Growth Performance and Meat Quality Evaluations in Three-Way

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Growth Performance and Meat Quality Evaluations in Three-Way Cross Cattle Developed for the Tibetan Plateau and their Molecular Understanding by Integrative Omics Analysis Xiu-Kai Cao,†,‡ Jie Cheng,†,‡ Yong-Zhen Huang,†,‡ Xiao-Gang Wang,‡ Yu-Lin Ma,§ Shu-Jun Peng,‡ Buren Chaogetu,§ Zhaxi Zhuoma,§ and Hong Chen*,‡ ‡

College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi 712100, China Animal Disease Control Center of Haixi Mongolian and Tibetan Autonomous Prefecture, Delingha, Qinghai 817000, China

J. Agric. Food Chem. Downloaded from pubs.acs.org by MOUNT ALLISON UNIV on 01/01/19. For personal use only.

§

S Supporting Information *

ABSTRACT: Despite of favorable characteristics of high protein, low fat, and free-pollution, yak meat has intrinsically poor performance in tenderness and color, which is ever challenging yak sector. To this end, a three-way cross system was first developed for high quality beef of the Tibetan Plateau using Angus cattle (Bos taurus) as terminal sire to mate with 1/2 yak (F1) generated from♂Qaidam cattle (Bos taurus) × ♀yak (Bos grunniens). The withers height, chest girth, and body weight of 1/4 yak (F2) were all great higher than that of yak and 1/2 yak (P < 0.01), especially at later period, suggesting the faster growth rate of 1/4 yak. Also the dressing percentage was much better in 1/4 yak (P < 0.01). Tenderness and meat color were both significantly improved in 1/4 yak with some unpleasant sacrifice of PUFAs, such as EPA and DHA, and meat protein, given the significantly lower shear force and higher L* (P < 0.01). A total of 769 genes, including SREBF1, GHR, and FASN, the widely recognized causal genes of meat quality, were identified from 11947 differently expressed genes by the data integration of transcriptome, GWAS and QTL. These genes were significantly enriched for important pathway and GO terms, such as insulin signaling pathway, fatty acid biosynthesis, calcium signaling pathway, metabolic pathway, and cellular response to stress (P < 0.01). And 12 promising candidates were exemplified with annotation of H3K4me3 data from divergent meat quality, such as OSTF1, NRAS1, and KCNJ11. Interestingly, 75 high-altitude adaptive candidate genes were also detected in the list. This study is a first step toward high quality beef of the Tibetan Plateau and provides useful information for their molecular understanding. KEYWORDS: yak, three-way cross, tenderness, fatty acid, omics

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INTRODUCTION The Tibetan Plateau, widely known as the “Roof of the World”, stands 5 km high over a region of approximately 3 million km2 and is therefore characterized by severe cold, hypoxia, and strong ultraviolet radiation.1 Yaks (Bos grunniens) developed special physiological features for their remarkable survival in these harsh conditions while providing a livelihood (such as food, transportation, fuel, and shelter) for indigenous pastoralists.2 They have been an iconic symbol and completely melted into Tibetans’ socio-cultural life.3 Genetic exploration and exploitation of these vital domesticated animals is a sure way to support the socio-economic development in the Tibetan Plateau.4 Yak meat has favorable characteristics of high protein, low fat, and being pollution free.5 The increasing demand for beef has highlighted that more than 14 million domestic yaks in China are an alternative meat source, which has greatly encouraged the regional economy. Now the yak industry, however, is increasingly challenged with the intrinsically poor characteristics of toughness and oxidative rancidity (responsible for deterioration of meat color) of yak meat which strongly correlate with consumer satisfaction.6,7 A number of attempts have been made to overcome the problems, including physical treatments, chemical additives, and proteolytic digestion for yak meat. These methods may not achieve their practices at industrial level because of the unfavorable taste, © XXXX American Chemical Society

residual effects of antioxidants on human health, overtenderization due to unequal distribution of proteolytic enzymes, and, of course, additional feed costs. These methods have been thoroughly described and excellently reviewed.6,8 The traits determining meat quality are difficult to improve by purebreeding because of their low to moderate heritability while heterosis may generate desirable improvement in them.9 There has been a relatively large amount of studies in China on two-way crosses of yak with Chinese native yellow cattle (Bos taurus) or wild yak. Most of these studies, however, were focused on production traits instead of meat quality, and there is no systematic comparison of meat quality between yak and its hybrids yet.10−13 The available data collected by Chinese CNKI (http://www.cnki.net/) suggested the inadequate improvement in meat quality of yak hybrids (see the supplementary references). To this end, a three-way cross system for yak has been practiced in Qinghai Province (average elevation of 4058 m) since 2012. Angus cattle (semen) was introduced as terminal sire and mated to 1/2 yak (F1) female generated from ♂Qaidam cattle (semen) × ♀yak. This system, to the best of our knowledge, is the first attempt for high Received: October 6, 2018 Revised: December 3, 2018 Accepted: December 17, 2018

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DOI: 10.1021/acs.jafc.8b05477 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

Amino Acid Analysis. Muscle samples (0.5 g) were homogenized with 5 mL of 0.01 M hydrochloric acid and centrifuged at 5000 rpm for 5 min, and then 0.5 mL of supernatants was mixed with 8% salicylsulfonic acid for one night under 4 °C. The mixtures were further centrifuged twice at 12000 rpm for 10 min.22 The final supernatants were used for amino acids analysis using high-speed amino acid analyzer. Fatty Acid Methyl Ester Analysis. According to published procedure, total lipids of muscle sample were extracted into fatty acid methyl esters (FAMEs) by a boron trifluoride, hexane, and methanol (35:20:45 v/v/v) mixture. Then gas chromatography equipped with a 60 m × 0.25 mm capillary column and 0.25 mm film thickness was used to determine the fatty acid composition.24 Finally, fatty acids were analyzed by GC ChemStation software. RNA-Seq Library Construction and Sequencing. An additional 9 animals (3 individuals per group) were used to collect longissimus dorsi samples between the 12th and 13th rib for RNA-seq Total RNA were extracted to prepare libraries, which were sequenced as paired-end, 2 × 100 bp on an Illumina HIseq2000.25 Notably, rRNA was depleted with a Ribo-Zero rRNA Removal Kit (Epicenter, USA) for RNA-seq library preparation. The quality of RNA-seq reads was examined using FastQC. The passed reads were mapped to the Bos taurus genome (UMD 3.1.1) using Tophat2, and transcripts were assembled using Cufflinks. EdgeR was used to normalize the data and extract differentially expressed genes (DEGs) with FDR < 0.05. All data have been uploaded to NCBI as BioProject (ID: PRJNA343359) and will be released on 09/18/2020. Functional Annotation and Principal Component Analysis. Gene ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses of DGEs were performed to annotate gene function systematically using KOBAS (http://kobas. cbi.pku.edu.cn/). The P values were corrected using Bonferroni correction. Principal component analysis (PCA) was performed by ade4 package in R. Integrative Omics Analysis. To obtain significant loci underlying the phenotypic improvements, public data of GWAS, QTL, and H3K4me3 were used for integration analysis with muscle RNA-seq GWAS and QTL data of two trait classes, meat and carcass traits and production traits, were retrieved from Animal QTLdb (http://www. animalgenome.org/) with systematic curation (available breed information, correct location, and P < 0.01). The called H3K4me3 peaks of GSE61936 were involved in integration. GSE61936 was generated from Angus cattle with divergent tenderness. And the 3385 high-altitude adaptive candidate genes were retrieved from PMID: 26538003. All data were remapped to bovine genome UMD3.1.1 and visualized by CIRCOS. We developed GWAScore to probe genomic region with phenotypic effects for GWAS data. Error of phenotypic measurement, array density and statistical method make a small overlap in the significant SNPs of GWAS.26−28 This method favors genomic region over those factors by concisely recapitulating SNPs of different studies or traits. Here, a 0.2 Mb window was used for GWAScore as

quality beef of the Tibetan Plateau. A systematic comparison of meat quality is currently necessary for this terminal sire system. Identifying the causal loci of meat quality is a subject of intense research, and only a fraction of these loci have been discovered (exquisite examples were detailed by Garrick and Ruvinsky).9 A further molecular understanding for the meat quality of our composite cattle will benefit a desirable enhancement by marker-assisted selection. Multiomics analysis is the most efficient method to this end, which, however, remains financially prohibitive for most research groups.14,15 Exploiting available omics data may be an alternative to uncover genotype−phenotype interactions, which performs better than the candidate gene approach and individual omics. The application of integrative analysis in genetics has been illustrated in many publications.16−19 In our study, nine muscle transcriptomes were generated and used for integration with the public data of quantitative trait loci (QTL), genomewide association study (GWAS), and histone H3 lysine 4 trimethylation (H3K4me3). Therefore, the purpose of this study was to systematically evaluate the effects of the three-way cross system on the meat quality of these developed alpine cattle. And, an integrative omics analysis was used to obtain its molecular atlas which will feed into the upstream breeding system and benefit the traditional selection schemes.

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MATERIALS AND METHODS

Experiment Design and Animal Management. In the terminal sire system, yaks were artificially inseminated by Qaidam cattle to produce 1/2 yak (F1). Then Angus cattle were used as terminal sire to artificially inseminate 1/2 yak to create 1/4 yak (F2). These animals grazed in Qinghai alpine pasture in summer and autumn. And in spring and winter, they were housed in a barn and fed the designed diet according to NY/T 815-2004 (Chinese recommended standard). Notably, all yaks used in our study were domestic animals and they belong to a well-known breed named Tibetan Plateau yak which has good performance in meat and growth. Ingredients and chemical composition of the diet are shown in Table S-1. Animals were selected randomly and penned at 25 months of age and serially slaughtered at 36 months of age for finishing experiment and meat quality analysis. Growth Performance and Dressing Percentage. Growth records of 112 yak (♀), 57 F1 hybrids (♀), and 40 F2 hybrids (♀), including withers height, chest girth, and body weight at different months (0, 12, 24) were collected for growth performance analysis. A total of 18 finished animals from the three populations (yak, 1/2 yak, and 1/4 yak) with 6 individuals per group were randomly selected for meat quality analysis. These animals were fasted for 24 h before slaughter (live weight), and carcasses were chilled 24 h at 4 °C. Dressing percentage was live weight/cold carcass weight ratio. And then longissimus dorsi between the 12th and 13th rib were sampled for the following analyses. Meat Chemical Analysis. Protein (Kjeldahl N × 6.25) was quantified according to GB/T 5009.5-2016 (Chinese recommended standards) by Kjeltec Auto Analyzer; fat was quantified in the light of GB 5009.6-2016 (Chinese recommended standards) by Soxhlet Extractor; water was quantified according to GB/T 5009.3-2016 (Chinese recommended standards) by direct drying method. Meat Quality Measurements. Color coordinates (lightness, L*; redness, a*; and yellowness, b*) were measured using colorimeter;20 the power of hydrogen (pH) was measured using pH meter; shear force was measured by tenderness tester after the muscle samples were cooked in plastic bags in a water bath at 70 °C for 1 h;21 the amount of water lost during cooking loss was calculated by mass balance, expressed as a percentage of the initial weight (%);22 the water loss of meat sample pressed by 2.5 kg for 5 min presented as a percentage of the initial weight (%) was defined as pressing loss.23

n

GWAScore(x , x + 0.2) =

x , x + 0.2) − ngwas(x , x + 0.2) × ∑i =snp( log P 1

0.2 × ngwas(x , x + 0.2)

Where ngwas(x,x+0.2) was the number of GWAS in the genomic region (x, x + 0.2), nsnp(x,x+0.2) was the number of SNPs in genomic region (x, x + 0.2), P was the P value of significant SNPs. Notably, a 200 Kb window pointed out the same number of long known causal genes as the 1 Mb window did. RT-qPCR Analysis. Total RNA was isolated from 6 biological replicates in each population using RNAiso Plus Reagent (TaKaRa, Japan) and treated with DNase (QIAGEN, USA) to remove any remaining genomic DNA. RNA quality was controlled by three key points: (1) the integrity of total RNA detected by agarose gel electrophoresis was marked by 5S band (the less the better); (2) the quantity of total RNA detected by NanoDrop ND-1000 spectrophotometer was >1000 ng/μL; (3) the purity of total RNA detected by NanoDrop ND-1000 spectrophotometer was marked by 1.80 < B

DOI: 10.1021/acs.jafc.8b05477 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry Table 1. Growth Performance of the Three Populations in the Terminal Sire System (mean ± SD) growth traits withers height (cm)

chest girth (cm)

body weight (kg)

dressing percentage (%)

age (numbera)

yak (♀b)

1/2 yakc (♀)

0 (112/57/40) 12 (112/57/40) 24 (112/57/40) 0 (112/57/40) 12 (112/57/40) 24 (112/57/40) 0 (112/57/40) 12 (112/57/40) 24 (112/57/40) 24 (6/6/6)

50.80 ± 3.21 b 88.90 ± 6.42 c 95.80 ± 9.52 c 56.80 ± 4.17 b 102.70 ± 11.22 b 128.60 ± 14.22 c 13.59 ± 1.87 c 122.78 ± 9.96 b 174.13 ± 14.30 c 45.00 ± 3.47 c

53.40 93.71 103.35 58.13 107.56 147.38 15.70 131.70 224.20 53.84

± ± ± ± ± ± ± ± ± ±

1/4 yakd (♀)

3.09 b 8.07 b 13.67 b 3.94 b 10.34 b 15.34 b 2.02 b 14.11 b 36.55 b 4.80 b

58.56 105.75 121.60 61.33 122.50 184.40 22.67 145.25 420.75 61.38

± ± ± ± ± ± ± ± ± ±

P

4.75 a 10.56 a 15.56 a 5.31 a 12.25 a 20.55 a 3.54 a 25.84 a 53.42 a 6.11 a

3.21 1.35 1.49 4.19 2.67 1.39 4.41 1.40 5.80 5.36

× × × × × × × × × ×

10−23 10−25 10−17 10−9 10−10 10−52 10−51 10−10 10−106 10−4

The first was the number of yak, the second was 1/2 yak, and the third was 1/4 yak. bFemale animal. cF1 population of Qaidam cattle (♂) and yak (♀). dF2 population of Angus cattle (♂) and the F1 (♀). Values without the same letters within the same line indicate a significant difference after multiple-test correction at P < 0.01 (P < 0.003). a

Table 2. Chemical Analysis of Fat, Water, and Protein of Longissimus Dorsi in Three Populations in the Terminal Sire System (mean ± SD, g/100g) component

yak (n = 6, ♀a)

1/2 yakb (n = 6, ♀)

1/4 yakc (n = 6, ♀)

P

protein fat water

23.83 ± 0.31 a 1.61 ± 0.30 c 74.02 ± 1.72 a

22.31 ± 0.26 b 5.02 ± 1.40 b 68.33 ± 1.47 b

22.08 ± 0.60 b 6.53 ± 1.41 a 66.38 ± 1.39 c

7.60 × 10−5 5.46 × 10−8 5.17 × 10−8

a

Female animal. bF1 population of Qaidam cattle (♂) and yak (♀). cF2 population of Angus cattle (♂) and the F1 (♀). Values without the same letters within the same line indicate a significant difference after multiple-test correction at P < 0.01 (P < 0.006).

Table 3. Meat Quality of Longissimus Dorsi in Three Populations in the Terminal Sire System (mean ± SD) meat quality trait

yak (n = 6, ♀a)

1/2 yakb (n = 6, ♀)

1/4 yakc (n = 6, ♀)

shear force (kg) pressing loss (%) cooking loss (%) pH24h L* a* b*

9.59 ± 0.11 a 33.49 ± 2.07 a 28.45 ± 1.13 b 5.57 ± 0.08 a 34.80 ± 1.24 b 18.98 ± 1.18 9.60 ± 0.55 b

5.84 ± 1.98 b 30.98 ± 0.57 ab 31.42 ± 1.73 a 5.48 ± 0.04 ab 32.11 ± 1.73 b 18.36 ± 1.81 9.24 ± 0.86 b

4.11 ± 0.87 c 29.51 ± 0.32 b 29.30 ± 1.96 ab 5.36 ± 0.05 b 38.21 ± 2.08 a 20.20 ± 1.95 10.22 ± 0.65 a

P 3.75 2.20 8.10 1.52 2.75 3.66 5.30

× × × × × × ×

10−6 10−2 10−4 10−2 10−4 10−1 10−4

a

Female animal. bF1 population of Qaidam cattle (♂) and yak (♀). cF2 population of Angus cattle (♂) and the F1 (♀). Values without the same letters within the same line indicate a significant difference after multiple-test correction at P < 0.01 (P < 0.004).

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OD260/280 < 2.0 and OD260/230 > 2.0. Two reference genes, HPRT1 and ACTB, were used and arithmetic mean of their Ct was calculated for relative expression analysis.29 We exemplified three novel candidate genes (TRPM6, CARNMT1, and OSTF1) about meat quality identified by integrative omics analysis. Primer information was detailed in Table S-2. The RT-qPCR analyses were carried out using the SYBR Premix Ex TaqII kit (TaKaRa) with a Bio-Rad CFX96 Real-Time PCR System. The gene TRPM6 was excluded from further analysis because of its Ct > 40. The standard curve method revealed similar amplification efficiencies (>96%) of the remaining four genes using four serial dilution points (pooled cDNA with concentrations ranging from 1000 ng to 1 ng) (Table S-3). The relative expression levels were presented as 2−ΔΔCt. Statistical Analysis. One-way ANOVA of SPSS was used for difference analysis with a significant level set at P < 0.01 (indicated when different). A modified Bonferroni correction was used to adjust for multihypotheses testing for type I errors as

α′ =

RESULTS Growth Performance and Dressing Percentage were Further Improved in 1/4 Yak (F2) Population. Body dimensions (withers height and chest girth) and body weight were recorded for the animals in the three-way cross system at different ages (birth/1-year/2-year). As Table 1 showed, the birth withers height of 1/4 yak was about 8 and 5 cm higher than that of yak and 1/2 yak, respectively (P < 0.01). Also the birth chest girth of 1/4 yak was larger than that of yak and 1/2 yak (P < 0.01). Subsequently, the birth weight increased 67% and 44% compared with that of yak and 1/2 yak populations, respectively (P < 0.01). And these growing differences were more impressive at 2 y old, for example, the body weight of 1/ 4 yak was 420 kg while that was less than 225 kg in other two populations. Animals after finishing experiments were used for dressing percentage analysis. The 1/4 yak dressing percentage was 61 and was raised by 36% compared with yak dressing percentage (P < 0.01). Trade-offs between Fat and Other Chemical Components in the Terminal Sire System. Chemical analyses of fat, water, and protein of longissimus dorsi were made before meat quality evaluation. Fat of 1/4 yak meat had a 306% increase from 1.61 g of yak meat (P < 0.01) and even a 30%

α n

where α′ is the Bonferroni-corrected significance level, α is 0.01 (indicated when different), and n is the number of hypothesis tests. Results were presented as the mean ± standard deviation (SD). C

DOI: 10.1021/acs.jafc.8b05477 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

Table 4. Estimated Amount of Amino Acid of Longissimus Dorsi in Three Populations in Terminal Sire System (mean ± SD, g/100g) amino acid

yak (n = 6, ♀a)

1/2 yakb (n = 6, ♀)

Ala Arg Asp Cys Glu Gly His Pro Ser Tyr total NEAAsd Ile Leu Lys Met Phe Thr Val total EAAse

1.06 ± 0.07 c 1.10 ± 0.05 b 1.77 ± 0.03 b 0.36 ± 0.01 b 3.07 ± 0.10 b 0.77 ± 0.03 b 0.98 ± 0.03 1.01 ± 0.06 a 1.01 ± 0.02 a 1.20 ± 0.09 a 12.53 ± 0.37 0.81 ± 0.05 b 1.57 ± 0.07 b 1.77 ± 0.10 b 0.39 ± 0.01 b 1.05 ± 0.05 c 1.10 ± 0.02 1.32 ± 0.02 a 8.03 ± 0.17 b

1.29 ± 0.01 b 1.50 ± 0.03 a 2.04 ± 0.06 a 0.49 ± 0.04 a 3.45 ± 0.1 0 a 0.89 ± 0.03 a 1.04 ± 0.07 0.63 ± 0.18 b 0.87 ± 0.03 b 0.81 ± 0.06 b 13.01 ± 0.62 0.98 ± 0.05 a 1.96 ± 0.03 a 1.98 ± 0.13 a 0.72 ± 0.02 b 1.15 ± 0.08 b 1.07 ± 0.06 1.04 ± 0.03 b 8.90 ± 0.21 a

1/4 yakc (n = 6, ♀) 1.30 1.50 2.03 0.47 3.49 0.89 1.00 0.45 0.86 0.81 12.8 1.00 1.92 2.02 0.78 1.26 1.05 1.06 9.13

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.04 0.04 0.02 0.03 0.05 0.01 0.02 0.02 0.02 0.03 0.28 0.03 0.07 0.07 0.01 0.08 0.02 0.01 0.14

a a a a a a c b b a a a a a b a

P 1.62 2.70 2.19 6.23 8.18 1.04 9.70 3.40 4.55 2.36 3.45 2.74 7.57 1.88 4.55 3.34 7.90 2.35 7.22

× × × × × × × × × × × × × × × × × × ×

10−7 10−11 10−8 10−6 10−7 10−6 10−1 10−6 10−9 10−8 10−1 10−6 10−9 10−3 10−19 10−4 10−2 10−12 10−3

a

Female animal. bF1 population of Qaidam cattle (♂) and yak (♀). cF2 population of Angus cattle (♂) and the F1 (♀). dNEAA, nonessential amino acids. eEAA, essential amino acids. Values without the same letters within the same line indicate a significant difference after multiple-test correction at P < 0.01 (P < 0.002).

Table 5. Estimated Amount of Fatty Acid of Longissimus Dorsi in Three Populations in Terminal Sire System (mean ± SD, g/ 100g) fatty acid

yak (n = 6, ♀a)

C12:0 C14:0 C15:0 C16:0 C17:0 C18:0 C21:0 C14:1 C16:1 9c C17:1 10c C18:1 9c C20:1 C18:2, linoleic C20:2 (n = 6) C18:3, α-linolenic C18:3, γ-linolenic C20:3 (n = 3) C30:3, dohomo C20:4, arachidonic C20:5, EPAd C22:6, DHAe Σomega-3 Σomega-6 ΣSFAsf ΣMUFAsg ΣPUFAsh omega-6/3i

0.51 ± 0.01 a 1.55 ± 0.14 b 0.50 ± 0.12 a 20.58 ± 0.91 a 1.37 ± 0.15 a 20.94 ± 1.41 a 0.17 ± 0.01 c 2.33 ± 1.06 b 32.04 ± 1.99 a 0.12 ± 0.01 b 3.96 ± 0.30 a 0.06 ± 0.01 1.52 ± 0.34 a 0.09 ± 0.01 0.05 ± 0.01 0.14 ± 0.02 b 1.12 ± 0.18 a 0.43 ± 0.11 0.13 ± 0.04 2.15 ± 0.31 a 1.35 ± 0.18 a 45.46 ± 1.57 a 34.67 ± 2.36 7.51 ± 0.44 a 0.65 ± 0.18 b

1/2 yakb (n = 6, ♀) 0.97 ± 0.22 c 0.25 ± 0.06 b 18.01 ± 1.13 b 0.99 ± 0.39 b 8.14 ± 1.29 c 0.24 ± 0.02 0.50 ± 0.01 b 6.05 ± 1.21 a 0.63 ± 0.06 b 28.28 ± 2.58 b 0.23 ± 0.02 a 3.72 ± 1.68 a j 0.43 ± 0.17 b

1/4 yakc (n = 6, ♀) 0.16 ± 0.07 b 1.81 ± 0.13 a 0.18 ± 0.03 c 12.74 ± 1.16 c 0.54 ± 0.03 c 14.16 ± 1.17 b 0.19 ± 0.07 3.28 ± 0.77 a 1.63 ± 0.85 b 2.91 ± 0.48 a 23.13 ± 5.80 b 0.09 ± 0.04 c 0.12 ± 0.02 c

P 2.46 7.09 1.05 6.98 1.21 1.40 1.84 5.14 5.13 3.76 4.07 2.99 7.74

× × × × × × × × × × × × ×

10−7 10−7 10−5 10−9 10−4 10−10 10−1 10−9 10−6 10−7 10−3 10−7 10−6

3.16 × 10−5

0.23 ± 0.02 a 1.47 ± 0.93 a

0.13 ± 0.08 b 0.08 ± 0.01 b

3.51 × 10−4 1.31 × 10−3

0.43 ± 0.16 b 1.70 ± 0.95 a 28.60 ± 1.65 b 35.69 ± 2.59 5.85 ± 1.60 b 4.39 ± 2.58 a

0.20 ± 0.08 b 29.81 ± 0.67 b 31.05 ± 5.86 0.33 ± 0.08 c

2.89 8.62 8.09 1.35 3.84 4.86

× × × × × ×

10−7 10−4 10−12 10−1 10−9 10−3

a

Female animal. bF1 population of Qaidam cattle (♂) and yak (♀). cF2 population of Angus cattle (♂) and the F1 (♀). dEPA, eicosapentaenoic acid. eDHA, docosahexaenoic acid. fSFAs, saturated fatty acids. gMUFA, monounsaturated fatty acids. hPUFAs, polyunsaturated fatty acids. i Omega-6/3, ratio of total omega 6/omega 3 fatty acids. jUndetectable. Values without the same letters within the same line indicate a significant difference after multiple-test correction at P < 0.01 (P < 0.002).

D

DOI: 10.1021/acs.jafc.8b05477 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

Figure 1. (A) Venn diagrams for annotated genes of multiomics. (B) Principal component analysis of selected genes for 1/4 yak, 1/2 yak, and yak.

Alteration of Fatty Acid Composition with Unpleasant Decrease in Healthy Fatty Acids in 1/4 Yak. Twentyone kinds of fatty acids were detected in the three groups, including seven kinds of saturated fatty acids (SFAs), five kinds of monounsaturated fatty acids (MUSFs), and nine kinds of polyunsaturated fatty acids (PUFAs) (Table 5). Only C18:2, C30:3, and C20:4 were detected among the nine kinds of PUFAs, and others were reduced to undetectable level such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in 1/4 yak. As a result, ΣPUFAs of 1/4 yak is 0.33 g much lower than 7.51 g of yak (P < 0.01) and 5.85 g of 1/2 yak (P < 0.01). There was no difference in both ΣSFAs and ΣMUFAs between the two hybrid groups. Notably, significant reduction was observed in ΣSFAs of the two hybrid groups compared with that of yak (P < 0.01). Excessive amounts of omega-6 PUFAs and a very high omega-6/omega-3 ratio is found in today’s diets, which promotes the pathogenesis of many diseases.30 Omega-3 PUFAs could exert suppressive effects on omega-6 PUFAs and therefore are desirable in our common diet. As Table 5 showed, yak naturally had high level of omega-6 and omega-3 with lower ratio of omega-6/omega3, but these advantages disappeared in 1/4 yak as well as 1/2 yak. Together, all these results indicated that the three-way cross system further improved growth performance and meat quality despite of the loss of some healthy fatty acids abundant in yak. Important Causal Genes of Meat Quality Differentially Expressed in 9 Transrciptomes. To understand the molecular basis of the improved meat quality, three longissimus dorsi samples per group were collected for RNAseq The information on the obtained reads was summarized in Table S-4. The passed reads mapped to 25 926 genes and 11 947 genes were differentially expressed, including 10 782 differentially expressed genes (DEGs) from 1/4 yak vs 1/2 yak, 1299 DEGs from 1/4 yak vs yak, and 7705 DEGs from 1/2 yak vs yak (Figure 1A). Principal component analysis was performed to test the ability to discriminate the 9 samples using these DEGs. As shown in Figure 1B, these samples were grouped into three sets according to their blood, suggesting the clear distinct expression patterns of the three populations. Functional annotation was performed using GO and KEGG analyses to recognize the association of these DEGs with phenotypic alteration. Collectively, 8379, 993, and 5873 DEGs, revealed in 1/4 yak vs 1/2 yak, 1/4 yak vs yak, and 1/2 yak vs yak, were found to be enriched in 9960, 2165, and 8647 GO terms, respectively. And 4997, 536, and 3593 DEGs, identified from 1/4 yak vs 1/2 yak, 1/4 yak vs yak, and 1/2 yak vs yak, were significantly enriched in 304, 247, and 299 pathways. The

increase compared with that of 1/2 yak meat (5.02 g) (P < 0.01) (Table 2). However, the protein and water of meat were significantly decreased in both 1/2 yak (P < 0.01) and 1/4 yak populations (P < 0.01). The high fat and low water characteristic of 1/4 yak meat may suggest its improved quality. Improved Meat Quality of 1/4 Yak Mainly Characterized by Remarkable Tenderness and Color. Meat quality is normally defined by a multitude of component traits, among which tenderness is recognized as the most important determinant of the consumer eating experience.30 The Warner−Bratzler shear force test is an objective measurement that quantifies meat tenderness.20 Lower shear force indicates higher tenderness. More than a 2-fold decrease was observed in the shear force of 1/4 yak meat compared with that of yak meat and even about 1.4-fold decrease compared with that of 1/2 yak (Table 3). However, the yak blood made the shear force of 1/4 yak meat (4.11 kg) still higher than the estimated range from 0.86 to 2.99 kg within different breeds of cattle ( Bos taurus).9 There were no differences in pressing loss and cooking loss, which are both negatively associated with the water-holding capacity and are used as indicators of meat juiciness, between two-way cross and three-way cross animals meat.5 Meat color, revolving around myoglobin, is of utmost importance in consumer choice. It is the bright cherry red color (oxymyoglobin) that consumers associate with freshness.30 The terminal sire Angus cattle made lightness (L*) (P < 0.01) higher than that of 1/2 yak meat (Table 3). The change of pH also favored the meat color improvement although no significant difference was observed between 1/2 yak and 1/4 yak groups (P > 0.01), given the fact that the higher he pH the darker the meat. Abundance of Two Essential Amino Acids Further Increased Based on 1/2 Yak (F1) Population. The essential amino acids (EAAs) are of great significance for human metabolism. These amino acids cannot be synthesized by the human body.31 In this study, we found total EAAs abundance of 1/4 yak meat was the highest among the three groups, despite of no statistical significance between 1/2 yak and 1/4 yak group (P > 0.05). However, the abundance of Met and Phe of 1/4 yak were both markedly increased from 1/2 yak (P < 0.01). Table 4 detailed the changes of abundance of 17 amino acids including nonessential amino acids (NEAAs). Notably, only the abundance of Pro was reduced with hybridization (P < 0.01). Amino acids with abundance less than 0.1 g were not present in Table 4. E

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Journal of Agricultural and Food Chemistry Table 6. Twelve Promising Candidate Genes of Meat Quality Revealed by Integrative Analysis

mean FPKM GeneID 506322, NRAS(NRAS proto-oncogene, GTPase) 532713, CAMK2D(calcium/calmodulin dependent protein kinase II delta) 281961, OSTF1(osteoclast stimulating factor 1) 541040, SEC63(homologue, protein translocation regulator) 514039, EPCAM(epithelial cell adhesion molecule) 785768, RANBP2(RAN binding protein 2) 616237, CHCHD7(coiled coil helix coiled coil helix domain containing 7) 532060, KCNJ11(potassium voltage-gated channel subfamily J member 11) 507095, SPRY1(sprouty RTK signaling antagonist 1) 282081, SOCS3(suppressor of cytokine signaling 3) 513878, BUD23(rRNA methyltransferase and ribosome maturation factor) 507834, ESRRA(estrogen related receptor alpha)

genome position (UMD3.1.1)

GWAScore QTL

H3K4me3 tender/tough

1/4 yak

1/2 yak

yak

3:28723874−28733774 6:12964522−13272871

26.51 32.30

1 10

93/125 195/148

9.53 74.06

31.00 33.61

12.42 40.57

8:51440275−51501480 9:42651150−42718789

34.33 26.30

2 2

188/102 93/0

20.66 16.18

96.04 72.81

23.72 20.27

11:29626087−29636774 11:44509055−44570959 14:25052885−25059871

32.42 26.15 36.85

2 1 3

25/0 40/29 135/60

1.37 16.81 84.44

65.59 24.25 72.33

0.12 12.99 101.18

15:35650715−35653365

29.43

1

0/28

33.94

0.23

43.92

17:34747564−34755963 19:54458150−54461241 25:34072619−34083970

38.57 35.67 28.29

1 3 2

102/67 98/106 25/0

19.81 5.70 41.38

30.16 63.07 52.63

30.68 10.80 38.55

29:43220220−43228313

24.60

1

0/29

108.38

24.25

64.78

Figure 2. Identification of OSTF1 gene with integrative omics. (A) Visualization of H3K4me3, GWAS, QTL, and DEGs by UCSC for OSTF1 gene. (B) Table browser of omics data surrounded the candidate gene. (C) RT-qPCR validation of OSTF1 and CARNMT1 genes.

most enriched GO and KEGG terms were summarized in Figure S-1. Notably, energy metabolism and environment adaption terms were markedly overrepresented in the three pairwise comparisons, such as mitochondrion (GO:0005739), oxidation−reduction process (GO:0055114), fatty acid metabolism (ko10258), and hypertrophic cardiomyopathy (ko10373). The functional annotation results supported the phenotypic association of some DEGs, such as SCD, EGR1, SREBF1, CAPN1, CAPN2, GHR, FASN, MSTN, ESRRB, LEP, and TG, which was dependent on rudimentary knowledge about gene function. Integrative analysis of available multilayer

omics data may be efficient to give more molecular information underlying the improved meat quality. Integrative Analysis Annotated Putative and Pleiotropic Candidate Genes of Meat Quality. QTL, GWAS, and H3K4me3 public data were integrated into DEGs to reveal novel candidates without annotation of meat quality yet. A total of 364 meat and carcass QTL, 1496 GWAS markers, and 21 171 H3K4me3 peaks were collected. GWAScore was designed to quantify the contribution of a given genomic region to phenotypic variation using independent GWAS. And 1110 regions with GWAScore ranging from 10 to 61 were mapped, which covered 222 Mb of genome. The integrated F

DOI: 10.1021/acs.jafc.8b05477 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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(body size and body weight) is recognized as a highly heritable trait in mammals, and the genetic architecture of cattle stature is similar to that of humans which consists of many polymorphisms of small effect.42,43 In contrast, ∼50% of variation in dogs or horses is attributed to a small number of genes. These results indicated that the genetic architecture and the phenotypic effect size of causal loci may be various among mammals.44−46 However, a set of common genes regulating body size in mammals with relative large effect has been proposed. Four genes of them were differently expressed in the three populations, including GHR, IGF1R, LCORL, and PLAG1. The PLAG1 is one putative regulator of expression of the IGF2 gene, which encodes a key hormone for fetal growth and development;47,48 GHR encodes receptor for pituitary gland growth hormone which stimulates the production of IGF1 and is involved in regulating postnatal body growth; the IGF1R could bind insulin-like growth factor with a high affinity, including IGF1 and IGF2; the LCORL is a transcription factor, and its molecular function in stature has not been determined.47 All these causal genes are developmentally regulated and have detectable expression levels in our study. Interestingly, gene pleiotropy, such as HMGA2 and LCORL with significant effects on subcutaneous fat thickness, makes muscle transcriptom informative for the explanation of stature variations. The expression levels of IGF1R and PLAG1 in the three populations were both 1/2 yak > yak >1/4 yak; the expression levels of LCORL were 1/2 yak >1/4 yak > yak; the expression levels of GHR were 1/4 yak >1/2 yak > yak. These unexpected expression levels collectively suggested the presence of nonadditive allelic effects which mainly contribute to heterosis in 1/2 yak stature. For a systemic analysis of heterosis in 1/2 yak and 1/4 yak, transcriptomes and phenotypic records of Angus and Qaidam are necessary, which were absent in the present study given the artificial insemination practice of the three-way cross system. The meat quality of 1/4 yak was much improved, especially tenderness and meat color, with the sacrifice of some nutritional value (Table 2). The protein and ΣPUFAs content were both significantly reduced in 1/4 yak meat. PUFAs have been intensely studied because of its healthy importance and the WHO has provided diet recommendations that focused on increasing the intake of omega-3 PUFA and on reducing the intake of SFA (considered to be associated with increased cholesterol). And omega-6/3 PUFA ratio was 0.65 in yak meat (Table 5) much lower than 4 according to the WHO recommendations.30 The reduction of total fatty acids content suggested the weak adaptability of 1/4 yak to the harsh conditions typical for yak, considering the potential protective effects of some trans fatty acids against the development of coronary heart diseases.49 The weaknesses of the nutritional value of 1/4 yak meat consequently benefited the lipid content and tenderness (Table 3). The appreciation of tenderness is generally positively associated with the beef lipid content which also plays an important role on the juiciness.50 Meat with more fat is always less dry than lean meat when chewing in the mouth. Our results supported these above positive associations. Additionally, early puberty and maturation were observed in 1/4 yak population which could promote fat deposition as well.9,51 Meat color, as the first characteristic perceived by the consumer, is often the only one trait considered at the time of purchase. The favorable red color of beef is mainly conferred by a pigment, myoglobin.52 It is not surprising in our study that 1/4 yak meat had a much higher

result is shown in Figure S-2 and could be visualized by UCSC (http://genome.ucsc.edu/) with the supplied tracker files as well. A total of 769 genes associated with meat quality were annotated by the integrative analysis (Table S-5), including the well-known gene FASN (Figure S-3). Further, we selected some genes for H3K4me3 annotation and top 12 genes were lists in Table 6. A systematic literature and database curation suggested their pleiotropic effects and uncharacterized role in meat quality. The OSTF1 gene, revealed by integrative analysis as shown in Figure 2, is involved in bone development (GeneCard: https://www.genecards.org/) and was found to be associated with glucose homeostasis (GWAS Catalog: http://www.ebi.ac.uk/gwas/), body mass index, and nonalcoholic fatty liver disease as well.32,33 The NRAS gene has intrinsic GTPase activity and knockout of mouse NRAS lead to short tibia and increased circulating glucose level (IMPC: http://www.mousephenotype.org/). Mutations in this gene have been associated with many diseases.34−36 The protein encoded by KCNJ11 is an integral membrane protein and inward-rectifier type potassium channel which participates in a wide range of physiologic responses (MGI: http://www. informatics.jax.org/). At present the role of KCNJ11 in growth traits and tenderness is beginning to be realized.37,38 Interestingly, 75 high-altitude adaptive candidate genes were also detected in the list (discussed below). These putative and pleiotropic candidates provided more molecular information on phenotypic effects of the three-way cross system.

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DISCUSSION Yaks, being able to maximize highland herbage resources, are the cornerstone of animal husbandry of the Tibetan Plateau. The last decade has seen a prosperous development of the yak industry which greatly encouraged the regional economy. However, it is a currently pressing need to evaluate the meat quality of yaks and their hybrids for consumer satisfaction. From the perspectives of genetics and breeding, introducing exogenous blood with favorable meat quality may be a better choice, given the quite low heritability of meat quality. Here, a three-way cross rather than two-way cross system was developed using Angus cattle which is a worldwide popular beef breed for its superior marbled appearance together with excellent tenderness and meat color.39 By introducing Qaidam bulls as parental F0, the three-way cross system could reduce the amount and degree of calving difficulty of the two-way cross system between Angus and yak. This native breed was distributed in Qinghai Province (average altitude of 4058 m) and has a medium-sized stature compared with Angus and yak.13,40 Their fine reproduction performance is preferred for yak hybridization in the Tibetan Plateau.41 It is the first threeway cross system developed for high-quality beef in the Tibetan Plateau, and our results supported the system’s fine compatibility of meat quality with alpine adaptability and reproductivity. F1 hybrids generally grow faster and become larger than yaks, especially in later periods, which agreed with our results (Table 1). By introducing Angus, inbreeding depression or the disappearance of heterosis resulted from backcrosses to the yaks or to local cattle breeds was not present and the growth performance was further improved in 1/4 yak population since the day of their birth compared with the 1/2 yak (Table 1). The knowledge of the structural genetic basis may give molecular insight into these phenotypic improvements. Stature G

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molecular understanding of the improvement in 1/4 yak meat quality from the perspective of high-altitude adaptation.

expression level of myoglobin gene than that of 1/2 yak and yak, suggesting the pleasant color. Notably, meat characteristics are complex and depend on multifactorial determinants, thus the 1/4 yak especially need better feeding, slaughtering conditions, and technological considerations for their high meat quality. The heritability of most quality traits is low to moderate, suggesting the significant nonadditive allelic effects on meat quality variations. Many transcriptomes have been conducted using Bos taurus × Bos indicus crosses or beef cattle × milk cattle crosses, where nonadditive effects and a very large difference in meat quality are known for candidate detection. However, it is difficult to select a list of promising candidate genes from a sea of DEGs produced by comparative transcriptomes. Nowadays, we are in a phase of unprecedented progress in health applications of multiple omics. Data integration of available omics is an emerging and efficient alternative to uncover genotype−phenotype interactions. Comparative genomics makes our integrative analysis plausible and informative in identifying genes about yak meat quality. Almost all yak genes have their roots in closely related cattle, and these genes in the two species probably share the same functions.3,53 This kind of integration based on comparative genomics of different species is widely used to find important genomic sequences for breeding, given the lack of multilayer data in livestock.19,54 Excitingly, the Functional Anotation of ANimal Genomes (FAANG) project is now working to help us understand the genotype to phenotype link in domesticated animals by producing original and invaluable data. Here, 11947 DEGs were detected among transcriptomes of yak, 1/2 yak, and 1/4 yak which differed in tenderness. To find genes with more prominent roles in meat quality, 3784 DEGs with 10 < mean FPKM < 100 were used for integrative analysis and finally 769 genes were pointed out. The identification of major genes underlying phenotypes depends on the presence of quantitative trait nucleotides (QTN). Empirically, low abundance genes have futile effects on muscle, and ubiquitous genes have much less QTN or individuals would be eliminated by natural selection. Most well-known genes about meat quality have moderate abundance in muscle, such as SCD, EGR1, SREBF1, CAPN1, CAPN2, GHR, FASN, FABP4, CAST, and DGAT1. The 769 genes (Table S-5) were significantly enriched for important pathway and GO terms, such as insulin signaling pathway, fatty acid biosynthesis, calcium signaling pathway, metabolic pathway, and cellular response to stress (data not shown). Interestingly, 75 candidates (Table S-5) in the list were reported to be differently expressed in heart, kidney, liver, or lung between yak and cattle indicating their potential functions in high-altitude adaptation.53 These genes were involved in adrenergic signaling in adrenergic signaling in cardiomyocytes, ATPase activity, cardiac muscle adaptation, and the hormone metabolic process (data not shown). For example, for platelet-derived growth factor receptor α (PDGFRα) and phospholipase C delta 3 (PLCD3), their expression levels in the three populations were both yak > 1/2 yak > 1/4 yak. PDGFRα, a receptor located on the surface of a wide range of cell types, and studies suggested its role in organ development, wound healing, and tumor progression.55 PLCD3, a member of the phospholipase C family, is associated with a variety of cellular responses to extracellular stimuli by inducing protein kinase C and increasing cytosolic Ca2+ concentrations.56 All this information together gave a different

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.8b05477. Dietary composition (as-fed) (Table S-1); primers for RT-qPCR analysis (UMD3.1.1) (Table S-2); raw data for standard curve (pooled cDNA) (Table S-3); summary of reads mapping to the reference genome (UMD3.1.1) (Table S-4); the 769 genes associated with beef quality revealed by integrative analysis including 75 high-altitude adaptive candidates (UMD3.1.1) (Table S5); conclusion-drawing references from CNKI (supplementary references); the most enriched GO and KEGG terms of DEGs (Figure S-1); visualization of integrative omics of QTL, GWAScore, DEGs, and H3K4me3 (Figure S-2); identification of FASN gene with integrative omics (Figure S-3) (PDF) Collected omics datasets for integrative analysis presented in 11 files (TXTs)

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AUTHOR INFORMATION

Corresponding Author

*Telephone: 86-029-87092102. Fax: 86-029-87092120. Email: [email protected]. ORCID

Hong Chen: 0000-0001-5128-6764 Author Contributions †

X.-K.C., J.C., and Y.-Z.H. contributed equally to this work. X.K.C. and Y.-Z.H. designed the research; J.C., Y.-L.M., B.C., and Z.Z. conducted the research; X.-K.C., X.-G.W., and S.-J.P. analyzed the data. The manuscript was written and edited by X.-K.C. and H.C. Funding

This work was supported by the Haixi Project of Qinghai Province: Identification of key genes of growth and highquality meat in Yak Multi hybrids, the Program of National Beef Cattle and Yak Industrial Technology System (CARS-38), and the National Natural Science Foundation of China (31772574, 31601926). Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED ACTB, actin beta; CARNMT1, carnosine N-methyltransferase 1; DEG, differently expressed gene; DHA, docosahexaenoic acid; EAA, essential amino acid; EPA, eicosapentaenoic acid; FAME, fatty acid methyl ester; FASN, fatty acid synthase; GO, gene ontology; GHR, growth hormone receptor; GWAS, genome-wide association study; H3K4me3, histone H3 lysine 4 trimethylation; HMGA2, high mobility group AT-hook 2; HPRT1, hypoxanthine phosphoribosyltransferase 1; IGF1, insulin like growth factor 1; IGF1R, insulin like growth factor 1 receptor; KCNJ11, potassium voltage-gated channel subfamily J member 11; KEGG, Kyoto Encyclopedia of Genes and Genomes; LCORL, ligand dependent nuclear receptor corepressor like; MUSF, monounsaturated fatty acid; NEAA, nonessential amino acid; NRAS1, NRAS protooncogene, GTPase; PDGFRα, platelet-derived growth factor H

DOI: 10.1021/acs.jafc.8b05477 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry receptor, alpha polypeptide; PLAG1, PLAG1 zinc finger; PLCD3, phospholipase C delta 3; PUFA, polyunsaturated fatty acid; OSTF1, osteoclast stimulating factor 1; QTN, quantitative trait nucleotide; QTL, quantitative trait loci; RTqPCR, real-time quantitative PCR; SFA, saturated fatty acid; SNP, single nucleotide polymorphism; SREBF1, sterol regulatory element binding transcription factor 1; TRPM6, transient receptor potential cation channel subfamily M member 6

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