Chan, Wing Hin. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 126-143). / Abstracts in English and Chinese. / Acknowledgement --- p.ii / Abstract --- p.iii / 摘要 --- p.vi / Table of Content --- p.viii / List of Figures --- p.xiv / List of Tables --- p.xvi / Abbreviations --- p.xviii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Food and herb authentication --- p.1 / Chapter 1.1.1 --- Background and definition --- p.1 / Chapter 1.1.2 --- Importance of species identification in food and herb authentication --- p.2 / Chapter 1.1.2.1 --- Primary health care --- p.2 / Chapter 1.1.2.2 --- Food and herb safety --- p.3 / Chapter 1.1.2.3 --- Conservation --- p.4 / Chapter 1.1.3 --- Methods for species identification in food and herb authentication --- p.4 / Chapter 1.1.3.1 --- Morphological identification --- p.5 / Chapter 1.1.3.2 --- Chemical analysis --- p.6 / Chapter 1.1.3.3 --- Molecular analysis --- p.9 / Chapter 1.1.4 --- Legislation --- p.11 / Chapter 1.1.4.1 --- Labeling ´ب --- p.11 / Chapter 1.1.4.2 --- Chinese medicine : --- p.12 / Chapter 1.1.4.3 --- Conservation --- p.12 / Chapter 1.2 --- Dongchongxiacao --- p.13 / Chapter 1.2.1 --- Background information of Dongchongxiacao --- p.13 / Chapter 1.2.2 --- Classification of fungal part of Dongchongxiacao --- p.14 / Chapter 1.2.3 --- Dongchongxiacao as a Traditional Chinese Medicine. --- p.15 / Chapter 1.2.4 --- The Dongchongxiacao market --- p.16 / Chapter 1.2.5 --- Adulteration and contamination of Dongchongxiacao --- p.18 / Chapter 1.2.6 --- Authentication of Dongchongxiacao --- p.19 / Chapter 1.2.6.1 --- Morphological identification --- p.19 / Chapter 1.2.6.2 --- Chemical analysis --- p.20 / Chapter 1.2.6.3 --- Molecular analysis --- p.22 / Chapter 1.2.6.3.1 --- "FINS analysis with genomic ITS, nrLSU, EF-lα and rpbl regions for fungal analyses" --- p.22 / Chapter 1.2.6.3.2 --- FINS analysis with mitochondrial CytB and COI regions for caterpillar analyses --- p.24 / Chapter 1.3 --- Abalone --- p.26 / Chapter 1.3.1 --- Background information of abalone --- p.26 / Chapter 1.3.2 --- Abalone as food --- p.27 / Chapter 1.3.3 --- The abalone market --- p.28 / Chapter 1.3.4 --- Adulteration of abalone --- p.31 / Chapter 1.3.5 --- Authentication of abalone --- p.32 / Chapter 1.3.5.1 --- Morphological identification --- p.32 / Chapter 1.3.5.2 --- Chemical analysis --- p.32 / Chapter 1.3.5.3 --- Molecular analysis --- p.33 / Chapter 1.3.5.3.1 --- FINS analysis with mitochondrial COI and 16S rDNA --- p.33 / Chapter 1.3.5.3.2 --- Haliotis-specific detection --- p.34 / Chapter 1.4 --- Aim and Objectives --- p.35 / Chapter Chapter 2 --- Materials and Methods --- p.36 / Chapter 2.1 --- Materials used in this sutdy --- p.36 / Chapter 2.1.1 --- Dongchongxiacao and Cordyceps samples --- p.36 / Chapter 2.1.2 --- Downloaded sequences from NCBI database included in Dongchongxiacao study. --- p.45 / Chapter 2.1.3 --- Abalone and gastropod samples --- p.48 / Chapter 2.1.4 --- Downloaded sequences from NCBI database included in abalone study --- p.54 / Chapter 2.2 --- Reagents and equipments : --- p.56 / Chapter 2.2.1 --- Chemical test on the presence of potassium alum in Dongchongxiacao --- p.56 / Chapter 2.2.2 --- Sample preparation and DNA extraction --- p.57 / Chapter 2.2.3 --- Polymerase Chain Reaction --- p.57 / Chapter 2.2.4 --- Agarose gel electrophoresis and Gene Clean --- p.57 / Chapter 2.2.5 --- Cloning --- p.58 / Chapter 2.2.6 --- Cycle sequencing --- p.58 / Chapter 2.3 --- Experimental procedures --- p.58 / Chapter 2.3.1 --- Morphological observation of Dongchongxiacao and abalone --- p.59 / Chapter 2.3.2 --- Chemical test of potassium in Dongchongxiacao --- p.59 / Chapter 2.3.3 --- Sample preparation and DNA extraction --- p.60 / Chapter 2.3.4 --- Polymerase Chain Reaction --- p.61 / Chapter 2.3.5 --- Agarose gel electrophoresis and Gene Clean --- p.64 / Chapter 2.3.6 --- Cloning --- p.65 / Chapter 2.3.7 --- Cycle sequencing --- p.67 / Chapter 2.3.8 --- Sequence analyses --- p.67 / Chapter 2.3.9 --- Haliotis-specific primer design and PCR test --- p.68 / Chapter Chapter 3 --- Results --- p.71 / Chapter 3.1 --- Dongchongxiacao --- p.71 / Chapter 3.1.1 --- Morphological observations --- p.71 / Chapter 3.1.2 --- Chemical test of potassium alum --- p.77 / Chapter 3.1.3 --- Sequence analyses --- p.79 / Chapter 3.1.4 --- The dendrograms --- p.81 / Chapter 3.2 --- Abalone --- p.91 / Chapter 3.2.1 --- Morphological observations --- p.91 / Chapter 3.2.2 --- Sequence analyses --- p.92 / Chapter 3.2.3 --- The dendrograms --- p.94 / Chapter 3.2.4 --- Haliotis-specific PCR --- p.96 / Chapter Chapter 4 --- Discussion --- p.98 / Chapter 4.1 --- Dongchongxiacao --- p.98 / Chapter 4.1.1 --- Species identification of Dongchongxiacao and related Cordyceps species --- p.98 / Chapter 4.1.1.1 --- Ophiocordyceps sinensis --- p.98 / Chapter 4.1.1.2 --- Cordyceps gunnii --- p.100 / Chapter 4.1.1.3 --- Metacordyceps taii --- p.102 / Chapter 4.1.1.4 --- Cordyceps militaris --- p.103 / Chapter 4.1.2 --- Adulteration of Dongchongxiacao and labeling --- p.104 / Chapter 4.1.3 --- Hosts of Dongchongxiacao fungi and relationship between them --- p.107 / Chapter 4.2 --- Abalone --- p.109 / Chapter 4.2.1 --- Species identification of abalones and other gastropod species by FINS analysis --- p.109 / Chapter 4.2.1.1 --- Haliotis species --- p.109 / Chapter 4.2.1.1.1 --- Haliotis diversicolor --- p.110 / Chapter 4.2.1.1.2 --- Haliotis discus --- p.110 / Chapter 4.2.1.1.3 --- Haliotis asinina --- p.111 / Chapter 4.2.1.1.4 --- Haliotis rufescens --- p.111 / Chapter 4.2.1.1.5 --- Haliotis midae --- p.111 / Chapter 4.2.1.1.6 --- Haliotis madaka --- p.112 / Chapter 4.2.1.1.7 --- Haliotis rubra --- p.113 / Chapter 4.2.1.1.8 --- Haliotis iris --- p.113 / Chapter 4.2.1.1.9 --- Haliotis corrugata --- p.114 / Chapter 4.2.1.2 --- Concholepas concholepas --- p.114 / Chapter 4.2.1.3 --- Hemifusus species --- p.115 / Chapter 4.2.1.4 --- """Dried abalone slice"" samples (D1 to D3) and canned top-shell (E5)" --- p.115 / Chapter 4.2.2 --- Haliotis-speciflc PCR --- p.115 / Chapter 4.2.3 --- Adulteration of abalone and labeling --- p.116 / Chapter 4.3 --- Significance and limitation of molecular approaches in authentication of food and herbs --- p.117 / Chapter 4.3.1 --- FINS analysis --- p.117 / Chapter 4.3.1.1 --- High interspecific variability but low intraspecific variations --- p.118 / Chapter 4.3.1.2 --- Amplification with universal primers --- p.118 / Chapter 4.3.1.3 --- Insufficient DNA sequence available in database --- p.119 / Chapter 4.3.1.4 --- Contamination by foreign DNA and amplification of undesirable DNA in sample mixture --- p.120 / Chapter 4.3.1.5 --- Amplification of degraded DNA --- p.121 / Chapter 4.3.1.6 --- Suggested regions for authentication of Dongchongxiacao and abalone based on FINS analysis results --- p.121 / Chapter 4.3.2 --- PCR with specific primers for targeted amplicons --- p.122 / Chapter 4.3.3 --- Other limitations of molecular approaches in authentication of food and herbs --- p.123 / Chapter 4.4 --- Further investigation --- p.124 / Chapter 4.5 --- Conclusion --- p.124 / References : --- p.126 / Chapter Appendix 1 --- Sequence alignment of 16S rDNA gene sequences of abalone for Haliotis-specific primer design --- p.144 / Chapter Appendix 2 --- Accession numbers of sequences of Dongchongxiacao and Cordyceps samples in this study --- p.149 / Chapter Appendix 3 --- Search results of CytB sequences of caterpillar host of Cordyceps samples based on BLAST search results from GenBank --- p.150 / Chapter Appendix 4 --- Search results of COI sequences of caterpillar host of Cordyceps samples based on BLAST search results from GenBank --- p.151 / Chapter Appendix 5 --- Search results of COI sequences of caterpillar host of Cordyceps samples based on BLAST search results from GenBank --- p.152 / Chapter Appendix 6 --- Sequence alignment of ITS sequences of Cordyceps samples and related sequences --- p.153 / Chapter Appendix 7 --- Sequence alignment of nrLSU sequences of Cordyceps samples and related sequences --- p.161 / Chapter Appendix 8 --- Sequence alignment of EF-lα sequences of Cordyceps samples and related sequences --- p.168 / Chapter Appendix 9 --- Sequence alignment of rpbl sequences of Cordyceps samples and related sequences --- p.173 / Chapter Appendix 10 --- "Sequence alignment of combined dataset of three regions (nrLSU, EF-lα and rpbl) of Cordyceps samples and related sequences" --- p.179 / Chapter Appendix 11 --- Sequences alignment of CytB sequences of caterpillar host of Cordyceps samples and related sequences --- p.188 / Chapter Appendix 12 --- Sequence alignment of COI sequences of caterpillar host of Cordyceps samples and related sequences --- p.191 / Chapter Appendix 13 --- Sequence alignment of COI sequences of Cordyceps samples D12-2 and D14 and related sequences --- p.195 / Chapter Appendix 14 --- Sequence distance matrix of ITS sequences of Cordyceps samples and related samples based on K2P algorithm --- p.196 / Chapter Appendix 15 --- Sequence distance matrix of nrLSU sequences of Cordyceps samples and related samples based on K2P algorithm --- p.203 / Chapter Appendix 16 --- Sequence distance matrix of EF-lα sequences of Cordyceps samples and related samples based on K2P algorithm --- p.208 / Chapter Appendix 17 --- Sequence distance matrix of rpbl sequences of Cordyceps samples and related samples based on K2P algorithm --- p.213 / Chapter Appendix 18 --- "Sequence distance matrix of combined dataset of three regions (nrLSU, EF-lα and rpbl) sequences of Cordyceps samples and related samples based on K2P algorithm" --- p.217 / Chapter Appendix 19 --- Sequence distance matrix of CytB sequences of caterpillar host of Cordyceps samples and related samples based on K2P algorithm --- p.219 / Chapter Appendix 20 --- Sequence distance matrix of COI sequences of caterpillar host of Cordyceps samples and related samples based on K2P algorithm --- p.223 / Chapter Appendix 21 --- Sequence alignment of chloroplast trnH-psbA sequences of Cordyceps sample D12-2 and related sequences --- p.226 / Chapter Appendix 22 --- Accession numbers of sequences of abalone and gastropod samples in this study --- p.227 / Chapter Appendix 23 --- Search results of 16S rDNA sequences of the abalone and gastropod samples based on BLAST search results from GenBank --- p.228 / Chapter Appendix 24 --- Search results of COI sequences of the abalone and gastropod samples based on BLAST search results from GenBank --- p.229 / Chapter Appendix 25 --- Search results of COI sequences of the abalone and gastropod samples based on BOLD-IDS --- p.230 / Chapter Appendix 26 --- Sequence alignment of 16S sequences of abalone samples and related sequences --- p.231 / Chapter Appendix 27 --- Sequence alignment of COI sequences of abalone samples and related sequences --- p.234 / Chapter Appendix 28 --- Sequence alignment of COI sequences of abalone product sample D2 and related sequences --- p.238 / Chapter Appendix 29 --- Sequence distance matrix of 16S sequences of abalone samples and related samples based on K2P algorithm --- p.239 / Chapter Appendix 30 --- Sequence distance matrix of COI sequences of abalone samples and related samples based on K2P algorithm --- p.243

Thesis (MScAgric)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: The species Haliotis midae is of great commercial value to the South African abalone industry and is mainly exported to Asian markets, specifically China. Up to 50% is sold as canned products with H. midae registering an average canning yield of approximately 35%. The species is presently genetically undomesticated and breeding programmes are being introduced to improve a range of production traits of which growth and yield is of primary importance. The objective of the study was to determine genetic parameters such as heritability, genotypic and phenotypic correlations of yield-related traits to assess the potential genetic improvement through selective breeding. A series of yield-related parameters were identified that is of relevance to the standard abalone canning procedure. Low to moderate heritabilities where recorded for most traits, including pre-shuck/live weight (0.20 ± 0.06), post-shuck weight (0.15 ± 0.05), post-gut weight (0.15 ± 0.05), post-brine weight (0.19 ± 0.06), pre-canning weight (0.19 ± 0.06), post-canning weight (0.21 ± 0.06), shell weight (0.16 ± 0.05), canning yield percentage (0.08 ± 0.03) and shell weight to post-gut weight ratio (SW: PGW) (0.09 ± 0.04). Weight related parameters are phenotypically highly correlated (0.86 ≤ r ≤ 0.99) but show negative correlation with canning yield percentage (-0.38 ≤ r ≤ 0.04). The nett yield of abalone shows a relatively strong positive correlation with the live weight (r = 0.66). Shell length is highly heritable (h2 ≈ 0.48) and show a strong positive correlation with live weight (r = 0.94). Shell weight is also highly correlated with live weight (r = 0.80) and the SW: PGW ratio does not show a significant correlate with live weight (r = 0.03). Weight-related traits show heritability values ranging from 0.15 to 0.20 that could allow a positive genetic response. Shell length (as a linear growth parameter) shows a high heritability (h2 ≈ 0.48) and a strong positive correlation with live weight (r = 0.94) which also makes it suitable for use as a selection criterion in breeding programmes for improved growth rate. Direct selection for canning yield is compromised by the destructive nature of measurement and the low heritability (h2 < 0.10). The negative correlations between yield as a percentage and growth traits (-0.38 ≤ r ≤ 0.04) further complicate its use as a direct breeding objective. Although the canning yield as a percentage shows a decrease with an increase in live weight, the nett canning yield increases (r = 0.66) with the live weight. It is therefore recommended to use shell length as a criterion for selection for increased growth rate and nett yield, thereby optimising profitability. / AFRIKAANSE OPSOMMING: Die spesie Haliotis midae is van groot kommersiёle waarde tot die Suid-Afrikaanse perlemoenindustrie en word meestal uitgevoer na markte in Asiё, spesifiek China. Tot 50% van die perlemoen wat in Suid-Afrika geproduseer en uitgevoer word, word verblik en huidiglik is die verblikkingsopbrengspersentasie ongeveer 35%. Haliotis midae is tans geneties onderontwikkeld en die gebruik van teelprogramme word nou geimplementeer met die doel om 'n verskeidenheid eienskappe te verbeter, waarvan groei en opbrengs van primêre belang is. Die doelwit van die studie was om genetiese parameters soos oorerflikheid en ook die genotipiese en fenotipiese korrelasies van obrengsverwante eienskappe te bepaal om sodoende die potensiёle genetiese verbetering as gevolg van selektiewe teeling te assesseer. 'n Reeks obrengsverwante eienskappe is geidentifiseer wat relevant is binne bestaande en standaard kommersiёle perlemoenverblikkingsprotokolle. Lae tot matige oorerflikheidswaardes is waargeneem en sluit in lewende, of voor-ontskulpingsgewig (0.20 ± 0.06), na-ontskulpingsgewig (0.15 ± 0.05), na-oopvlekkingsgewig (0.15 ± 0.05), na-pekelgewig (0.19 ± 0.06), voor-verblikkingsgewig (0.19 ± 0.06), na-verblikkingsgewig (0.21 ± 0.06), skulpgewig (0.16 ± 0.05), verblikkingsopbrengspersentasie (0.08 ± 0.03) en 'n skulpgewig tot na-oopvlekkingsgewig verhouding (SW: PGW) (0.09 ± 0.04). Gewigsverwante parameters is fenotipies hoogs gekorreleerd met mekaar (0.86 ≤ r ≤ 0.99) maar toon 'n negatiewe korrelasie met die verblikkingsopbrengspersentasie (-0.38 ≤ r ≤ 0.04). Die netto opbrengs van perlemoen dui op 'n relatiewe sterk positiewe korrelasie met lewende gewig (r = 0.66). Skulplengte is hoogs oorerflik (h2 ≈ 0.48) en toon 'n sterk positiewe korrelasie met lewende gewig (r = 0.94). Skulpgewig is ook hoogs gekorreleerd met lewende gewig (r = 0.80) en die SW: PGW verhouding toon geen beduidende korrelasie met lewende gewig nie (r = 0.03). Gewigsverwante eienskappe toon oorerflikheidswaardes wat varieer tussen 0.15 en 0.20 en kan 'n moontlike genetiese respons lewer. Skulplengte (as 'n liniêre groeiparameter) toon 'n hoё oorerflikheid (h2 ≈ 0.48) en 'n sterk positiewe korrelasie met lewende gewig (r = 0.94) wat dit gepas maak vir gebruik as 'n seleksiekriterium in 'n teelprogram met verbeterde groeitempo as doel. Direkte seleksie in terme van verblikkingsopbrengs word ingeboet danksy die destruktiewe natuur van die metingsmetodiek asook 'n lae oorerflikheid (h2 < 0.10). Die negatiewe korrelasies tussen verblikkingsopbrengs (uitgedruk as 'n persentasie) en groeieienskappe (-0.38 ≤ r ≤ 0.04) dien as 'n verdere komplikasie in die gebruik van dié eienskap as direkte teeldoelwit. Alhoewel die verblikkingopbrengs 'n afname toon soos lewende gewig toeneem, is daar steeds 'n positiewe korrelasie tussen die netto verblikkingsopbrengs en die lewende gewig (r = 0.66). Dit word dus aangeraai om skulplengte as seleksiekriterium vir verbeterde groeitempo en netto opbrengs te gebruik om sodoende wins te maksimaliseer.