Analisis Pewarisan Marka Molekuler dan Ekspresi Gen Imunitas Udang Vaname Tahan White Spot Syndrome Virus

Abstract

Udang vaname (Litopenaeus vannamei) merupakan komoditas ekspor utama di Indonesia, namun budidaya udang vaname masih terkendala serangan penyakit, akibat infeksi white spot syndrome virus (WSSV). Seleksi udang vaname yang secara genetik memiliki sifat tahan WSSV melalui seleksi marka single nucleotide polymorphism (SNP) pada gen anti-lipopolysaccharide factor (ALF) adalah salah satu cara pencegahan infeksi WSSV yang efektif. Penelitian ini bertujuan untuk mendeteksi SNP g.455 A>G pada gen ALF, menganalisis korelasi marka SNP pada gen ALF dengan daya tahan udang terhadap infeksi WSSV, mengukur ekspresi gen dan frekuensi SNP pada populasi yang tahan WSSV, serta menganalisis pewarisan marka SNP pada keturunan pertama (G1) udang vaname hasil seleksi. Marka molekuler SNP dideteksi menggunakan metode tetra-primer amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) pada 120 ekor udang vaname yang berasal dari 4 famili. Frekuensi SNP GG (q2) yang diperoleh selanjutnya dibandingkan dengan tingkat kelangsungan hidup (TKH) udang setelah dilakukan uji tantang WSSV dengan dosis LD70 untuk menilai korelasi antara SNP g.455 A>G pada gen ALF dengan daya tahan udang vaname terhadap WSSV. Analisis ekspresi gen ALF dan gen imunitas dilakukan pada famili yang memiliki TKH tertinggi dan terendah pasca infeksi WSSV. Analisis pewarisan marka molekuler SNP g.455 A>G pada gen ALF dilakukan dengan cara membandingkan frekuensi SNP pada G0 dan G1 berdasarkan perhitungan kesetimbangan Hardy-Weinberg. SNP g.455 A>G pada gen ALF berhasil dideteksi menggunakan metode ARMS-PCR dan memiliki korelasi yang tinggi antara genotipe GG (q2) dan TKH (p = 0.001 and R-square = 82.52%). Tingkat ekspresi gen ALF dan SOD serta frekuensi genotipe AA pada populasi udang vaname tahan WSSV lebih tinggi secara signifikan dibandingkan populasi udang vaname rentan WSSV.Frekuensi genotipe populasi induk dan benih udang vaname tidak berbeda nyata berdasarkan analisis chi-square (χ2 = 0.46 and p-value = 0.497), yang menunjukkan marka molekuler diwariskan sesuai persamaan Hardy-Weinberg. Penelitian ini menunjukkan bahwa populasi udang vaname tahan WSSV yang memiliki genotipe homozigot AA perlu diproduksi melalui seleksi marka molekuler.

The Pacific white shrimp (Litopenaeus vannamei) is Indonesia’s main export commodity, but its production is constrained by the white spot syndrome virus (WSSV). Selective breeding of disease-resistant broodstock based on single nucleotide polymorphism (SNP) in the anti-lipopolysaccharide factor (ALF) gene is an alternative strategy for solving the disease problem. This study aimed to detect the SNP g.455 A>G in the anti-lipopolysaccharide factor (ALF) shrimp gene, evaluate the correlation of SNP with WSSV-resistance trait, analyze the expression level of immunity genes and genotype frequencies of the WSSV-resistance population shrimp and analyze the SNP inheritance in the first generation of selected shrimp. A total of 120 individuals from 4 families were used to detect the SNP marker using tetra-primer amplification refractory mutation system-polymerase chain reaction (ARMS-PCR). The correlation between molecular markers and resistance to the WSSV trait was obtained by comparing the survival rate (SR) after the LD70 WSSV challenge test with the SNP frequency GG (q2) obtained before. The expression level of immunity genes was analyzed in families with the highest and lowest SR after LD70 WSSV infection. Molecular markers inheritance was analyzed by comparing the SNP frequencies in G0 and G1 generation based on the Hardy-Weinberg equilibrium. SNP g.455 A>G was successfully detected using the ARMS-PCR method and had a strong correlation between the marker GG (q2) and SR (p = 0.001 and R-square = 82.52%). The resistant population has significantly higher ALF and SOD gene expression levels and AA genotype frequency. The SNP marker was inherited, so the broodstock and pedigree have the same genotype frequencies according to chi-square analysis (χ2 = 0.46 and p-value = 0.497). These results suggested that the g.455 genotype AA could be selected to produce WSSV-resistant Pacific white shrimp.