits. The development of new EST-SSR markers represents an important tool for genetic analysis. In sugarcane breeding programs, functional markers can be used to accelerate the process and select important agronomic traits, especially in the mapping of quantitative traits loci (QTL) and plant resistant pathogens or qualitative resistance loci (QRL). The aim of this work was to develop new simple sequence repeat (SSR) markers in sugarcane using the sugarcane expressed sequence tag (SUCEST database). Findings A total of 365 EST-SSR molecular markers with trinucleotide motifs were developed and evaluated in a collection of 18 genotypes of sugarcane (15 varieties and 3 species). In total, 287 of the EST-SSRs markers amplified fragments of the expected size and were polymorphic in the analyzed sugarcane varieties. The number of alleles ranged from 2-18, with an average of 6 alleles per locus, while polymorphism information content values ranged from 0.21-0.92, with an average of 0.69. The discrimination power was high for the majority of the EST-SSRs, with an average value of 0.80. Among the markers characterized in this study some have particular interest, those that are related to bacterial defense responses, generation of precursor metabolites and energy and those involved in carbohydrate metabolic process. Conclusions These EST-SSR markers presented in this work can be efficiently used for genetic mapping studies of segregating sugarcane populations. The high Polymorphism Information Content (PIC) and Discriminant Power (DP) presented facilitate the QTL identification and marker-assisted selection due the association with functional regions of the genome became an important tool for the sugarcane breeding program.

Identificazione di un QTL principale per resistenza a ruggine bruna sul cromosoma 7B di frumento duro

Leaf rust caused by Puccinia triticina is a serious disease of durum wheat (Triticum durum) worldwide. However, genetic and molecular mapping studies aimed at characterizing leaf rust resistance genes in durum wheat have been only recently undertaken. The Italian durum wheat cv. Creso shows a high level of resistance to P. triticina that has been considered durable and that appears to be due to a combination of a single dominant gene and one or more additional factors conferring partial resistance. In this study, the genetic basis of leaf rust resistance carried by Creso was investigated using 176 recombinant inbred lines (RILs) from the cross between the cv. Colosseo (C, leaf rust resistance donor) and Lloyd (L, susceptible parent). Colosseo is a cv. directly related to Creso with the leaf rust resistance phenotype inherited from Creso, and was considered as resistance donor because of its better adaptation to local (Emilia Romagna, Italy) cultivation environment. RILs have been artificially inoculated with a mixture of 16 Italian P. triticina isolates that were characterized for virulence to seedlings of 22 common wheat cv. Thatcher isolines each carrying a different leaf rust resistance gene, and for molecular genotypes at 15 simple sequence repeat (SSR) loci, in order to determine their specialization with regard to the host species. The characterization of the leaf rust isolates was conducted at the Cereal Disease Laboratory of the University of Minnesota (St. Paul, USA) (Chapter 2). A genetic linkage map was constructed using segregation data from the population of 176 RILs from the cross CL. A total of 662 loci, including 162 simple sequence repeats (SSRs) and 500 Diversity Arrays Technology markers (DArTs), were analyzed by means of the package EasyMap 0.1. The integrated SSR-DArT linkage map consisted of 554 loci (162 SSR and 392 DArT markers) grouped into 19 linkage blocks with an average marker density of 5.7 cM/marker. The final map spanned a total of 2022 cM, which correspond to a tetraploid genome (AABB) coverage of ca. 77% (Chapter 3). The RIL population was phenotyped for their resistance to leaf rust under artificial inoculation in 2006; the percentage of infected leaf area (LRS, leaf rust susceptibility) was evaluated at three stages through the disease developmental cycle and the area under disease progress curve (AUDPC) was then calculated. The response at the seedling stage (infection type, IT) was also investigated. QTL analysis was carried out by means of the Composite Interval Mapping method based on a selection of markers from the CL map. A major QTL (QLr.ubo-7B.2) for leaf rust resistance controlling both the seedling and the adult plant response, was mapped on the distal region of chromosome arm 7BL (deletion bin 7BL10-0.78-1.00), in a gene-dense region known to carry several genes/QTLs for resistance to rusts and other major cereal fungal diseases in wheat and barley. QLr.ubo-7B.2 was identified within a supporting interval of ca. 5 cM tightly associated with three SSR markers (Xbarc340.2, Xgwm146 e Xgwm344.2), and showed an R2 and an LOD peak value for the AUDPC equal to 72.9% an 44.5, respectively. Three additional minor QTLs were also detected (QLr.ubo-7B.1 on chr. 7BS; QLr.ubo-2A on chr. 2AL and QLr.ubo-3A on chr. 3AS) (Chapter 4). The presence of the major QTL (QLr.ubo-7B.2) was validated by a linkage disequilibrium (LD)-based test using field data from two different plant materials: i) a set of 62 advanced lines from multiple crosses involving Creso and his directly related resistance derivates Colosseo and Plinio, and ii) a panel of 164 elite durum wheat accessions representative of the major durum breeding program of the Mediterranean basin. Lines and accessions were phenotyped for leaf rust resistance under artificial inoculation in two different field trials carried out at Argelato (BO, Italy) in 2006 and 2007; the durum elite accessions were also evaluated in two additional field experiments in Obregon (Messico; 2007 and 2008) and in a green-house experiment (seedling resistance) at the Cereal Disease Laboratory (St. Paul, USA, 2008). The molecular characterization involved 14 SSR markers mapping on the 7BL chromosome region found to harbour the major QTL. Association analysis was then performed with a mixed-linear-model approach. Results confirmed the presence of a major QTL for leaf rust resistance, both at adult plant and at seedling stage, located between markers Xbarc340.2, Xgwm146 and Xgwm344.2, in an interval that coincides with the supporting interval (LOD-2) of QLr.ubo-7B.2 as resulted from the RIL QTL analysis. (Chapter 5). The identification and mapping of the major QTL associated to the durable leaf rust resistance carried by Creso, together with the identification of the associated SSR markers, will enhance the selection efficiency in durum wheat breeding programs (MAS, Marker Assisted Selection) and will accelerate the release of cvs. with durable resistance through marker-assisted pyramiding of the tagged resistance genes/QTLs most effective against wheat fungal pathogens.

Different approaches to identify markers for meat quality and production traits in pigs: analysis of the transcriptome in post mortem skeletal muscle and investigation of candidate genes

Due to the growing attention of consumers towards their food, improvement of quality of animal products has become one of the main focus of research. To this aim, the application of modern molecular genetics approaches has been proved extremely useful and effective. This innovative drive includes all livestock species productions, including pork. The Italian pig breeding industry is unique because needs heavy pigs slaughtered at about 160 kg for the production of high quality processed products. For this reason, it requires precise meat quality and carcass characteristics. Two aspects have been considered in this thesis: the application of the transcriptome analysis in post mortem pig muscles as a possible method to evaluate meat quality parameters related to the pre mortem status of the animals, including health, nutrition, welfare, and with potential applications for product traceability (chapters 3 and 4); the study of candidate genes for obesity related traits in order to identify markers associated with fatness in pigs that could be applied to improve carcass quality (chapters 5, 6, and 7). Chapter three addresses the first issue from a methodological point of view. When we considered this issue, it was not obvious that post mortem skeletal muscle could be useful for transcriptomic analysis. Therefore we demonstrated that the quality of RNA extracted from skeletal muscle of pigs sampled at different post mortem intervals (20 minutes, 2 hours, 6 hours, and 24 hours) is good for downstream applications. Degradation occurred starting from 48 h post mortem even if at this time it is still possible to use some RNA products. In the fourth chapter, in order to demonstrate the potential use of RNA obtained up to 24 hours post mortem, we present the results of RNA analysis with the Affymetrix microarray platform that made it possible to assess the level of expression of more of 24000 mRNAs. We did not identify any significant differences between the different post mortem times suggesting that this technique could be applied to retrieve information coming from the transcriptome of skeletal muscle samples not collected just after slaughtering. This study represents the first contribution of this kind applied to pork. In the fifth chapter, we investigated as candidate for fat deposition the TBC1D1 [TBC1 (tre-2/USP6, BUB2, cdc16) gene. This gene is involved in mechanisms regulating energy homeostasis in skeletal muscle and is associated with predisposition to obesity in humans. By resequencing a fragment of the TBC1D1 gene we identified three synonymous mutations localized in exon 2 (g.40A>G, g.151C>T, and g.172T>C) and 2 polymorphisms localized in intron 2 (g.219G>A and g.252G>A). One of these polymorphisms (g.219G>A) was genotyped by high resolution melting (HRM) analysis and PCR-RFLP. Moreover, this gene sequence was mapped by radiation hybrid analysis on porcine chromosome 8. The association study was conducted in 756 performance tested pigs of Italian Large White and Italian Duroc breeds. Significant results were obtained for lean meat content, back fat thickness, visible intermuscular fat and ham weight. In chapter six, a second candidate gene (tribbles homolog 3, TRIB3) is analyzed in a study of association with carcass and meat quality traits. The TRIB3 gene is involved in energy metabolism of skeletal muscle and plays a role as suppressor of adipocyte differentiation. We identified two polymorphisms in the first coding exon of the porcine TRIB3 gene, one is a synonymous SNP (c.132T> C), a second is a missense mutation (c.146C> T, p.P49L). The two polymorphisms appear to be in complete linkage disequilibrium between and within breeds. The in silico analysis of the p.P49L substitution suggests that it might have a functional effect. The association study in about 650 pigs indicates that this marker is associated with back fat thickness in Italian Large White and Italian Duroc breeds in two different experimental designs. This polymorphisms is also associated with lactate content of muscle semimembranosus in Italian Large White pigs. Expression analysis indicated that this gene is transcribed in skeletal muscle and adipose tissue as well as in other tissues. In the seventh chapter, we reported the genotyping results for of 677 SNPs in extreme divergent groups of pigs chosen according to the extreme estimated breeding values for back fat thickness. SNPs were identified by resequencing, literature mining and in silico database mining. analysis, data reported in the literature of 60 candidates genes for obesity. Genotyping was carried out using the GoldenGate (Illumina) platform. Of the analyzed SNPs more that 300 were polymorphic in the genotyped population and had minor allele frequency (MAF) >0.05. Of these SNPs, 65 were associated (P<0.10) with back fat thickness. One of the most significant gene marker was the same TBC1D1 SNPs reported in chapter 5, confirming the role of this gene in fat deposition in pig. These results could be important to better define the pig as a model for human obesity other than for marker assisted selection to improve carcass characteristics.

Genetische Kartierung und QTL-Analyse agronomischer Eigenschaften der Weinrebe unter besonderer Berücksichtigung der Pilzresistenz

153 Nachkommen einer Kreuzung aus der pilzresistenten Rebsorte ‘Regent‘ und ‘Lemberger‘ als klassischer pilzsensitiver Sorte zeigen quantitative Merkmalsvariation bezüglich der Resistenz gegen Plasmopara viticola und Uncinula necator sowie für weitere Eigenschaften, die z.B. das Eintreten der Beerenreife betreffen. Auf dem Weg über die genetische Kartierung mit molekularen Markern und der Lokalisierung von QTL-Effekten konnten Hinweise auf weinbaulich relevante Genomregionen gewonnen werden; dies liefert z.B. die Basis für markergestützte Selektion bei Zuchtvorhaben mit dem Resistenzträger ‘Regent’ (vgl. auch FISCHER et al., 2004). Ein Major-QTL für die Resistenz gegen den Echten Mehltau Uncinula necator sowie zwei Major QTL für die Resistenz gegen den Erreger des Falschen Mehltau, Plasmopara viticola, traten mit hoher Signifikanz auf drei verschiedenen Kopplungsgruppen von ‘Regent‘ auf. Auch Regionen mit Relevanz für das Eintreten der Beerenreife wurden beschrieben. Über die Isolierung, Sequenzierung und anschließende Analyse einzelner Markerfragmente mit Methoden der Bioinformatik ist es gelungen, ein putatives T10P12.4-Ortholog der Weinrebe (ein thioredoxinähnliches Protein) in enger Kopplung zu einem Major-QTL-Maximum für Plasmopara viticola-Resistenz zu identifizieren, das als Kandidat für die Beteiligung an der Pathogenantwort in Frage kommt. Es konnte exemplarisch gezeigt werden, dass die eingesetzten Methoden der Kartierung und QTL-Analyse unter Verwendung PCR-basierter Markertypen wie SSR und AFLP und einer beschleunigten Analyse über computergestützte Kapillargelelektrophorese in vertretbarem Zeitrahmen bis zur Isolation potentieller Schlüsselgene führen können. Die grundsätzliche Eignung der QTL-Analyse als effizientes Werkzeug gezielter Züchtungsplanung für den Weinbau bestätigte sich. Ihre Anwendung im Rahmen der vorliegenden Dissertation hat die Basis für die Nutzung von QTL-Information bei dem Vergleich etablierter und der Entwicklung neuer Sorten gelegt und zum Verständnis von Prozessen beigetragen, die den betrachteten Eigenschaften wie der Pilzresistenz möglicherweise zu Grunde liegen. Ein großer Teil der gewonnenen Daten bringt auch die Untersuchungen anderer Kultivare voran und ist intervarietal übertragbar. Darüber hinaus haben sich Chancen für vergleichende Studien zwischen der Weinrebe einerseits und der Modellpflanze Arabidopsis thaliana sowie weiteren Kulturpflanzen andererseits abgezeichnet. Die Hinweise auf die zentrale Rolle und universelle Natur des Redox-Signalling haben interessante Perspektiven zum Verständnis organismenübergreifender physiologischer Zusammenhänge eröffnet. Dies betrifft z.B. auch die Reaktion auf Verwundung oder die Pathogenantwort.

Comparative expression profiling of E. coli and S. aureus inoculated primary mammary gland cells sampled from cows with different genetic predispositions for somatic cell score

BACKGROUND: During the past ten years many quantitative trait loci (QTL) affecting mastitis incidence and mastitis related traits like somatic cell score (SCS) were identified in cattle. However, little is known about the molecular architecture of QTL affecting mastitis susceptibility and the underlying physiological mechanisms and genes causing mastitis susceptibility. Here, a genome-wide expression analysis was conducted to analyze molecular mechanisms of mastitis susceptibility that are affected by a specific QTL for SCS on Bos taurus autosome 18 (BTA18). Thereby, some first insights were sought into the genetically determined mechanisms of mammary gland epithelial cells influencing the course of infection. METHODS: Primary bovine mammary gland epithelial cells (pbMEC) were sampled from the udder parenchyma of cows selected for high and low mastitis susceptibility by applying a marker-assisted selection strategy considering QTL and molecular marker information of a confirmed QTL for SCS in the telomeric region of BTA18. The cells were cultured and subsequently inoculated with heat-inactivated mastitis pathogens Escherichia coli and Staphylococcus aureus, respectively. After 1, 6 and 24 h, the cells were harvested and analyzed using the microarray expression chip technology to identify differences in mRNA expression profiles attributed to genetic predisposition, inoculation and cell culture. RESULTS: Comparative analysis of co-expression profiles clearly showed a faster and stronger response after pathogen challenge in pbMEC from less susceptible animals that inherited the favorable QTL allele 'Q' than in pbMEC from more susceptible animals that inherited the unfavorable QTL allele 'q'. Furthermore, the results highlighted RELB as a functional and positional candidate gene and related non-canonical Nf-kappaB signaling as a functional mechanism affected by the QTL. However, in both groups, inoculation resulted in up-regulation of genes associated with the Ingenuity pathways 'dendritic cell maturation' and 'acute phase response signaling', whereas cell culture affected biological processes involved in 'cellular development'. CONCLUSIONS: The results indicate that the complex expression profiling of pathogen challenged pbMEC sampled from cows inheriting alternative QTL alleles is suitable to study genetically determined molecular mechanisms of mastitis susceptibility in mammary epithelial cells in vitro and to highlight the most likely functional pathways and candidate genes underlying the QTL effect.

The genomic Echinococcus microsatellite EmsB sequences: from a molecular marker to the epidemiological tool

In the field of molecular and epidemiological parasitology, characterization of fast evolving genetic markers appears as an important challenge to consider the diversity and genetic structure of parasites. The study of respective populations can help us to understand their adaptive strategies to survive and perpetuate the species within different host populations, all trying to resist infection. In the past, the relative monomorphic features of Echinococcus multilocularis, the causative agent of alveolar echinococcosis and a severe human parasitic disease, did not stimulate studies dealing with the genetic variability of Echinococcus species or respective populations. A recently developed, characterized and validated original multilocus microsatellite, named EmsB, tandemly repeated in the genome, offered an additional opportunity for this line of investigation. We have compiled in this review new insights brought by this molecular tracker on the transmission activity of Echinococcus among different hosts and at different geographical scales.

Future trends in Animal Breeding due to new genetic tecnologies

The Darwin theory of evolution by natural selection is based on three principles: (a) variation; (b) inheritance; and (c) natural selection. Here, I take these principles as an excuse to review some topics related to the future research prospects in Animal Breeding. With respect to the first principle I describe two forms of variation different from mutation that are becoming increasingly important: variation in copy number and microRNAs. With respect to the second principle I comment on the possible relevance of non-mendelian inheritance, the so-called epigenetic effects, of which the genomic imprinting is the best characterized in domestic species. Regarding selection principle I emphasize the importance of selection for social traits and how this could contribute to both productivity and animal welfare. Finally, I analyse the impact of molecular biology in Animal Breeding, the achievements and limitations of quantitative trait locus and classical marker-assisted selection and the future of genomic selection

Artificial analysis of molecular marker loci linked to tree resistance response by an artificial neural network

One of the biggest challenges that software developers face is to make an accurate estimate of the project effort. Radial basis function neural networks have been used to software effort estimation in this work using NASA dataset. This paper evaluates and compares radial basis function versus a regression model. The results show that radial basis function neural network have obtained less Mean Square Error than the regression method.

Cartografía de QTL asociados a la tolerancia a estrés hídrico en frijol común (Phaseolus vulgaris L.)

Phaseolus vulgaris L. (frijol común o judía) es una leguminosa de gran demanda para la nutrición humana y un producto agrícola muy importante. Sin embargo, la producción de frijol se ve limitada por presiones ambientales como la sequía. En México, el 85% de la cosecha de frijol se produce en la temporada de primavera-verano, principalmente en las regiones del altiplano semiárido con una precipitación anual entre 250 y 400 mm. A pesar del implemento de tecnología en el campo, los factores naturales impiden al agricultor llegar a los rendimientos deseados. El Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias (INIFAP), como instituto de investigación gubernamental en México, tiene como objetivo la mejora de cultivos estratégicos, uno de ellos, P. vulgaris. Los estudios en relación a la sequía se enfocan especialmente en la selección de genotipos tolerantes, los cuales son sometidos en condiciones de estrés y monitoreando parámetros como el rendimiento y peso de semilla, además de algunos indicadores tales como índice de cosecha. El resultado de estos trabajos ha sido la obtención de variedades con mayor tolerancia a la sequía, tales como Pinto Villa y Pinto Saltillo. En los últimos años se ha avanzado notablemente en el conocimiento de las bases moleculares en las respuestas de las plantas al estrés. De acuerdo a diversos estudios se ha demostrado que las plantas bajo estrés por sequía experimentan cambios en la expresión de genes involucrados en la señalización, regulación de la transcripción y la traducción, transporte de agua y la función directa en la protección celular. También se ha observado que el déficit de agua es causado por las temperaturas extremas y la alta concentración de sales, por lo que al nivel molecular, las respuestas al estrés tienen puntos de especificidad y puntos de entrecruzamiento. La sequía puede generar estreses secundarios, tales como el nutricional, oxidativo y osmótico. Sin embargo, es necesario identificar y caracterizar muchos de los componentes involucrados en las respuestas al déficit hídrico, la caracterización de estos genes permitirá tener una mejor comprensión de los mecanismos bioquímicos y fisiológicos involucrados en la tolerancia al estrés. Actualmente, con el apoyo de la biología molecular se han identificado algunos genes que otorgan ventajas para la adaptación a ambientes desfavorables. Por lo que el objetivo del presente trabajo es identificar marcadores genéticos asociados a rasgos fenotípicos con énfasis a la tolerancia a estrés hídrico en P. vulgaris. Una vez establecidos los marcadores asociados al estrés hídrico, es factible considerar su uso para la selección asistida por marcadores en líneas o variedades de frijol de interés para los mejoradores. Se evaluaron 282 familias F3:5 derivadas de la cruza entre los cultivares Pinto Villa y Pinto Saltillo. Las familias se sembraron bajo un diseño simple de látice 17x17, el experimento se llevo acabo en el ciclo primavera-verano del 2010 y 2011, y otoñoinvierno de 2010 en el Campo Experimental Bajío del INIFAP con dos repeticiones para cada tratamiento de humedad (riego completo y sequía terminal). En todos los genotipos se realizó el fenotipado (variables fenotípicas) y el genotipado a través de marcadores moleculares. Los análisis estadísticos se basaron en el análisis de componentes principales (Eigen Analysis Selection Index Method, ESIM), la asociación entre marcadores SNP y el fenotipado (paquete SNPassoc para R) y el análisis de varianza (ANOVA). Los valores ESIM mostraron que las variables de Rendimiento, Días a floración, Días a madurez fisiológica e Índice de cosecha fueron sobresalientes en sequía terminal, por lo que se sugieren tomarse en consideración para los estudios de sequía en P. vulgaris como monitores de evaluación a la resistencia. Se identificaron nueve familias sobresalieron por sus valores ESIM (PV/PS6, 22, 131, 137, 149, 154, 201, 236 y 273), además de presentar valores superiores para el rendimiento en comparación con los parentales. Estos genotipos son candidatos interesantes para realizar estudios de identificación de loci asociados con la respuesta al estrés, y como potenciales parentales en el desarrollo de nuevas variedades de frijol. En los análisis de asociación SNPassoc se identificaron 83 SNPs significativos (p<0,0003) asociados a los rasgos fenotípicos, obteniendo un total de 222 asociaciones, de las cuales predomina el modelo genético de codominancia para las variables Días a floración, Periodo reproductivo y Biomasa total. Treinta y siete SNPs se identificaron a diferentes funciones biológicas a través del análisis de anotación funcional, de los cuales 12 SNPs (9, 18, 28, 39, 61, 69, 80, 106, 115, 128, 136 y 142) sobresalen por su asociación al fenotipado, y cuya anotación funcional indica que se encuentran en genes relacionados a la tolerancia a la sequía, tales como la actividad kinasa, actividad metabólica del almidón, carbohidratos y prolina, respuesta al estrés oxidativo, así como en los genes LEA y posibles factores de transcripción. En el caso de los análisis ANOVA, se identificaron 72 asociaciones entre los SNPs y las variables fenotípicas (F< 3,94E-04). Las 72 asociaciones corresponden a 30 SNPs y 7 variables fenotípicas, de las que predomina Peso de 100 semillas y Periodo reproductivo. Para los rasgos de Rendimiento, Índice de cosecha y Días a madurez fisiológica se presentaron asociaciones con seis SNPs (17, 34, 37, 50, 93 y 107), de los cuales, a los SNP37 y SNP107 fueron identificados a la anotación biológica de protein binding. Por otro lado, los SNP106 y SNP128 asociados al Periodo reproductivo, son genes con actividad kinasa y actividad metabólica del almidón, respectivamente. Para los marcadores tipo AFLP, se identificaron 271 asociaciones (F<2,34E-04). Las asociaciones corresponden a 86 AFLPs con todas las variables fenotípicas evaluadas, de las que predomina peso de 100 semillas, Días a floración y Periodo reproductivo. Debido a que los en los AFLPs no es posible determinar su anotación biológica, se proponen como marcadores potenciales relacionados a la resistencia a la sequía en frijol. Los AFLPs candidatos requieren más estudios tales como la secuenciación de los alelos respectivos, así como la identificación de éstas secuencias en el genoma de referencia y su anotación biológica, entre otros análisis, de esta manera podríamos establecer aquellos marcadores candidatos a la validación para la selección asistida. El presente trabajo propone tanto genotipos como marcadores genéticos, que deben ser validados para ser utilizados en el programa de mejoramiento de P. vulgaris, con el objetivo de desarrollar nuevas líneas o variedades tolerantes a la sequía. ABSTRACT Phaseolus vulgaris L. (common bean or judia) is a legume of great demand for human consumption and an important agricultural product. However, the common bean production is limited by environmental stresses, such as drought. In Mexico, 85% of the common bean crop is produced in the spring-summer season mainly in semiarid highland regions with a rainfall between 250 and 400 mm per year. In spite of the improvement of crop technology, the natural factors hamper getting an optimal yield. The National Institute for Forestry, Agriculture and Livestock (INIFAP) is a government research institute from Mexico, whose main objective is the genetic breeding of strategic crops, like P. vulgaris L. The drought tolerance studies particularly focus on the selection of bean tolerant genotypes, which are subjected to stress conditions, by means of monitoring parameters such as yield and seed weight, plus some agronomic indicators such as harvest index. The results of these works have led to obtain cultivars with higher drought tolerance such as Pinto Villa and Pinto Saltillo. Significant achievements have been recently made in understanding the molecular basis of stress plant responses. Several studies have shown that plants under drought stress present changes in gene expression related to cell signalling, transcriptional and translational regulation, water transport and cell protection. In addition, it has been observed that the extreme temperatures and high salt concentrations can cause a water deficiency so, at the molecular level, stress responses have specific and crossover points. The drought can cause secondary stresses, such as nutritional, oxidative and osmotic stress. It is required the identification of more components involved in the response to water deficit, the characterization of these genes will allow a better understanding of the biochemical and physiological mechanisms involved in stress tolerance. Currently, with the support of molecular biology techniques, some genes that confer an advantage for the crop adaptation to unfavourable environments have been identified. The objective of this study is to identify genetic markers associated with phenotypic traits with emphasis on water stress tolerance in P. vulgaris. The establishment of molecular markers linked to drought tolerance would make possible their use for marker-assisted selection in bean breeding programs. Two hundred and eighty two F3:5 families derived from a cross between the drought resistant cultivars Pinto Villa and Pinto Saltillo were evaluated. The families were sowed under a 17x17 simple lattice design. The experiment was conducted between spring-summer seasons in 2010 and 2011, and autumn-winter seasons in 2010 at the Bajio Experimental Station of INIFAP with two treatments (full irrigation and terminal drought). All families were phenotyped and genotyped using molecular markers. Statistical analysis was based on principal component analysis (Eigen Analysis Selection Index Method, ESIM), association analysis between SNP markers and phenotype (SNPassoc package R) and analysis of variance (ANOVA). The ESIM values showed that seed yield, days to flowering, days to physiological maturity and harvest index were outstanding traits in terminal drought treatment, so they could be considered as suitable parameters for drought-tolerance evaluation in P. vulgaris. Nine outstanding families for the ESIM values were identified (PV/PS6, 22, 131, 137, 149, 154, 201, 236 and 273), in addition, these families showed higher values for seed yield compared to the parental cultivars. These families are promising candidates for studies focused on the identification of loci associated to the stress response, and as potential parental cultivars for the development of new varieties of common bean. In the SNPassoc analysis, 83 SNPs were found significantly associated (p<0.0003) with phenotypic traits, obtaining a total of 222 associations, most of which involved the traits days to flowering, reproductive period and total biomass under a codominant genetic model. The functional annotation analysis showed 37 SNPs with different biological functions, 12 of them (9, 18, 28, 39, 61, 69, 80, 106, 115, 128, 136 and 142) stand out by their association to phenotype. The functional annotation suggested a connection with genes related to drought tolerance, such as kinase activity, starch, carbohydrates and proline metabolic processes, responses to oxidative stress, as well as LEA genes and putative transcription factors. In the ANOVA analysis, 72 associations between SNPs and phenotypic traits (F<3.94E- 04) were identified. All of these associations corresponded to 30 SNPs markers and seven phenotypic traits. Weight of 100 seeds and reproductive period were the traits with more associations. Seed yield, harvest index and days to physiological maturity were associated to six SNPs (17, 34, 37, 50, 93 and 107), the SNP37 and SNP107 were identified as located in protein binding genes. The SNP106 and SNP128 were associated with the reproductive period and belonged to genes with kinase activity and genes related to starch metabolic process, respectively. In the case of AFLP markers, 271 associations (F<2.34E-04) were identified. The associations involved 86 AFLPs and all phenotypic traits, being the most frequently associated weight of 100 seeds, days to flowering and reproductive period. Even though it is not possible to perform a functional annotation for AFLP markers, they are proposed as potential markers related to drought resistance in common bean. AFLPs candidates require additional studies such as the sequencing of the respective alleles, identification of these sequences in the reference genome and gene annotation, before their use in marker assisted selection. This work, although requires further validation, proposes both genotypes and genetic markers that could be used in breeding programs of P. vulgaris in order to develop new lines or cultivars with enhanced drought-tolerance.