Molecular analysis of a RAPD marker (B20) reveals two microsatellites and differential mRNA expression in Penaeus vannamei.
We previously reported a population-specific DNA fragment (B20) in Penaeus vannamei shrimp, fragment found using the randomly amplified polymorphic DNA (RAPD) procedure, that was present in Population 2 but not in Populations 1 and 4. The specific objectives of this study were to clone and sequence this genetic marker, determine if all or part of this cloned sequence could be found in any of the other populations in which this marker could not be amplified, and examine if this marker represents a functional gene by examining the steady-state levels of mRNA expression using Northern blot hybridization. Sequence information of the 1259-bp B20 clone revealed two microsatellites and two candidate open reading frames. Although the entire B20 sequence could only be amplified in Population 2 (from Ecuador), Population 3 (a hybrid of Populations 1 and 2), and a few individuals from wild Ecuadorian shrimp samples, portions of the B20 DNA could be amplified in individuals from Populations 1, 2, 3, candidate Population 4, and wild Ecuadorian samples. These microsatellites vary in size between populations and families. Northern blot hybridization analysis using radiolabeled B20 probe detected two mRNA transcripts of approximately 1.5 and 2.0 kb. Expression data throughout development indicated that these transcripts were present at low levels in nauplii from two of the three crosses examined using broodstocks of Population 1. Higher levels were observed in postlarvae (PL) 6, PL8, and PL10 in one of the three crosses. Individuals from all crosses showed higher levels of expression in the juvenile tail muscle. The mRNA transcript levels were undetected in zoea 3, PL2, and PL4 stages of development and broodstock tail muscle. The levels of expression of B20 mRNA transcripts varied significantly between Populations 1, 2, 3, 4, and wild Ecuadorian individuals as well as between families and within individuals representative of seven families from Population 1. In summary, the B20 clone revealed the presence of two microsatellites that vary in size between populations. These microsatellites will be useful for estimating genetic diversity within and between populations, identifying family-specific markers, and mapping loci responsible for economically important traits in penaeid shrimp. The mRNA levels detected by the B20 clone showed differential expression during development, and the pattern of expression was influenced by the genetic background of the parental crosses used.
Garcia
D K
DK
Department of Comparative Medicine, Tufts University School of Veterinary Medicine, North Grafton, Massachusetts 01536, USA.
Dhar
A K
AK
Alcivar-Warren
A
A
D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D003433:Crosses, Genetic; D005260:Female; D018507:Gene Expression Regulation, Developmental; D005819:Genetic Markers; D018895:Microsatellite Repeats / Q000235:genetics*; D008969:Molecular Sequence Data; D009132:Muscles / Q000737:chemistry; D016366:Open Reading Frames / Q000235:genetics; D033561:Penaeidae / Q000196:embryology / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis; D019105:Random Amplified Polymorphic DNA Technique; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013623:Tail / Q000737:chemistry
D005819:Genetic Markers; D012333:RNA, Messenger