by Jaime R. Alvarado Bremer, Brad L. Smith, Ching-Ping Lu, Jennifer Atchison and Alex Chapman

Transcription

1 The use of High Resolution Melting (HRM) to identify marine species, avian gender determination, and in studies of population genetics of tunas and billfish by Jaime R. Alvarado Bremer, Brad L. Smith, Ching-Ping Lu, Jennifer Atchison and Alex Chapman Department of Marine Biology, Texas A&M University at Galveston and Department of Wildlife and Fisheries Sciences, Texas A&M University

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4 High resolution melting analysis (HRMA) is a closedtube highly sensitive genotyping alternative capable of distinguishing single nucleotide polymorphisms (SNPs) and short insertion-deletion events by examining the melting profile of the PCR products

5 In this presentation I will talk about the impact of HRMA on three areas of study in my laboratory, namely...

6 Forensic Identification

7 Correct species ID of early stages is a prerequisite to properly characterize recruitment patterns What factors limit the numbers of marine invertebrates & fishes? Benthic vs Planktonic processes limiting s recruitment Hypotheses * Pre-settlement events *1) Lottery (chance) *2) Recruitment limitation + Post-settlement events +3) Predation-Disturbance +4) Competition

8 Forensic identification of Penaeid shrimp from the northern Gulf of Mexico nauplii

9 mtdna 16S rrna sequence identifies each of the species of Penaeid shrimp

10 However, sequencing involves numerous steps and thus is not practical when characterizing hundreds of samples. As an alternative, a multiplex PCR assay that incorporate diagnostic primers, was developed in my laboratory.

11 Still, although more efficient and cost effective than sequencing, multiplex PCR still requires numerous primers and involves multiple steps (PCR, gel electrophoresis, and photodocumentation) As an alternative, an unlabelled probe HRMA can distinguish all five species of shrimp in a closed-tube, fast (20 min), highly sensitive and inexpensive ($0.20/sample) assay

12 The unlabeled probe is specific to brown shrimp

13 Photo credit: Rafael Estrada Anaya (FishBase)

14 While adult grunts are easily identified their early stages are cryptic H. carbonarium-l108 (9.9mm TL, 8.0mm SL) Caesar grunt H. plumieri-l95 (10.1 mm TL, 8.2 mm SL) White grunt French grunt H. flavolineatum-l152 (9.3 mm TL, 7.9 mm SL)

15 Grunt species ID with unlabelled probe HRM Probe matches 44 nt of the mtdna COI sequence of H. carbonarium Segment is PCR-amplified using a universal COI forward primer and a grunt-specific reverse primer

16 Melting profiles of nine grunts (Haemulon spp.) from La Parguera, Puerto Rico

17 Recruitment at Collado, La Parguera, Puerto Rico in 2008

18 Recruitment at Collado, La Parguera, Puerto Rico in 2009

19 Species ID of tunas and billfish

20 HRM Billfish Species ID Assay

21 Short amplicon HRM Billfish ID Assay (Normalized Derivative Plot)

22 Forensic identification of tunas Melting Difference Plot using bluefin tuna as the reference

23 2. Avian Gender Determination Background Female birds are heterogametic (ZW) whereas males are homogametic (ZZ). Similar to approximately 50% of all avian species, Caribbean Flamingo lacks sexual dimorphism and behavior can often be misleading unless mating is observed at a breeding site.

24 ID gender of birds: conventional approaches Gender in birds can be determined using: Laparoscopy Morphometrically Behaviorally Hormone levels However, these methods are time-consuming and intrusive, particularly for chicks and juveniles

25 Molecular approaches Conserved Chromo-helicase-DNA binding genes (CHD) CHD1-W and CHD1-Z are located respectively on the W and Z chromosomes. Introns of CHD-W and CHD-Z differ in length Thus, a using single primer set that primes in orthologous sections of CHD-Z and CHD W, a size polymorphism (males 1-band, females 2-bands) can be visualized through gel electrophoresis

26 Prior research: A DNA test to sex most birds Richard Griffiths used DNA samples of 28 birds across the class Aves, along with two primers called P2 and P8, in a polymerase chain reaction (PCR). PCR products were separated by gel electrophoresis. Known sex is indicated of each individual.

27 Prior research: A DNA test to sex most birds However, when intron size difference between W and Z introns is small <12 bp long, agarose gels cannot be used to ID gender 2.5% Ethidium bromidestained agarose gel comparing 8 male (top) and 6 female (bottom) Caribbean Flamingo. Only a single band is visualized in both Sequencing of CHD-Z and CHD-W revealed an 8 bp difference in intron size in Caribbean Flamingo

28 HRMA to identify gender of Caribbean flamingo An HRM assay was designed after aligning and comparing the CHD- Z and CHD-W sequences of Caribbean Flamingo. DNA of 17 Caribbean Flamingo of known gender was isolated and amplified. DNA was isolated from dry blood blotted onto filter paper without organic extractions using the Hot Sodium Hydroxide Tris (Hot- SHOT) method.

29 Gender determination of flamingos

30 Universal application of HRMA for avian gender ID Since SA-Primers were designed based on the alignment of other avian W and Z sequences in GenBank, the potential for universal avian application was tested. Additional species were also tested: Greater Roadrunner Lesser Flamingo Saddle-billed Stork Scarlet Ibis Roseate Spoonbill White-bellied Stork

31 The use of High Resolution Melting Analysis (HRMA) in population genetics studies of marine fishes

32 Development of Markers for Population Genetic Studies 1. Amplification using multiple exon-primed intron primers for many different loci 2. Successful amplification was followed by sequencing 1. SNPs and polymorphic loci are identified for genotyping 2. Appropriate HRM method is developed for the SNPs of interest (e.g., unlabeled probe or short amplicon)

33 HRM profile of swordfish Calmodulin (CAM) gene

34 Melting profiles of swordfish ldha gene using an unlabelled probe

35 Locus SRP54 Short amplicon HRM

36 Locus ATPase Beta

37 Anonymous Loci originally targeted with universal OTY1 primers

38 Conclusion HRMA is a powerful, highly sensitive, and inexpensive genotyping alternative that can be tailored to a wide variety of applications Future research in my lab is focusing on the development of universal primers sets for Avian gender ID, species ID, and additional markers for population studies of fishes We are also interested in determining how the different mutations affect the shape of the heteroduplex curves

39 Acknowledgements Special thanks to Brandon Saxton of the Department of Wildlife and Fisheries Sciences, Texas A&M University, for assistance in the laboratory. Thanks to IDAHO Technology Inc., and Roche Applied Science for technical advise to optimize assays. Research was supported by the NOAA-SK program, the McDaniel Foundation, and the Texas Institute of Oceanography (TIO).