Dissecting the Heat-Stress Tolerance QTLome in Durum Wheat

Heat stress negatively affects wheat performance during its entire cycle, particularly during the reproductive stage. In view of the climate change and the prediction of a continued increase in temperature in the new future, it is urgent to concentrate efforts to discover novel genetic sources able to improve the resilience of wheat to heat stress. In this direction, this study addressed two different experiments in durum wheat to identify novel QTLs suitable to be applied in marker-assisted selection for heat tolerance. Chlorophyll fluorescence (ChlF) is a valuable indicator of plant response to environmental changes allowing a detailed assessment of PSII activity in view of its non-invasive measurement and high-throughput phenotyping. In the first study (Chapter 2), the Light-Induced Fluorescence Transient (LIFT) method was used to access ChlF data to map QTLs for ChlF-related traits during the vegetative growth stage in durum wheat under heat stress condition. Our results provide evidence that LIFT consistently measures ChlF at the level of high-throughput phenotyping combined with high accuracy which is required for Genome-Wide Association Study (GWAS) aimed at identifying genomic regions affecting PSII activity. The 50 QTLs identified for ChlF-related traits under heat stress mostly clustered into five chromosomes hotspots unrelated to phenology, a feature that makes these QTLs a valuable asset for marker-assisted breeding programs across different latitudes. In the second study (Chapter 3), a set of 183 accessions suitable for GWAS, was exposed to optimal and high temperature during two crop seasons under field conditions. Important agronomic traits were evaluated in order to identify valuable QTLs for GY and its components. The GWAS analysis identified several QTLs in the single years as well as in the joint analysis. From the total QTLs identified, 13 QTL clusters can be highlighted to be affecting heat tolerance across different years and/or different traits.

Gwas analysis for the identification of molecular basis responsible for yield related traits and disease resistance in durum wheat

The PhD thesis was developed in the framework of Innovar H2020 project. This project aimed at using genomics, transcriptomics and phenotyping techniques to update varietal registration procedure used in Europe for Value of Cultivation and Use (VCU) and Distinctiness Uniformity and Stability (DUS) protocols. The phenotypic and genotypic diversity of a durum wheat panel were assessed for different agronomic traits, connected with wheat development, disease resistance and spike fertility. A panel of 253 durum wheat varieties was characterized for VCU and DUS traits and genotyped with Illumina 90K SNP Chip array (Wang et al., 2014). GWAS analysis was performed, detecting strong QTLs confirmed also by literature review. Candidate genes were identified for each trait and molecular markers will be developed to be used for marker assisted selection in breeding programs. As for disease resistance, the panel was evaluated for resistance to Soil-Borne-Cereal-Mosaic-Virus (SBCMV). A major QTL, sbm2, was detected on chromosome 2B responsible for durum wheat resistance (Maccaferri et al., 2011). The sbm2 interval was explored by fine mapping on segregant population using KASP markers and by RNASeq analysis, detecting candidate genes involved in plant-pathogen reaction. As regards yield related traits, detailed analysis was performed on the GNI-2A QTL (Milner et al., 2016), responsible for increased number spike fertility. Fine mapping analysis was performed on durum panel identifying hox2 a strong candidate gene, codifying for transcription factor protein. The gene is paralogue of GNI-1 (Sakuma et al., 2019), and it has a 4 kbp deletion responsible for increased number of florets per spikelet. To conclude, the herein reported thesis shows a complete characterization of agronomic and disease resistance traits in modern durum wheat varieties. The results obtained will augment available information for each variety, identifying informative molecular markers for breeding purposes and QTLs/candidate genes responsible for different agronomic traits.

Exploring mediterranean durum wheat wild relatives and elite diversity panel for future breeding

Durum wheat (Triticum durum) is an important crop that has been used for millennia for human consumption, and modern breeding can take advantage of the wide variability useful for the adaptation to new challenges. Novel beneficial alleles can be found in wild relatives and landraces thus enhancing crop adaptation to many biotic and abiotic stresses. This dissertation considers the source of variability from both before and after wheat domestication, that caused a loss of potentially useful alleles. Chapter 1. is the thesis introduction, which outlines the importance of wheat in the world, providing an historical overview of the domestication, the evolution mechanisms that led to the current forms of durum wheat and the use of wild relatives as a source of germplasm for future breeding programs is crucial. Moreover, the emergence of Z. tritici has been considered as the main pathogen of wheat since it contains extremely high levels of genetic variability and is thus difficult to control. Chapter 2. Considers the contribution of the phenotypic diversity of 242 accessions of Aegilops tauschii from the Open Wild Wheat Consortium, involved in wheat domestication, provided with whole-genome resequencing. The accessions were phenotyped both in the field and in controlled conditions and A k-mer-based GWAS was performed to identify genomic regions involved in useful traits. Chapter 3. Describes the genetic basis of resistance to Z. tritici in a durum wheat elite diversity panel representative of the germplasm bred in Mediterranean. Quantitative trait loci (QTL) analysis results revealed several loci involved in the STB response that were found in several chromosome regions with a high infection rate. The genomic regions associated with STB resistance identified in this study could be of interest for marker assisted selection (MAS) in durum wheat breeding programs.

Development of SCAR marker linked to stem canker resistance gene in soybean

Stem canker caused by the fungus Diaporthe phaseolorum f. sp. meridionalis is a disease that limits soybean cultivation. Phenotypic evaluations aiming at disease resistance require labor-intensive processes, as for instance handling and transport of phytopathogens. The use of DNA markers in the selective procedures eases certain phases, besides being practical, safe and reliable. A RAPD fragment of 588pb was identified among bulks of resistant and susceptible plants in the cross BR92-15454 (R) x IAC-11 (S). Through co-segregation, the distance between the resistance locus and the fragment was estimated at 7.4 ± 2.1 cM, with a Lodmax. of 23.072 (first year) and at 6.0 ± 3.4 cM with a Lodmax. of 7.806 (second year). The fragment was converted into a SCAR marker and digested with enzyme Hinc II, which made the classification in homozygous resistant, heterozygous resistant and susceptible plants possible. This SCAR marker is suitable for use in the improvement program conducted in Jaboticabal.

Characterization of bovine transcripts preferentially expressed in testis and with a putative role in spermatogenesis

Although the number of genes known to be associated with bovine spermatogenesis has increased in the past few years, regulation of this biological process remains poorly understood. Therefore, discovery of new male fertility genetic markers is of great value for assisted selection in commercially important cattle breeds, e.g., Nelore, that have delayed reproductive maturation and low fertility rates. The objective of the present study was to identify sequences associated with spermatogenesis that could be used as fertility markers. With RT-PCR, the following five transcripts preferentially expressed in adult testis were detected: TET(656) detected only in adult testis; TET(868) and TET(515) expressed preferentially in adult testis but also detected in fetal gonads of both sexes; and TET(456) and TET(262). expressed primarily in the testis, but also present in very low amounts in somatic tissues. Based on their homologies and expression profiles, we inferred that they had putative roles in spermatogenesis. Detection of sequences differentially expressed in testis, ovary, or both, was a useful approach for identifying new genes related to bovine spermatogenesis. The data reported here contributed to discovery of gene pathways involved in bovine spermatogenesis, with potential for prediction of fertility. (C) 2011 Elsevier Inc. All rights reserved.

Quantitative trait loci for agronomic and seed quality traits in an F-2 and F-4 : 6 soybean population

Molecular breeding is becoming more practical as better technology emerges. The use of molecular markers in plant breeding for indirect selection of important traits can favorably impact breeding efficiency. The purpose of this research is to identify quantitative trait loci (QTL) on molecular linkage groups (MLG) which are associated with seed protein concentration, seed oil concentration, seed size, plant height, lodging, and maturity, in a population from a cross between the soybean cultivars 'Essex' and 'Williams.' DNA was extracted from F-2 generation soybean leaves and amplified via polymerase chain reaction (PCR) using simple sequence repeat (SSR) markers. Markers that were polymorphic between the parents were analyzed against phenotypic trait data from the F-2 and F-4:6 generation. For the F-2 population, significant additive QTL were Satt540 (MLG M, maturity, r(2)=0.11; height, r(2)=0.04, seed size, r(2)=0.061, Satt373 (MLG L, seed size, r(2)=0.04; height, r(2)=0.14), Satt50 (MLG A1, maturity r(2)=0.07), Satt14 (MLG D2, oil, r(2)=0.05), and Satt251 (protein r(2)=0.03, oil, r(2)=0.04). Significant dominant QTL for the F-2 population were Satt540 (MLG M, height, r(2)=0.04; seed size, r(2)=0.06) and Satt14 (MLG D2, oil, r(2)=0.05). In the F-4:6 generation significant additive QTL were Satt239 (MLG I, height, r(2)=0.02 at Knoxville, TN and r(2)=0.03 at Springfield, TN), Satt14 (MLG D2, seed size, r(2)=0.14 at Knoxville, TN), Satt373 (MLG L, protein, r(2)=0.04 at Knoxville, TN) and Satt251 (MLG B I, lodging r(2)=0.04 at Springfield, TN). Averaged over both environments in the F-4:6 generation, significant additive QTL were identified as Satt251 (MLG B 1, protein, r(2)=0.03), and Satt239 (MLG 1, height, r(2)=0.03). The results found in this study indicate that selections based solely on these QTL would produce limited gains (based on low r(2) values). Few QTL were detected to be stable across environments. Further research to identify stable QTL over environments is needed to make marker-assisted approaches more widely adopted by soybean breeders.

Characterization of bovine transcripts preferentially expressed in testis and with a putative role in spermatogenesis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Marcadores RAPD para detecção de resistência à ferrugem-asiática-da-soja

Os objetivos deste trabalho foram confirmar a herança da resistência da PI 459025 (Rpp4) à ferrugem-asiática-da-soja e identificar marcadores moleculares do tipo RAPD, ligados a este gene de resistência, em populações de soja. Pelo cruzamento dos genitores contrastantes PI 459025 x Coodetec 208 obteve-se uma população, cujas populações das gerações F2 e F2:3 foram artificialmente infectadas e avaliadas quanto à reação ao fungo Phakopsora pachyrhizi, pelo tipo de lesão (RB - resistente e TAN - suscetível). Com os resultados da avaliação fenotípica, dois bulks foram obtidos com DNA de plantas homozigóticas resistentes e suscetíveis, respectivamente, pela análise de bulks segregantes. de 600 iniciadores RAPD aleatórios, foram identificados três com fragmentos polimórficos entre os bulks e parentais contrastantes quanto à resistência. Pela análise do qui-quadrado, confirmaram-se: a herança monogênica, com dominância completa quanto à resistência ao patógeno, e a segregação 3:1 para a presença de banda dos três marcadores. Os três marcadores são ligados respectivamente a 5,1, 6,3 e 14,7 cM de distância do loco de resistência, em fase de repulsão no grupo de ligação G, o que foi confirmado pela utilização do marcador microssatélite Satt288. Estes marcadores são promissores na seleção assistida para resistência à ferrugem-asiática-da-soja.

Aplicação de ferramentas moleculares e convencionais no melhoramento genético da soja

Pós-graduação em Agronomia (Genética e Melhoramento de Plantas) - FCAV

Mapeamento molecular do loco Rpp5 de resistência à ferrugem asiática da soja

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

QTL mapping for yield components in a tropical maize population using microsatellite markers

QTL mapping provides usefull information for breeding programs since it allows the estimation of genomic locations and genetic effects of chromossomal regions related to the expression of quantitative traits. The objective of this study was to map QTL related to several agronomic important traits associated with grain yield: ear weight (EW), prolificacy (PROL), ear number (NE), ear length (EL) and diameter (ED), number of rows on the ear (NRE) and number of kernels per row on the ear (NKPR). Four hundred F-2:3 tropical maize progenies were evaluated in five environments in Piracicaba, Sao Paulo, Brazil. The genetic map was previously estimated and had 117 microssatelite loci with average distance of 14 cM. Data was analysed using Composite Interval Mapping for each trait. Thirty six QTL were mapped and related to the expression of EW (2), PROL (3), NE (2), EL (5), ED (5), NRE (10), NKPR (5). Few QTL were mapped since there was high GxE interaction. Traits EW, PROL and EN showed high genetic correlation with grain yield and several QTL mapped to similar genomic regions, which could cause the observed correlation. However, further analysis using apropriate statistical models are required to separate linked versus pleiotropic QTL. Five QTL (named Ew1, Ne1, Ed3, Nre3 and Nre10) had high genetic effects, explaining from 10.8% (Nre3) to 16.9% (Nre10) of the phenotypic variance, and could be considered in further studies.

Congenic strains provide evidence that four mapped loci in chromosomes 2, 4, and 16 influence hypertension in the SHR

Aneas I, Rodrigues MV, Pauletti BA, Silva GJ, Carmona R, Cardoso L, Kwitek AE, Jacob HJ, Soler JM, Krieger JE. Congenic strains provide evidence that four mapped loci in chromosomes 2, 4, and 16 influence hypertension in the SHR. Physiol Genomics 37: 52-57, 2009. First published January 6, 2009; doi: 10.1152/physiolgenomics.90299.2008. - To dissect the genetic architecture controlling blood pressure (BP) regulation in the spontaneously hypertensive rat (SHR) we derived congenic rat strains for four previously mapped BP quantitative trait loci (QTLs) in chromosomes 2, 4, and 16. Target chromosomal regions from the Brown Norway rat (BN) averaging 13 - 29 cM were introgressed by marker-assisted breeding onto the SHR genome in 12 or 13 generations. Under normal salt intake, QTLs on chromosomes 2a, 2c, and 4 were associated with significant changes in systolic BP (13, 20, and 15 mmHg, respectively), whereas the QTL on chromosome 16 had no measurable effect. On high salt intake (1% NaCl in drinking water for 2 wk), the chromosome 16 QTL had a marked impact on SBP, as did the QTLs on chromosome 2a and 2c (18, 17, and 19 mmHg, respectively), but not the QTL on chromosome 4. Thus these four QTLs affected BP phenotypes differently: 1) in the presence of high salt intake (chromosome 16), 2) only associated with normal salt intake (chromosome 4), and 3) regardless of salt intake (chromosome 2c and 2a). Moreover, salt sensitivity was abrogated in congenics SHR. BN2a and SHR. BN16. Finally, we provide evidence for the influence of genetic background on the expression of the mapped QTLs individually or as a group. Collectively, these data reveal previously unsuspected nuances of the physiological roles of each of the four mapped BP QTLs in the SHR under basal and/or salt loading conditions unforeseen by the analysis of the F2 cross.

Genetic polymorphisms related to meat traits in purebred and crossbred Nelore cattle

The objective of this work was to estimate the allelic and genotypic frequencies of CAST/XmnI, a calpastatin gene polymorphism, and CAPN530, a calpain 1 large subunit gene polymorphism, in different beef genetic groups (Nelore and Nelore x Bos taurus), and to investigate associations between these polymorphisms and carcass and meat traits. Three hundred animals - comprising 114 Nelore, 67 Angus x Nelore, 44 Rubia Gallega x Nelore, 41 Canchim, 19 Brangus three-way cross and 15 Braunvieh three-way cross- were genotyped by PCR-RFLP and phenotyped for rib-eye area (REA), back-fat thickness (BT), intramuscular fat (IF), shear force (SF) and myofibrillar fragmentation index (MFI). The occurrence of the two alleles of the CAST/XmnI and CAPN530 single nucleotide polymorphisms (SNPs) in a B. indicus breed, which permitted association studies in purebred and crossbred Nelore cattle, was first shown in the present work. No relationship was found between the CAST or CAPN1 SNPs and growth-related traits (REA) or fat deposition (BT and IF), since calpastatin and µ-calpain are not physiologically involved with these traits. Moreover, the association results between genotypes and aged meat tenderness (assessed by SF and MFI) showed that these markers are useless in assisted selection for purebred Nelore and their crosses with B. taurus.

Polymorphisms in candidate genes and their association with carcass traits and meat quality in Nellore cattle

The objective of this work was to estimate the allele polymorphism frequencies of genes in Nellore cattle and associate them with meat quality and carcass traits. Six hundred males were genotyped for the following polymorphisms: DGAT1 (VNTR with 18 nucleotides at the promoter region); ANK1, a new polymorphism, identified and mapped here at the gene regulatory region NW_001494427.3; TCAP (AY428575.1:g.346G>A); and MYOG (NW_001501985:g.511G>C). In the association study, phenotype data of hot carcass weight, ribeye area, backfat thickness, percentage of intramuscular fat, shear force, myofibrillar fragmentation index, meat color (L*, a*, b*), and cooking losses were used. Allele B from the ANK1 gene was associated with greater redness (a*). Alleles 5R, 6R, and 7R from the DGAT1 VNTR gene were associated with increased intramuscular fat, reduced cooking losses and increased ribeye area, respectively. The single nucleotide polymorphism (SNP) of the TCAP gene was not polymorphic, and MYOG alleles were not associated with any of the evaluated characteristics. These results indicate that ANK1 and DGAT1 genes can be used in the selection of Nellore cattle for carcass and meat quality.

Breeding, genetic and genomic of citrus for disease resistance

Although the citriculture is one of the most important economic activities in Brazil, it is based on a small number of varieties. This fact has contributed for the vulnerability of the culture regarding the phytosanitary problems. A higher number of varieties/genotypes with potential for commercial growing, either for the industry or fresh market, has been one of the main objectives of citrus breeding programs. The genetic breeding of citrus has improved, in the last decades, due to the possibility of an association between biotechnological tools and classical methods of breeding. The use of molecular markers for early selection of zygotic seedlings from controlled crosses resulted in the possibility of selection of a high number of new combination and, as a consequence, the establishment of a great number of hybrids in field experiments. The faster new tools are incorporated in the program, the faster is possibility to reach new genotypes that can be tested as a new variety. Good traits should be kept or incorporate, whereas bad traits have to be excluded or minimized in the new genotype. Scion and rootstock can not be considered separately, and graft compatibility, fruit quality and productivity are essential traits to be evaluated in the last stages of the program. The mapping of QTLs has favored breeding programs of several perennial species and in citrus it was possible to map several characteristics with qualitative and quantitative inheritance. The existence of linkage maps and QTLs already mapped, the development of EST and BAC library and the sequencing of the Citrus complete genome altogether make very demanding and urgent the exploration of such data to launch a wider genetic study of citrus. The rising of information on genome of several organisms has opened new approaches looking for integration between breeding, genetic and genome. Genome assisted selection (GAS) involves more than gene or complete genome sequencing and is becoming an import support in breeding programs of annual and perennial species. An huge information amount can be derivate from genome analysis. The use and benefit of such informations will depend on the genetic basis of the breeding program.