SÃO PAULO STATE UNIVERSITY SCHOOL OF AGRICULTURAL AND VETERINARIAN SCIENCES CAMPUS OF JABOTICABAL GENOMIC STUDY FOR FEMALE SEXUAL PRECOCITY, CARCASS, AND MEAT QUALITY TRAITS IN NELLORE CATTLE Leonardo Machestropa Arikawa Animal Scientist 2022 SÃO PAULO STATE UNIVERSITY SCHOOL OF AGRICULTURAL AND VETERINARIAN SCIENCES CAMPUS OF JABOTICABAL GENOMIC STUDY FOR FEMALE SEXUAL PRECOCITY, CARCASS, AND MEAT QUALITY TRAITS IN NELLORE CATTLE Leonardo Machestropa Arikawa Advisor: Prof.ª Dra. Lucia Galvão de Albuquerque Co-Advisor: Prof.ª Dra. Ana Fabrícia Braga Magalhães Dissertation presented to the School of Agricultural and Veterinarian Sciences – São Paulo State University, Campus of Jaboticabal in partial fulfillment of requirements for the degree of master in Genetics and Animal Breeding. A699g Arikawa, Leonardo Machestropa Genomic study for female sexual precocity, carcass, and meat quality traits in Nellore cattle / Leonardo Machestropa Arikawa. -- Jaboticabal, 2022 151 p. : il., tabs. Dissertação (mestrado) - Universidade Estadual Paulista (Unesp), Faculdade de Ciências Agrárias e Veterinárias, Jaboticabal Orientadora: Lucia Galvão de Albuquerque Coorientadora: Ana Fabrícia Braga Magalhães 1. Beef cattle. 2. Carcass. 3. Genetic parameters. 4. GWAS. 5. Meat quality. I. Título. Sistema de geração automática de fichas catalográficas da Unesp. Biblioteca da Faculdade de Ciências Agrárias e Veterinárias, Jaboticabal. Dados fornecidos pelo autor(a). Essa ficha não pode ser modificada. UNIVERSIDADE ESTADUAL PAULISTA Câmpus de Jaboticabal GENOMIC STUDY FOR FEMALE SEXUAL PRECOCITY, CARCASS, AND MEAT QUALITY TRAITS IN NELLORE CATTLE TÍTULO DA DISSERTAÇÃO: CERTIFICADO DE APROVAÇÃO AUTOR: LEONARDO MACHESTROPA ARIKAWA ORIENTADORA: LUCIA GALVÃO DE ALBUQUERQUE COORIENTADORA: ANA FABRÍCIA BRAGA MAGALHÃES Aprovado como parte das exigências para obtenção do Título de Mestre em GENÉTICA E MELHORAMENTO ANIMAL, pela Comissão Examinadora: Profa. Dra. LUCIA GALVÃO DE ALBUQUERQUE (Participaçao Virtual) Departamento de Zootecnia / FCAV Unesp Jaboticabal Prof.Dr. FERNANDO SEBASTIAN BALDI REY (Participaçao Virtual) Departamento de Zootecnia / FCAV / UNESP - Jaboticabal Ph.D. LÚCIO FLÁVIO MACÊDO MOTA (Participaçao Virtual) Department of Agronomy Food Natural Resources Animals and Environment - DAFNAE / University of Padova - Campus Agripolis Jaboticabal, 24 de fevereiro de 2022 Faculdade de Ciências Agrárias e Veterinárias - Câmpus de Jaboticabal - Via de Acesso Professor Paulo Donato Castellane, s/n, 14884900, Jaboticabal - São Paulo https://www.fcav.unesp.br/#!/pos-graduacao/programas-pg/genetica-e-melhoramento-animalCNPJ: 48.031.918/0012-87. ABOUT THE AUTHOR Leonardo Machestropa Arikawa – born on May 8, 1995, in Taquaritinga, São Paulo, Brazil, son of Hisanori Arikawa and Sueli Machestropa. He started his undergraduate in Animal Science in March 2013 at School of Agricultural and Veterinarian Sciences, UNESP, campus of Jaboticabal. He performed an extracurricular internship at the Department of Animal Science in the area of Genetics and Animal Breeding, from August 2018 to August 2019, under the supervision of Prof. Dr. Ana Fabrícia Braga Magalhães. From September to November 2019, he completed the mandatory curricular internship at the “Instituto de Zootecnia – Centro Avançado de Pesquisa Tecnológica dos Agronegócios de Bovinos de Corte (APTA)”, in the area of Animal Breeding with Prof. Dr. Lucia Galvão de Albuquerque and Prof. Dr. Maria Eugenia Zerlotti Mercadante as advisors. He obtained his bachelor's degree in Animal Science in February 2020. In March 2020, he started his Master of Science (MSc.) studies in the Graduate Program in Genetics and Animal Breeding at School of Agricultural and Veterinarian Sciences, UNESP, campus of Jaboticabal, under the supervision of Prof. Dr. Lucia Galvão de Albuquerque and co-supervision of Prof. Dr. Ana Fabrícia Braga Magalhães. His M.Sc studies have been granted with a scholarship from “Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)”, from March 2020 to February 2022. “Who among you can at the same time laugh and be exalted? He who climbeth on the highest mountains, laugheth at all tragic plays and tragic realities. Courageous, unconcerned, scornful, coercive - so wisdom wisheth us; she is a woman, and ever loveth only a warrior.” Friedrich Nietzsche (Thus Spake Zarathustra) I dedicate this work to all Brazilian researchers who remain resilient, fighting for value and working to promote advances in knowledge, critical thinking, and development, even in the midst of a country that devalues and contests scientific thinking. ACKNOWLEDGMENTS Firstly, I thank my family, especially my parents Hisanori and Sueli, for their support at all times, for the education they provided me, and for never measuring efforts to help me, whatever the circumstances. I can only thank you, saying that this achievement belongs to you. To my sister, Amanda, and my nephews, Davi, Henrique, and Nicolas, for their love and affection, and for being an inspiration to me. To my advisor Prof. Dr. Lucia Galvão de Albuquerque, first of all for having received me and for the opportunity to work in her research group. For all the availability, support, patience, and help offered for the development of this project. Thank you for your extensive professional and personal advice, for believing in my potential and my work. You are a great inspiration to me. To my co-advisor Dr. Ana Fabricia Braga Magalhães and also Dr. Larissa Fernanda Simielli Fonseca, for all the attention, support, and help they've been offering since undergraduate. I am extremely grateful for being excellent professors and for awakening in me an interest in the area of Genetics and Animal Breeding. I would like to express my gratitude to Prof. Dr. Roberto Carvalheiro and Dr. Delvan Alves da Silva, for their support, attention, suggestions to improve this work and for dedicating their time to transmit valuable knowledge to me. To the members of my Master's dissertation and general qualification exam committee, Prof. Dr. Fernando Sebastian Baldi Rey, Dr. Lucio Flavio Macedo Mota, and Dr. Gerardo Alves Fernandes Júnior, for the valuable suggestions that contributed to the improvement of the work. To the “Albuquerque” team, to the friends and co-workers from graduate program in Genetics and Animal Breeding, who welcomed me, helped, and taught me from the first day I came to the research group. I thank everyone who gave me strength and encouragement in difficult times and also celebrated the happy times, especially my great friend Daiane. I was very lucky to be around amazing people who were fundamental to my personal growth. To my brothers from the eternal “Casa Verde”, André (Reditube), Felipe (Falo), Jorge (Pran-xana), Lindomar (Libumba), and Matheus (Télson), for the companionship, the coexistence, and all the good times we had living together in Jaboticabal. To the School of Agricultural and Veterinarian Sciences – UNESP, for the support and structure provided to us, and all the professors from graduate program in Genetics and Animal Breeding, for all the help, classes, and knowledge offered. To Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) for granting the scholarship. I am very grateful to everyone who contributed directly or indirectly, either with knowledge or support, so that I could succeed during this stage. This study was financially supported by FAPESP #2009/16118-5; #2017/10630- 2; and #2018/20026-8, and in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001. This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001. SUMMARY pages RESUMO...................................................................................................................... i ABSTRACT ................................................................................................................ iii Chapter 1 – General Considerations ....................................................................... 1 1. INTRODUCTION ..................................................................................................... 1 2. LITERATURE REVIEW .......................................................................................... 3 2.1. Reproductive traits ............................................................................................ 3 2.2. Carcass traits .................................................................................................... 5 2.3. Meat quality traits .............................................................................................. 7 2.4. Genetic correlations .......................................................................................... 9 2.5. Genome-wide association studies .................................................................. 12 3. OBJECTIVES ....................................................................................................... 18 3.1. General objective ............................................................................................ 18 3.2. Specific objectives .......................................................................................... 18 4. REFERENCES ...................................................................................................... 19 Chapter 2 – Genetic parameters estimates using genomic information for female sexual precocity, carcass and meat quality traits in Nellore cattle .................... 33 1. INTRODUCTION ................................................................................................... 34 2. MATERIAL AND METHODS ................................................................................ 36 2.1. Database ........................................................................................................ 36 2.1.1. Phenotypic data ........................................................................................ 36 2.1.2. Genotypic data ......................................................................................... 39 2.2. Estimates of (co)variance components and genetic parameters .................... 39 3. RESULTS AND DISCUSSION ............................................................................. 41 3.1. Genetic parameters for carcass and meat quality traits .................................. 41 3.2. Heritability estimates for female sexual precocity and their genetic correlations with carcass and meat quality traits ....................................................................... 43 4. CONCLUSIONS .................................................................................................... 45 5. REFERENCES ...................................................................................................... 46 Chapter 3 – Genome-wide scans for carcass and meat quality traits in Nellore cattle ......................................................................................................................... 53 1. INTRODUCTION ................................................................................................... 54 2. MATERIAL AND METHODS ................................................................................ 56 2.1. Phenotypic data .............................................................................................. 56 2.2. Genotypic data ................................................................................................ 58 2.3. Genome-wide association analysis ................................................................. 58 2.4. Functional analysis ......................................................................................... 60 3. RESULTS AND DISCUSSION ............................................................................. 60 3.1. GWAS for carcass traits .................................................................................. 60 3.2. GWAS for meat quality traits ........................................................................... 70 4. CONCLUSIONS .................................................................................................... 80 5. REFERENCES ...................................................................................................... 81 6. SUPPLEMENTARY INFORMATION .................................................................. 100 i ESTUDO GENÔMICO PARA CARACTERÍSTICAS DE PRECOCIDADE SEXUAL DE FÊMEAS, CARCAÇA E QUALIDADE DA CARNE EM BOVINOS DA RAÇA NELORE RESUMO – O Brasil é o maior exportador de carne bovina do mundo e o país com o maior rebanho bovino comercial. O Nelore é a principal raça de gado de corte do país; entretanto, os animais desta raça tendem a produzir carcaças e carne de qualidade inferior às raças Bos taurus. Além disso, as características de carcaça obtidas no post- mortem e a qualidade da carne são atributos de expressão tardia e de difícil mensuração e, consequentemente, são difíceis de selecionar pelos métodos convencionais. Em termos reprodutivos, os zebuínos atingem a puberdade tardiamente e essas características, principalmente as observadas nas fêmeas, são altamente influenciadas por fatores ambientais. Assim, o uso de abordagens genômicas torna-se uma alternativa para contornar esses desafios. Nesse contexto, os objetivos deste estudo foram: i) estimar parâmetros genéticos para características de precocidade sexual de fêmeas, carcaça e qualidade da carne, utilizando informação genômica; ii) conduzir um estudo de associação genômica ampla (GWAS) para características de carcaça e qualidade da carne em bovinos Nelore. A base de dados utilizada é composta por informações de características de carcaça obtidas no post-mortem (AOL: área de olho de lombo, EGS: espessura de gordura subcutânea e PCQ: peso da carcaça quente); qualidade da carne (MAC: maciez, MARM: marmoreio e LIP: teor de lipídios); e precocidade sexual (IPP: idade ao primeiro parto e PE: perímetro escrotal). No Capítulo 2, o conjunto de dados utilizado para estimação de parâmetros genéticos foi de 602.122 registros para características de precocidade sexual e 6.910 para características de carcaça/carne, e registros genotípicos de 15.000 animais Nelore genotipados ou imputados com o Illumina Bovine HD Beadchip. Os componentes de (co)variância e os parâmetros genéticos foram obtidos considerando a abordagem single-step (ssGBLUP) por dois métodos: 1) para características de carcaça e qualidade de carne, um modelo multi-característica e inferência bayesiana foram aplicados usando o software GIBBS2F90, e o peso ao sobreano (PS) foi incluído na análise como característica âncora; 2) modelos bi- característica e inferência frequentista foram adotados utilizando o software AIREMLF90 para estimar as correlações genéticas entre as características de precocidade sexual com as de carcaça e qualidade da carne. As estimativas de herdabilidade variaram de 0,13 a 0,34 para as características de carcaça e qualidade de carne, e foram de 0,06 e 0,45 para IPP e PE, respectivamente. Correlações genéticas favoráveis foram estimadas entre PS–PCQ (0,79±0,03), PS–AOL (0,28±0,05), PCQ-AOL (0,44±0,05), MARM–LIP (0,90±0,07), MAC–LIP (-0,20±0,11), EGS–MARM (0,29±0,08), EGS–LIP (0,22±0,09), PCQ–MAC (-0,22±0,09) e EGS–IPP (-0,26±0,11). No Capítulo 3, um total de 6.910 animais Nelore fenotipados e 25.000 genotipados foram utilizados para o estudo de GWAS. Os efeitos dos SNP foram estimados com base na abordagem weighted single-step GBLUP (WssGBLUP). As 10 principais regiões genômicas explicaram 8,79%, 12,06% e 9,01% da variância genética aditiva e abrigaram um total de 134, 158 e 93 genes candidatos posicionais para AOL, EGS e PCQ, respectivamente. Para as características de qualidade da carne, as janelas de maior efeito foram responsáveis por 14,72%, 14,79% e 14,13% da variância aditiva, e 137, 163 e 89 genes candidatos foram encontrados para MAC, MARM e LIP, respectivamente. Entre os genes candidatos encontrados, estão ii PPARGC1A, AQP3, AQP7, MYLK2, PLAGL2, PLAG1, XKR4, MYOD1, KCNJ11, WWOX, CARTPT, RAC1, PSAP, PLA2G16 e PLCB3, genes que foram anteriormente associados a diversas características produtivas, como de crescimento, carcaça, qualidade da carne, ingestão alimentar e reprodutivas em Nelore e outras raças de bovinos. Palavras-chave: bovinos de corte, carcaça, GWAS, parâmetros genéticos, precocidade sexual, qualidade da carne iii GENOMIC STUDY FOR FEMALE SEXUAL PRECOCITY, CARCASS, AND MEAT QUALITY TRAITS IN NELLORE CATTLE ABSTRACT – Brazil is the largest beef exporter in the world and the country with the largest commercial bovine herd. Nellore is the main beef cattle breed in Brazil; however, animals of this breed tend to produce carcasses and beef of lower quality than Bos taurus. In addition, carcass traits obtained in post-mortem and meat quality are attributes that are late expressed and difficult to measure, consequently, they are difficult to select by conventional methods. In reproductive terms, Zebu cattle reach puberty late and these traits, especially those observed in females, are highly influenced by environmental factors. Thus, the use of a genomic approach becomes an alternative to overcome these challenges. In this context, the aims of this study were: i) to estimate genetic parameters for female sexual precocity, carcass, and meat quality traits, using genomic information; ii) to perform a genome-wide association study (GWAS) for carcass and meat quality traits in Nellore cattle. The database used is composed of information for carcass traits obtained in the post-mortem (LMA: longissimus muscle area, BF: backfat thickness, and HCW: hot carcass weight); meat quality (SF: shear-force tenderness, MARB: marbling, and IMF: intramuscular fat content); and sexual precocity traits (AFC: age at first calving and SC: scrotal circumference). In Chapter 2, the dataset used to estimate genetic parameters consisted of 602,122 records for sexual precocity traits and 6,910 for carcass/meat traits, and genotypic records of 15,000 Nellore animals genotyped or imputed to the Illumina Bovine HD Beadchip. The (co)variance components and genetic parameters were obtained considering a Single-step approach (ssGBLUP) in two methods: 1) for carcass and meat quality traits, a multi-trait model and Bayesian inference were applied using the GIBBS2F90 software, and yearling weight (PW) was included in the analysis as an anchor trait; 2) bi-trait models and frequentist inference were adopted using the AIREMLF90 software to estimate the genetic correlations of sexual precocity traits with carcass and meat quality traits. Heritability estimates ranged from 0.13 to 0.34 for carcass and meat quality traits, and were 0.06 and 0.45 for AFC and SC, respectively. Favorable genetic correlations were estimated between YW–HCW (0.79±0.03), YW–LMA (0.28±0.05), HCW–LMA (0.44±0.05), MARB–IMF (0.90±0.07), SF–IMF (-0.20±0.11), BF–MARB (0.29±0.08), BF–IMF (0.22±0.09), HCW–SF (- 0.22±0.09), and BF–AFC (-0.26±0.11). In Chapter 3, a total of 6,910 phenotyped and 25,000 genotyped Nellore animals were used for GWAS. The effects of SNPs were estimated based on the weighted single-step GBLUP (WssGBLUP) approach. The top 10 genomic regions explained 8.79, 12.06, and 9.01% of the additive genetic variance and harbored a total of 134, 158, and 93 positional candidate genes for LMA, BF, and HCW, respectively. For meat quality traits, the windows of greatest effect accounted for 14.72, 14.79, and 14.13% of the additive variance, and 137, 163, and 89 candidate genes were found for SF, MARB, and IMF, respectively. Among the candidate genes found, there are PPARGC1A, AQP3, AQP7, MYLK2, PLAGL2, PLAG1, XKR4, MYOD1, KCNJ11, WWOX, CARTPT, RAC1, PSAP, PLA2G16, and PLCB3, genes that were previously associated with several production traits, such as growth, carcass, quality of meat, feed intake and reproductive traits in Nellore and other cattle breeds. Keywords: beef cattle, carcass, genetic parameters, GWAS, meat quality, sexual precocity 1 Chapter 1 – General Considerations 1. INTRODUCTION With the globalization process combined with the growing demand for safe food, Brazil has become one of the largest producers and exporters of beef, due to its high technological potential, high level of production and, mainly, the quality of its production (Almeida and Michels, 2012; Gomes et al., 2017). Currently, the country stands out on the world stage as the largest exporter of meat, in addition to having the largest cattle herd. In 2018, the Brazilian cattle herd reached about 214.7 million heads. In total, 44.2 million heads were slaughtered, producing, approximately, 10.9 million tons of carcass weight equivalent (CWE). From the total meat produced, 20.2% was exported and the rest supplied the domestic market, guaranteeing average consumption per capita of 49.12 kg/year. Beef exports totaled 1,600 tons, with a value of US$ 6,572.30 million, representing 3.5% of agribusiness exports (ABIEC, 2019). The Brazilian bovine population consists of a variety of Taurine (Bos taurus taurus) and Zebu (Bos taurus indicus) breeds. Approximately 80% of the national herd is made up of zebu animals of different breeds, with Nellore being the most expressive with an aptitude for beef (Costa et al., 2015; Lopes et al., 2016; Magalhães et al., 2016). Thus, Brazilian beef cattle are kept, predominantly, with the genetics of zebu cattle. Brazilian Nellore has undergone an intense process of genetic improvement over time, becoming the most important national beef cattle breed. Animals of this breed have productive and reproductive attributes that best adapt to tropical climatic conditions. Its rusticity, natural resistance to ecto and endoparasites and to heat, are examples of favorable traits of Nellore in Brazil. Moreover, cows have outstanding reproductive longevity and excellent maternal ability (Santos, 2000; Albuquerque et al., 2006; Lopes et al., 2016; Feitosa et al., 2017). However, Bos indicus animals tend to show moderate growth rate and adult weight, late testicular development, and reach puberty at older ages (Cundiff et al., 2004). Compared to Taurine breeds, Nellore animals produce leaner meat, with less marbling index and less tenderness, besides finishing late, partially due to the extensive production system adopted in Brazil (Cundiff et al., 2004; Albuquerque et al., 2006). Albuquerque et al. (2006), in a review 2 paper, concluded that in order to increase animal productivity and, consequently, the production of quality food, besides of selecting Zebu cattle raised extensively, it is important to be concerned with animal welfare and food safety conditions. In order to reach production levels that meet consumer demand, it is essential to deepen the understanding of the genetic mechanisms involved in the expression of carcass and meat traits in Nellore cattle. So that, it will be possible to identify genetic variants that control these traits, and to assist ranchers in planning breeding programs and achieving their goals related to the final product (Xia et al., 2016; Bhuiyan et al., 2017). However, information for these characteristics obtained post-mortem are still insufficient, due to the fact that they are late expressed and difficult to measure, requiring progeny tests since the candidate animals cannot be directly evaluated, which increases the costs and the length of generation intervals (Fernandes Júnior et al., 2016a; Fonseca et al., 2017; Leal-Gutiérrez et al., 2019; Magalhães et al., 2019). The reproductive traits also are important for the production system, as they have a great economic impact on the production of beef cattle. In this context, sexual precocity has been increasingly explored by breeding programs with the objective of reducing production costs and generation intervals, as well as increasing genetic gain rates (Moorey and Biase, 2020). Furthermore, these traits guarantee a greater number of born calves throughout the productive life of precocious heifers (Eler et al., 2014). Thus, as it is a decisive factor in the total production efficiency, traits associated with female sexual precocity must be included in the indexes as a selection criterion. However, reproductive problems in Brazilian beef cattle are the main limiting on the productive efficiency of a herd, due to the sexual precocity traits, especially those observed in females, are highly influenced by environmental factors and, thus, are little inheritable (Cardoso et al., 2015). Furthermore, considering that Brazilian cattle are predominantly composed of Zebu breeds, heifers tend to reach puberty later than Bos taurus animals (Albuquerque et al., 2006; Nascimento et al., 2016). Thus, in the face of such difficulties in the breeding of Zebu breeds, it is necessary to search for alternative methods, such as those that use genomic approaches, which allow for accurate genetic evaluations, which can be used to improve female sexual precocity, carcass and meat quality traits (Fernandes Júnior et al., 2016a; Magalhães et al., 2019). In this sense, the inclusion of molecular tools in 3 genetic analysis can improve the scanning of the genetic architecture of these traits, which in turn are of complex inheritance since their variation is determined by many genes with small effects. Genome-wide association studies (GWAS) aim to associate genomic regions with the phenotypes of interest through statistical analysis, to identify variations in the genome (mainly single nucleotide polymorphisms - SNP) linked to regions of great effect on a given characteristic, providing a better understanding of biological functions and genetic influence on phenotypic expression (Zhang et al., 2012; Yang et al., 2013; Magalhães et al., 2016; Magalhães et al., 2019; Pegolo et al., 2020). The basic principle of GWAS is that a set of phenotypes of a target trait in a sample of animals, is tested for a panel of SNP markers across the genome, in order to identify statistical associations between the trait and all markers, simultaneously, and quantify the size of the effect that each marker contributes to the expression of the characteristic (Goddard and Hayes, 2009). Therefore, whether in the application of estimates of genetic parameters and/or GWAS, the trend is that, increasingly, the use of molecular tools in genetic analyzes will become effective and routine in animal breeding programs. Thus, due to the small number of studies and the difficulty in improving sexual precocity rates and selecting carcass and meat quality traits using the traditional method, it is essential to develop genomic studies aiming to better understand the expression of these traits in Nellore cattle. 2. LITERATURE REVIEW 2.1. Reproductive traits Reproductive efficiency plays a key role in the economic sustainability of the livestock production system, especially traits related to sexual precocity in heifers (Brumatti et al., 2011). These traits are directly related to the availability of animals in the herd, influencing, consequently, the intensity of the selection, in order to guarantee greater genetic gains, shorter generation intervals, and the economic success of production (Kluska et al., 2018; Ramos et al., 2020). 4 In reproductive terms, compared to the Taurine breeds, Zebu cattle reach puberty at a later age (Albuquerque et al., 2006; Nascimento et al., 2016), probably due to low human interference in the selection process of Zebu herds, and may delay the beginning of a cow's reproductive life, impairing the production chain and the profitability of the system. In addition, the sexual precocity traits are generally highly influenced by environmental factors, such as nutrition and heat stress (Samadi et al., 2014; Nepomuceno et al., 2017; Ferraz et al., 2018), which affect the genetic gain per generation (Cardoso et al., 2015; Mota et al., 2020). The age at first calving (AFC) is one of the main traits used to assess female fertility and sexual maturity. AFC is an economically important trait in beef cattle, as it is directly linked to the reproductive longevity of the cow and the interval between generations, and, in this context, the longevity of beef cows ensures a lower replacement rate and greater utilization of the herd (Perotto et al., 2006). The inclusion of heifers at young ages in reproduction is strongly important so that genetic differences in reproductive capacity are detected early (Boligon et al., 2015), providing a reduction in production costs and increasing rates of genetic gain. However, producing breeding stock that has these attributes is one of the biggest challenges for beef cattle breeders, since reproductive traits, especially those evaluated in females, generally have low heritability, implying the difficulty of direct selection for younger ages at first calving (Boligon et al., 2010). Several studies conducted on the Nellore breed in recent years have reported heritability for AFC ranging from 0.08 to 0.24 (Boligon et al., 2015; Terakado et al., 2015; Buzanskas et al., 2017; Kluska et al., 2018; Lacerda et al., 2018; Schmidt et al., 2018; Brunes et al., 2020; Costa et al., 2020). According to Buzanskas et al. (2017), the increase in heritability estimates for AFC could be due to the increase in genetic variability that has been introduced through the selection of sexually precocious heifers and through greater control of environmental factors. Due to the problems associated with female sexual performance, for many years, the inclusion of easy-to-measure traits, genetically correlated to female reproductive events as selection criteria, was an excellent alternative to decrease the generation interval and increase the genetic gain for sexual precocity in females (Boldt et al., 2016; Kluska et al., 2018). The scrotal circumference (SC), although it has no 5 economic value, has been widely used to improve sexual precocity and the reproductive performance of cattle herds, since males and females share genes that are involved in physiological mechanisms linked to reproductive events (Toelle and Robinson, 1985). This trait is easy to measure with relatively low cost and it is favorably correlated with young female reproductive traits (Terakado et al., 2015; Buzanskas et al., 2017; Soares et al., 2017). Lacerda et al. (2018) report that SC has been widely used also to improve sexual precocity and reproductive performance in males, such as early onset of spermatogenesis, in addition to being a good indirect indicator of puberty in females, provided it is measured at a young age. Heritability estimates for SC are generally of moderate to high magnitudes. When measured at yearling, heritability estimates found in the literature for the Nellore breed range from 0.33 to 0.52 (Boligon et al., 2015; Terakado et al., 2015; Buzanskas et al., 2017; Kluska et al., 2018; Lacerda; et al., 2018; Brunes et al., 2020). Based on the results of a meta-analysis study, which grouped several other studies with Nellore cattle, Oliveira et al. (2017) reported a heritability estimate of 0.56 for SC measured at yearling, indicating that the trait must respond quickly to selection events. 2.2. Carcass traits Attributes such as carcass are economically important traits, since the beef industry pays producers based on some conditions of the carcass, such as weight and finish (Vaz et al., 2013; Fernandes Júnior et al., 2016a; Mcphee et al., 2020). Measuring carcass traits is necessary to analyze qualitative and quantitative parameters related to the composition of the final product (Tonussi et al., 2015). Besides, selection for these characteristics can lead to improvements in carcass composition, increasing the proportion of edible body parts (Tonussi et al., 2015; Kluska et al., 2018), ensuring a higher percentage of yield from commercial cuts. The longissimus muscle area (LMA) is a carcass trait expressed in square centimeters (cm2) and measured in the Longissimus thoracis dorsal muscle, between the 12th and 13th ribs. LMA is an efficient indicator of muscle mass, carcass composition and edible portion (Caetano et al., 2013; Gordo et al., 2018), and this trait is positively associated with the amount of muscle, growth rates, carcass yield and, mainly, with the proportion of cuts that add commercial value for meat products (Bertrand et al., 6 2001; Caetano et al., 2013; Tonussi et al., 2015; Gordo et al., 2018). The heritability coefficients observed in the literature for LMA in Nellore cattle are of moderate to high magnitude, ranging from 0.10 to 0.28 for LMA measured post-mortem (Tonussi et al., 2015; Fernandes Júnior et al., 2016a; Gordo et al., 2018;), and from 0.29 to 0.44 when was obtained by ultrasonography (Ceacero et al., 2016; Buzanskas et al., 2017; Kluska et al., 2018; Silva Neto et al., 2020) indicating that selection for LMA can promote a rapid genetic progress. The backfat thickness (BF) is directly related to the quality of the final product. BF indicates the degree of finishing of the carcass (Tonussi et al., 2015; Gordo et al., 2018). It influences the cooling speed after slaughtering, acting as a thermal insulator, protecting the carcass against the stiffness and darkening of muscles, in addition to avoiding the reduction of weight and tenderness caused by dehydration during the cooling process (Caetano et al., 2013; Baldassini et al., 2017; Silva-Vignato et al., 2017). Scarce fat covering causes problems in the carcass, devaluing its quality but, when in excess, it is undesirable, as it provides a negative look to the consumer, in addition to reducing the edible portion. The heritability estimates for BF in the Nellore breed assume values with wide variation in the literature, between 0.08 and 0.21 for BF obtained post-mortem (Fernandes Júnior et al., 2016a; Feitosa et al., 2017; Gordo et al., 2018), and 0.17 to 0.59 for BF measured by ultrasound (Yokoo et al., 2015; Ceacero et al., 2016; Silva Neto et al., 2020). The hot carcass weight (HCW) is a phenotypic measure expressed in kilograms (kg) and is related to the weight of the newly slaughtered animal, which is obtained from the weighing of the carcass after skinning, evisceration, and carcass toilet processes. This is a classificatory trait used by slaughterhouses and is directly related to the commercial value of the animal, since the amount paid to cattle breeders is, mainly, based on the carcass weight (Fernandes Júnior et al., 2016a; Gordo et al., 2018). Studies with Nellore animals showing HCW genetic parameter estimates are still scarce. The heritability estimates found in the literature for the breed range from 0.11 to 0.39 (Tonussi et al., 2015; Fernandes Júnior et al., 2016a; Gordo et al., 2018; Carvalho et al., 2019). In a review work, Utrera and Van Vleck (2004) reported a mean value of 0.40 for heritability estimates for HCW for several bovine breeds, ranging from 0.09 to 0.92. The authors reported that the wide variation may be associated with 7 differences in racial groups, estimation methods, model effects, number of observations, measurement errors, animal sex, and farm management. 2.3. Meat quality traits Meat quality traits are fundamental to guarantee consumers satisfaction (Gordo et al., 2018). The concept of "quality meat" most demanded by buyers includes a series of sensory factors, such as tenderness, juiciness, and flavor, which together contribute to a better palatability of the meat, in addition to a more attractive visual appearance that includes attributes such as color and distribution of the fat (Magnabosco et al., 2016; Xia et al., 2016; Gordo et al., 2018; Leal-Gutiérrez et al., 2019). Among the meat attributes, tenderness is considered the most important sensory parameter for consumers. This trait is usually determined by shear force (SF) and is influenced by genetic and environmental factors, such as genotype, age, sex, management, post-mortem pH drop, and carcass composition (Zhao et al., 2012; Fonseca et al., 2017; Mwangi et al., 2019). Considering the high percentage of Zebu animals in the Brazilian bovine population, the improvement of meat tenderness is essential to meet the quality demanded by buyers, since animals belonging to this genetic group present unfavorable genes for tenderness (Ferraz and Felício, 2010; Magnabosco et al., 2016; Fonseca et al., 2017). Heritability estimates for SF range from low to moderate (0.09 to 0.21) in studies carried out for the Nellore breed (Castro et al., 2014; Tonussi et al., 2015; Gordo et al., 2018; Magalhães et al., 2018; Bonin et al., 2021), suggesting the possibility of obtaining relatively slow genetic progress through selection. Marbling fat contributes to the juiciness of the meat, providing the necessary lubrication between the muscle fibers, increasing the perception of juiciness, in addition to preventing the loss of water by cooking (Mwangi et al., 2019). Another factor influenced by this trait is the flavor through a complex interaction between precursors of fatty and lean meat components (Arshad et al., 2018; Mwangi et al., 2019). Studies with Nellore animals have reported heritability estimates ranging from 0.11 to 0.32 (Neves et al., 2014; Tonussi et al., 2015; Gordo et al., 2018; Magalhães et al., 2018). In a search to estimate genetic parameters for quality meat traits of Nellore evaluated at different anatomical points of the Longissimus thoracis muscle, Bonim et al. (2021) 8 found heritability estimates of 0.15 and 0.16 for MARB, obtained from samples taken from longissimus in the 5th and 12th ribs, respectively. In addition, the authors found a high genetic association (0.89±0.33) between the different MARB measures used in the study, suggesting that the collection region does not influence the estimates, and any of the measures can be used for selection purposes, with similar direct and correlated responses. The percentage of lipids can also be associated with the same attributes as the marbling score and it is also used to assess meat quality. The intramuscular fat content (IMF) represents the content of accumulated lipids between fibers or inside muscle cells (Cesar et al., 2015). It is a polygenic trait influenced by several factors (such as sex, age, race, nutrition, and genetics) and is directly associated with the texture and quality of meat, and its quantity in the meat tends to influence acceptability by consumers (Jiang et al., 2017). Intramuscular fat content consists of a variety of fats, including omega-3 long-chain polyunsaturated fatty acids, which are beneficial to the brain and retinal development, in maternal and fetal health during pregnancy, cognitive system, and psychological state in humans (Williams, 2007; Mwangi et al., 2019). In addition, IMF also contains fatty acids resulting from the ruminal biohydrogenation of lipids, such as conjugated linoleic acid (CLA), which is an isomer of linoleic acid from food and has anticarcinogenic properties (Ferraz and Felício, 2010). The lipid content of meat is also rich in monounsaturated fatty acids (MUFA) which influence the melting point of fat, thus reducing the levels of bad cholesterol (LDL) in the bloodstream in humans (Jakobsen et al., 2008; Cesar et al., 2014). Although the many benefits to human health, animal fat consumption levels should be moderated, as beef also has saturated fatty acids (SFA), which, in excess, significantly increase the plasma concentration of low-density lipoprotein cholesterol (LDL), potentially increasing the risk of cardiovascular problems (Feitosa et al., 2017; Nettleton et al., 2017). For the Nellore breed, there are few studies that estimate genetic parameters for IMF. Feitosa et al. (2017) and Magalhães et al. (2018) observed relatively low values of heritability, 0.07 and 0.13, respectively. In a review, Utrera and Van Vleck (2004) summarized heritability estimates for IMF from works published up to 2004 and the mean estimate was 0.51, ranging from moderate (0.35) to high (0.65) in different taurine breeds. 9 2.4. Genetic correlations Researches that seek to investigate genetic associations between sexual precocity and meat quality traits are scarce in the literature. Studying animals Red Angus, McAllister et al. (2011) described estimates of genetic correlation between SC with marbling (MARB) and intramuscular fat content (IMF) of 0.01 and 0.05, respectively, suggesting that selection for SC will not promote a genetic gain in intramuscular fat deposition in the studied breed. In a work carried out in Japan with Wagyu cattle, Oyama et al. (1996) reported a negative and moderate genetic relationship between AFC and MARB (-0.39), higher than that found by Oyama et al. (2004) for the same breed (-0.24). Despite the difference in magnitude, these results indicate the existence of genes acting together in early sexual maturation and intramuscular adipogenesis. Scrotal circumference was moderately correlated with LMA and BF (0.31 and 0.25, respectively) in the study by Buzanskas et al. (2017), while Kluska et al. (2018) found lower genetic association values for SC–LMA and SC–BF (both 0.17). Despite the differences in magnitude in the works, the results suggest that selection for greater SC, in the long or medium time, should genetically increase LMA and BF. Some studies with Nellore animals showed negative and desirable genetic correlation estimates of moderate magnitude between BF and AFC (Caetano et al., 2013; Buzanskas et al., 2017; Kluska et al., 2018). Pires et al. (2016) reported an association of -0.69±0.35 between the same traits in Canchim animals, indicating that greater fat deposition can result in benefits in sexual precocity. These findings are biologically expected since lipid production or fat deposition is directly associated with the metabolism of certain hormones (such as steroids and eicosanoids) that modulate reproductive events, in addition to having a direct effect on the transcription of encoding genes of proteins essential for reproduction (Mattos et al., 2000). Also in Nellore, low and close to zero genetic correlations were estimated between LMA and AFC (Buzanskas et al., 2017; Kluska et al., 2018; Caetano et al., 2013) indicating that the progress of one trait doesn't tend to interfere with the other. Genetic correlations between carcass traits vary greatly in studies found in the literature. Between LMA and BF, some authors found genetic associations low or close to nullity in the Nellore breed (Ceacero et al., 2016; Buzanskas et al., 2017; Kluska et 10 al., 2018) indicating that selection for one of these traits will not imply in correlated response in the other. On the other hand, other studies have found positive correlations of moderate magnitude in the same breed (Gordo et al., 2012; Caetano et al., 2013). In a meta-analysis study in Nellore, Oliveira et al. (2017) reported a genetic association of 0.1694 between these traits, a result similar to the estimates of the aforementioned authors. Genetic correlation estimates of HCW with LMA and BF show a wide range from practically null to 0.62. Elzo et al. (2017), studying a multibreed Angus-Brahman population, observed genetic correlations of 0.57±0.08 and 0.12±0.13 of HCW with LMA and BF, respectively. Working with Hanwoo cattle, Do et al. (2016) obtained an association similar to that of Elzo et al. (2017) between HCW and LMA (0.62±0.003), however, they found a higher estimate between HCW and BF (0.31±0.005). Savoia et al. (2019) reported absence of genetic correlation between HCW and LMA (0.003±0.116) in young Piemontese bulls. In general, the marbling index can be considered a determining factor in the tenderness of beef (Warner et al., 2021). This relationship is supported by the strong and favorable genetic correlation estimates reported by Wheeler et al. (2010) and Mateescu et al. (2014), which were -0.52 and -0.50, respectively, suggesting that a lower shear force is genetically associated with higher marbling scores. Similar, genetic correlations between IMF and SF were estimated by the same authors to be - 0.52 (Wheeler et al., 2010) and -0.47 (Mateescu et al., 2014). However, the role of intramuscular fat content on beef tenderness is still quite controversial. Reverter et al. (2003), studying bovine breeds of temperate climate (TEMP) and breeds adapted to tropical climate (TROP), observed genetic associations between IMF and SF of -0.38 and -0.09, for TEMP and TROP, respectively, concluding that the inconsistencies between the estimates are likely due to differences in genetic architecture between breeds, or to other factors such as age differences and environmental influence on traits. In the literature, genetic associations between intramuscular fat content and marbling scores are high and positive. Several studies using pure or composite Taurine breeds have obtained estimates ranging from 0.56 to 1.00 (MacNeil et al., 2010; Wheeler et al., 2010; McAllister et al., 2011; Mateescu et al., 2014), which is expected 11 since these two traits are different ways of measuring the amount of fat in the meat. Working with Nellore animals, Bonin et al. (2021) reported estimates of the genetic correlation between MARB and IMF measured in meat samples taken from the Longissimus muscle in the 5th and 12th ribs, of 0.74 and 0.78, respectively, results consistent with the findings for the Bos taurus breeds and their crosses. Estimates of genetic correlations found in the literature between carcass and meat quality traits vary between studies, from different magnitudes to opposite signs of association. Gordo et al. (2018) reported a correlation of -0.47 between SF and LMA for Nellore cattle, indicating that selection for a higher yield of meat cuts should lead to favorable correlated responses in meat tenderness. In contrast, Wheeler et al. (2010) reported a correlation of 0.28 between SF and LMA, similar to that estimated by Reverter et al. (2003), that obtained a value of 0.27 for the group of Taurine animals (TEMP), and a low relationship between the traits (-0.14) for the group of animals of tropical climate (TROP). Between SF and HCW, Gordo et al. (2018) estimated a genetic correlation of -0.27, in agreement with the findings by Reverter et al. (2003), who reported similar estimates between the two groups of animals studied, of -0.20 (TEMP) and -0.21 (TROP). In turn, Wheeler et al. (2010) reported an association of 0.46, contrary to the results found by the authors previously mentioned. Between SF and BF, the traits were not genetically correlated in the study carried out by Gordo et al. (2018), suggesting that selection for one trait should not imply genetic gains in the other. However, Smith et al. (2007) reported correlation estimates between BF and tenderness at 7- and 14-day of maturation of -0.82 and -0.36, respectively. These divergences may be due to differences between the studied populations, adopted managements, and applied methodologies. Among the associations between marbling index and carcass composition traits, Gordo et al. (2018) obtained positive correlations of low and moderate magnitude, between MARB–BF (0.14) and MARB–LMA (0.38), respectively, and close to zero between MARB–HCW (-0.04), suggesting that in the long and medium term, the production of carcasses with greater finish or ribeye area should promote genetic progress towards marbling. Smith et al. (2007) estimated a genetic correlation of 0.17 between MARB and LMA, supporting, partially, the one reported by Gordo et al. (2018), however, obtained discordant estimates for MARB–BF (0.04) and MARB–HCW (0.51), 12 indicating that part of genes expressing HCW have an influence on MARB. Wheeler et al. (2010), contrary to the studies cited, reported estimates of -0.13±0.18 and - 0.28±0.26 between MARB–LMA, and MARB–HCW, respectively. However, the genetic correlation estimates in the study by Wheeler et al. (2010) showed high standard errors, indicating low predictive reliability, requiring caution in interpreting the results. The associations of intramuscular fat content and ribeye area indicate an antagonism in the action of shared genes between traits, with correlation estimates ranging from -0.15 to -0.22 (Reverter et al., 2003; Wheeler et al., 2010). For HCW, the genetic associations with IMF were -0.03 and -0.30, according to the studies carried out by Reverter et al. (2003) and Wheeler et al. (2010), suggesting nullity or an antagonism in the expression of characteristics. Torres-Vazquéz et al. (2018), working with an Australian Angus herd, found divergent estimates from the aforementioned authors, of 0.06 for IMF–LMA and 0.21 for IMF–HCW. The authors also reported a low, negative genetic correlation of -0.11 between IMF and BF. Differently, in a recent study with Nellore cattle, Bonin et al. (2021) obtained genetic correlations between IMF and BF of 0.17 and 0.41, when IMF was measured in muscle samples taken from the 5th and 12th rib, respectively, indicating that both IMF and BF must have genes in common acting in the same direction on fat metabolism. Considering the scarcity of information in the literature, it is necessary to design studies, as well as to develop methodologies and tools that seek to improve the accuracy of genetic evaluations. Moreover, it will be useful to better elucidate the relationship between female sexual precocity, carcass composition and meat quality traits, in order to find economically viable alternatives to improve them and thus achieve sustainable and high-quality production of Nellore meat. 2.5. Genome-wide association studies Genome-wide association studies (GWAS) aim to associate genomic regions with the phenotypes of interest through statistical analysis. Identification of variations in the genome (mainly single nucleotide polymorphisms - SNP) linked to or in regions (QTL - Quantitative Trait Loci) with great effect on a given characteristic, can provide a better understanding of biological functions and genetic influence on phenotypic 13 expression (Zhang et al., 2012; Yang et al., 2013; Magalhães et al., 2016; Magalhães et al., 2019; Pegolo et al., 2020). GWAS studies explore the existence of linkage disequilibrium (LD), which is an existing correlation structure between SNP and variants in the genome, resulting from evolutionary events, such as mutation, drift, and selection (Visscher et al., 2012; Visscher et al., 2017). Thus, LD structures arise because, when molecular markers are passed from one generation to another, in the absence of recombination, markers close to each other tend to be inherited together, causing alleles or SNP to correlate with each other in close regions in the DNA sequence (Lee et al., 2017). SNP are sequence polymorphisms, caused by the mutation of a single nucleotide at a specific locus in the DNA sequence. For a variation to be considered a molecular marker of the SNP type, the least frequent allele (MAF) must be present in at least 1% in the population (Brookes, 1999; Vignal et al., 2002). Currently, SNP are the most used markers in association studies, as they are widely distributed across the genome, with the necessary density for fine mapping (Vignal et al., 2002), in addition to the possibility of being in LD with the regions responsible for the expression of economically important traits (Zhang et al., 2012). With the emergence and availability of high-density SNP panels, it became possible to measure the variability found within the bovine genome in studies of genomic similarity, in order to characterize genomic regions and genetic profiles associated with several phenotypes (Mudado et al., 2016). One of the methods widely used in genomic evaluations is the single-step GBLUP (ssGBLUP), which was initially proposed by Misztal et al. (2009) and adapted for association analysis (ssGWAS) by Wang et al. (2012). This procedure, simultaneously, combines pedigree information, phenotypes and genotypes in a single step analysis, through the construction of relationship matrix H which encompass genomic (G) and pedigree-based (A) relationship matrices (Wang et al., 2012). The advantages of ssGWAS are that the method allows obtaining more accurate genomic evaluations in a simple way and fast computation, it can incorporate data from non- genotyped animals without the need to use pseudophenotypes, besides calculating the effects of each SNP and to estimate variations of these effects in the genome (Aguilar et al., 2010; Wang et al., 2012). 14 In beef cattle, few studies have identified genetic variants associated with carcass traits and meat quality in Nellore breed (Tizioto et al., 2013; Espigolan et al., 2015; Fernandes Júnior et al., 2016b; Lemos et al., 2016; Magalhães et al., 2016; Castro et al., 2017; Oliveira Silva et al., 2017). Using high-density (~770k) SNP genotyping data in Nellore cattle, Fernandes Júnior et al. (2016b) confirmed the polygenic nature of carcass traits, reporting important genomic regions distributed in 16 of 29 autosome chromosomes. In the study, the authors highlighted regions on chromosomes 5, 7, 8, 10, 12, 20, and 29 explaining 8.72% of the additive genetic variance for the eye area of the Longissimus muscle (LMA). Among the genes found, TSHR, CDKN2A/CDKN2B, SLC38A1/SLC38A2 and WWC1 stand out (Fernandes Júnior et al.; 2016b). These genes are involved in thyroid cell metabolism, cell cycle regulation, amino acid transport, and cell proliferation, respectively (van den Heuvel, 2005; Klimienė et al., 2008; Schiöth et al., 2013; Wennmann et al., 2014; Fernandes Júnior et al., 2016b), functions that can influence muscle growth. Oliveira Silva et al. (2017), identified genomic regions located on chromosomes 1, 6, 7, 8, 14, 15, 21, 24, and 28 associated with LMA in Nellore cattle and highlighted the genes ALKBH3 and HSD17B12 both playing a role in DNA repair, being related to fibroblast death (Nay et al., 2012) and steroid metabolism (Visus et al., 2011), respectively. Saatchi et al. (2014) conducted a series of genomic association studies using 50k panel genotyped data for different Bos taurus breeds. The authors identified QTL in the genomic regions in BTA6 and BTA14, regions later identified in the study by Oliveira Silva et al. (2017) and in BTA5, later identified by Fernandes Júnior et al. (2016b). These QTL, also harbor genes that have been associated with birth weight, weaning weight, yearling weight, mature weight, carcass weight, carcass yield, lipid content, and calving ease, indicating that QTL have pleiotropic properties (Saatchi et al., 2014). Liu et al. (2019) performed a GWAS to systematically detect additive and dominance variants for different traits in Chinese Simmental cattle. The authors detected FGF5 gene, in additive association with carcass weight (HCW), which is a gene of the Fibroblast Growth Factor (FGF) family, a group that is involved in embryonic development, cell growth, morphogenesis, tissue repair, tumor growth, and invasion (Ornitz and Itoh, 2015; Liu et al.; 2019). Working with a Nellore cattle 15 population, Espigolan et al. (2015) identified the EFCAB8 and VSTM2L genes, both related to skeletal muscle formation and development (Maki et al., 2002; Rossini et al., 2011; Espigolan et al., 2015), and might be excellent candidates for HCW. Furthermore, EFCAB8 belongs to the EF-hand calcium binding family, a set of genes that act directly on calpain and sorcin, proteins related to muscle synthesis and modulation of cellular Ca2+ channels, respectively (Espigolan et al., 2015). Through haplotype block analysis, Utsunomiya et al. (2017) reported that a mutation in the PLAG1 gene was associated with body size, weight, and reproduction in cattle. Functional evidences report that PLAG1 expresses a transcription factor that regulates IGF-2, insulin-like growth factor 2 (Van Dyck et al., 2007; Fortes et al., 2013; Utsunomiya et al., 2017). Fernandes Júnior et al. (2016b) identified and highlighted PLAG1 (BTA14) as the most promising gene associated with carcass weight, as it has a pleiotropic effect on several traits of economic interest in livestock (Fortes et al., 2013). Oliveira Silva et al. (2017) reported the same genomic region in BTA14, explaining 1.89% of the additive variance, linked to the expression of subcutaneous fat thickness (BF) in Nellore cattle. Comparing different approaches for GWAS studies (ssGWAS, Bayes A and Bayes B), Hay and Roberts (2018) identified LYN and RPS20 in BTA14 associated with BF in the three methods used, consistent with the reports by Oliveira Silva et al. (2017), showing the high influence of the 24Mb region on chromosome 14 for carcass traits. In the study by Oliveira Silva et al. (2017), 16 windows were associated with BF explaining more than 14% of the total genetic variance. The authors found the XKR4 gene, located on chromosome 14, previously identified as a candidate for rump fat thickness (Porto Neto et al., 2012). The gene XKR4 has been linked to the regulation of prolactin secretion in cattle (Bastin et al., 2014). In research conducted in hamsters, Cincotta and Meier (1987) observed a reduction in abdominal fat stores due to inhibition of prolactin secretion, concluding that the hormone plays an important role in the regulation of fat metabolism. In the search for candidates related to variation in meat and carcass traits in Nellore cattle, Tizioto et al. (2013) found a cluster of 20 genes associated with BF, acting in neuroactive ligand-receptor interaction pathway, indicating that the genes connected to this pathway play a role in the deposition of fat in beef cattle. Investigating 16 the regulation of adipogenesis in a transcriptome study, Khan et al. (2020) confirmed the evidence of Tizioto et al. (2013) by identifying differentially expressed genes involved in the fat deposition process in cattle, acting in neuroactive ligand-receptor interaction pathway. Also, in the study by Tizioto et al. (2013), genes for calpain and calpastatin associated with measures of meat tenderness at different stages of maturation were identified. The proteolytic system of calpain (CAPN1) is the main factor responsible for myofibrillar proteolysis during the post-mortem period, providing tenderness to meat. Calpastatin (CAST) is a regulator of post-mortem proteolysis, acting in the inhibition of CAPN1 and its increased activity leads to reduced tenderization of the meat (Koohmaraie, 1996). In this sense, it can be said that calpain and calpastatin genes are excellent candidates based on their biological functions and associations with beef tenderness. Several studies have reported CAPN1 and CAST associated with the tenderness of Bos taurus meat (Smith et al., 2000; Gill et al., 2009; Bolormaa et al., 2011; McClure et al., 2012; Ramayo-Caldas et al., 2016). However, few studies have observed the same genes related to meat quality traits in Bos indicus populations and their crosses (Tizioto et al., 2013; Magalhães et al., 2016; Leal-Gutiérrez et al., 2018). In a study associating μ-Calpain and Calpastatin polymorphisms with meat tenderness in a multibreed Angus–Brahman herd, Leal-Gutiérrez et al. (2018) found a significant effect on meat tenderness (p<0.0001) in animals with more than 80% Angus composition, thus it is expected that crossbreds tend to segregate a higher percentage of tenderness related alleles than pure Zebu breeds. These references show that genetic polymorphisms discovered in Bos taurus animals cannot be predictive in Bos indicus populations (Leal-Gutiérrez et al., 2019). Given the genetic differences between zebu and taurine animals, different regions of QTL have been reported in the literature associated with the marbling index (MARB). For example, Bedhane et al. (2019), when performing a genome-wide scanning study for meat quality traits in Hanwoo cattle, identified the GALR1 gene close to the most significant SNP in BTA24 for MARB. GALR1 is responsible for binding neuropeptides and peptide hormones (Jurkowski et al., 2013), in addition to being associated with the synthesis of bioactive lipids, lipids that affect cell functions due to changes in their concentration (Contos et al., 2002; Bedhane et al., 2019). 17 Several bioactive lipids were associated with the marbling index in Wagyu–dairy cross beef cattle (Bermingham et al., 2018) and, considering that all Wagyu's fame is attributed to the high degree of marbling of its meat, GALR1 becomes a candidate gene option for MARB. In the Nellore breed, Magalhães et al. (2016) located genomic regions associated with marbling, on chromosomes 5, 15, 16, and 25, explaining 3.89% of the total additive genetic variance. The authors observed that marbling and tenderness of the meat shared the same QTL in the BTA5, an interesting result since there is evidence that the marbling score gives the meat a feeling of tenderness (Wheeler et al., 2010; Mateescu et al., 2017; Luo et al., 2018). In the same study, the TNFRSF12A (BTA25) gene was identified, a TNF receptor superfamily member, associated with MARB. There are no reports in the literature on the role of this family of genes in regulating pathways associated with intramuscular fat deposition. However, Fonseca et al. (2020) through transcriptomics analysis, identified the TNFRSF12A gene differentially expressed in muscle tissue of Nellore cattle, associated with a low marbling score, corroborating with the finding by Magalhães et al. (2016). Another way to assess the intramuscular fat content of beef is by determining the percentage of lipids (IMF) through chemical analysis. However, studies investigating genomic regions associated with this measure are scarce in the literature, especially for Zebu breeds. In a survey conducted in Taurine animals, Hay and Roberts (2018) identified LYN and LYPLA1 associated with intramuscular fat content and subcutaneous fat thickness, genes that were previously related to feeding intake and growth in cattle (Lindholm-Perry et al., 2011). In addition, the region of chromosome 14 that harbors these genes has been associated with several other phenotypes of economic interest in livestock (Lindholm-Perry et al., 2011; Fortes et al., 2013; Fernandes Júnior et al., 2016b; Oliveira Silva et al., 2017; Magalhães et al., 2016; Hay and Roberts, 2018). In the same study, Hay and Roberts (2018) identified the SCD5 gene in a QTL on BTA6 that explained 1.47% of the additive genetic variance for IMF. SCD5 is a member of the family of genes that encode stearoyl-coenzyme A desaturase (SCD), an integral membrane protein of the endoplasmic reticulum, associated with increased fat accumulation, which catalyzes the conversion of saturated to monounsaturated fatty acids in several body tissues (Zheng et al., 2001; Lengi and 18 Corl, 2008). Considering the important role that these genes play in lipid biosynthesis, SCD5 may be of interest to improve the quality and accumulation of fat in beef. In general, traits of economic importance in beef cattle are polygenic in nature and are under the control of genetic and environmental factors. Therefore, detecting variations within the genome associated with carcass composition and meat quality traits can be a great challenge, mainly because these traits are controlled by numerous genes with small effects. Thus, conducting genome-wide association studies, aiming to investigate regions of great effect on carcass and meat quality traits, is of importance to identifying new genes, as well as validating those already found in the literature, in order to better understand the biological processes involved in the expression and, consequently, the phenotypic variability of these traits. 3. OBJECTIVES 3.1. General objective To estimate genetic parameters for female sexual precocity, carcass and meat quality traits and carry out a genomic-wide association study (GWAS) for carcass and meat quality traits in Nellore cattle, aiming to better understand the genetic inheritance of these traits in order to contribute for the genetic evaluation including genomic information in beef cattle in Brazil. 3.2. Specific objectives • Estimate (co)variance components and genetic parameters for female sexual precocity (age at first calving and scrotal circumference), carcass (longissimus muscle area, backfat thickness, and hot carcass weight), and meat quality traits (tenderness, marbling, and intramuscular fat content) in Nellore cattle, using genomic information; • Conduct GWAS in order to identify genomic regions and potential candidate genes acting in biological processes and metabolic pathways of meat and carcass traits. 19 4. REFERENCES Aguilar, I.; Misztal, I.; Johnson, D. L.; Legarra, A.; Tsuruta, S.; Lawlor, T. J. Hot topic: A unified approach to utilize phenotypic, full pedigree, and genomic information for genetic evaluation of Holstein final score. Journal of Dairy Science, v. 93, n. 2, p. 743-752, 2010. Albuquerque, L. G. Mercadante, M. E. Z.; Eler, J. P. Recents studies on the genetic basis for the selection of Bos indicus for beef production. 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Genomics, v. 71, n. 2, p. 182-191, 2001. 33 Chapter 2 – Genetic parameters estimates using genomic information for female sexual precocity, carcass and meat quality traits in Nellore cattle ABSTRACT – The objective of the present study was to estimate genetic parameters for longissimus muscle area (LMA), backfat thickness (BF), hot carcass weight (HCW), shear-force tenderness (SF), marbling score (MARB), intramuscular fat content (IMF), age at first calving (AFC), and scrotal circumference (SC) in Nellore cattle. The dataset available for this study were from 602,122 animals phenotyped for sexual precocity traits and 6,910 for carcass/meat traits and a total of 15,000 genotyped Nellore animals. The animals were genotyped using the Illumina Bovine HD Beadchip and the GeneSeek® Genomic Profilers HDi 75K and Low-Density 35K. The animals genotyped with GGP panels were imputed to the HD panel by the FImpute v3 software, using the ARS-UCD1.2 reference map. The (co)variance components and genetic parameters were estimated in two different ways, considering the Single-step (ssGBLUP) approach: 1) in a multi-trait analysis, performed for carcass and meat quality traits, by Bayesian inference using the GIBBS2F90 software; 2) and bi-trait analyzes, performed for sexual precocity traits with carcass and meat quality traits, by frequentist inference using the AIREMLF90 software. The animal model included additive and residual genetic effects as random; the fixed effects of GC (for all traits) and date of analysis as classes (for BF, SF, and MAR