Integrated morphometric and molecular approaches to screen hybrid from wild Labeo rohita and Labeo catla parent populations.

BACKGROUND: Interspecific hybrids of rohu (Labeo rohita) and catla (Labeo catla) are common, especially in India due to constrained breeding. These hybrids must segregate from their wild parents as part of conservational strategies. This study intended to screen the hybrids from wild rohu and catla parents using both morphometric and molecular approaches. METHODS & RESULTS: The carp samples were collected from Jharkhand and West Bengal, India. The correlation and regression analysis of morphometric features are considered superficial but could be protracted statistically by clustering analysis and further consolidated by nucleotide variations of one mitochondrial and one nuclear gene to differentiate hybrids from their parents. Out of 21 morphometric features, 6 were used for clustering analysis that exhibited discrete separation among rohu, catla, and their hybrids when the data points were plotted in a low-dimensional 2-D plane using the first 2 principal components. Out of 40 selected single nucleotide polymorphism (SNP) positions of the COX1 gene, hybrid showed 100% similarity with catla. Concerning SNP similarity of the 18S rRNA nuclear gene, the hybrid showed 100% similarity with rohu but not with catla; exhibiting its probable parental inheritance. CONCLUSIONS: Along with morphometric analysis, the SNP comparison study together points towards strong evidence of interspecific hybridization between rohu and catla, as these hybrids share both morphological and molecular differences with either parent. However, this study will help screen the hybrids from their wild parents, as a strategy for conservational management.

Utility of indels for species-level identification of a biologically complex plant group: a study with intergenic spacer in Citrus.

The Consortium of Barcode of Life plant working group proposed to use the defined portion of plastid genes rbcL and matK either singly or in combination as the standard DNA barcode for plants. But DNA barcode based identification of biologically complex plant groups are always a challenging task due to the occurrence of natural hybridization. Here, we examined the use of indels polymorphism in trnH-psbA and trnL-trnF sequences for rapid species identification of citrus. DNA from young leaves of selected citrus species were isolated and matK gene (~800 bp) and trnH-psbA spacer (~450 bp) of Chloroplast DNA was amplified for species level identification. The sequences within the group taxa of Citrus were aligned using the ClustalX program. With few obvious misalignments were corrected manually using the similarity criterion. We identified a 54 bp inverted repeat or palindrome sequence (27-80 regions) and 6 multi residues indel coding regions. Large inverted repeats in cpDNA provided authentication at the higher taxonomic levels. These diagnostics indel marker from trnH-psbA were successful in identifying different species (5 out of 7) within the studied Citrus except Citrus limon and Citrus medica. These two closely related species are distinguished through the 6 bp deletion in trnL-trnF. This study demonstrated that the indel polymorphism based approach easily characterizes the Citrus species and the same may be applied in other complex groups. Likewise other indels occurring intergenic spacer of chloroplast regions may be tested for rapid identification of other secondary citrus species.

Identification of ethnomedicinal plants (Rauvolfioideae: Apocynaceae) through DNA barcoding from northeast India.

BACKGROUND: DNA barcode-based molecular characterization is in practice for plants, but yet lacks total agreement considering the selection of marker. Plant species of subfamily Rauvolfioideae have long been used as herbal medicine by the majority of tribal people in Northeast (NE) India and at present holds mass effect on the society. Hence, there is an urgent need of correct taxonomic inventorization vis-à-vis species level molecular characterization of important medicinal plants. OBJECTIVE: To test the efficiency of matK in species delineation like DNA barcoding in Rauvolfiadae (Apocynaceae). MATERIALS AND METHODS: In this study, the core DNA barcode matK and trnH-psbA sequences are examined for differentiation of selected ethnomedicinal plants of Apocynaceae. DNA from young leaves of selected species was isolated, and matK gene (~800 bp) and trnH-psbA spacer (~450 bp) of Chloroplast DNA was amplified for species level identification. RESULTS: The ~758 bp matK sequence in comparison to the trnH-psbA showed easy amplification, alignment, and high level of discrimination value among the medicinal Rauvolfioidae species. Intergenic spacer trnH-psbA is also exhibited persistent problem in obtaining constant bidirectional sequences. Partial matK sequences exhibited 3 indels in multiple of 3 at 5 end. Evidently, generated matK sequences are clustered cohesively, with their conspecific Genbank sequences. However, repeat structures with AT-rich regions, possessing indels in multiple of 3, could be utilized as qualitative molecular markers in further studies both at the intra-specific and shallow inter-specific levels like the intergenic spacers of CpDNA. CONCLUSION: matK sequence information could help in correct species identification for medicinal plants of Rauvolfioideae.

The species dilemma of Northeast Indian Mahseer (Actinopterygii: Cyprinidae): DNA barcoding in clarifying the riddle.

BACKGROUND: The taxonomic validity of Northeast Indian endemic Mahseer species, Tor progeneius and Neolissochilus hexastichus, has been argued repeatedly. This is mainly due to disagreements in recognizing the species based on morphological characters. Consequently, both the species have been concealed for many decades. DNA barcoding has become a promising and an independent technique for accurate species level identification. Therefore, utilization of such technique in association with the traditional morphotaxonomic description can resolve the species dilemma of this important group of sport fishes. METHODOLOGY/PRINCIPAL FINDINGS: Altogether, 28 mahseer specimens including paratypes were studied from different locations in Northeast India, and 24 morphometric characters were measured invariably. The Principal Component Analysis with morphometric data revealed five distinct groups of sample that were taxonomically categorized into 4 species, viz., Tor putitora, T. progeneius, Neolissochilus hexagonolepis and N. hexastichus. Analysis with a dataset of 76 DNA barcode sequences of different mahseer species exhibited that the queries of T. putitora and N. hexagonolepis clustered cohesively with the respective conspecific database sequences maintaining 0.8% maximum K2P divergence. The closest congeneric divergence was 3 times higher than the mean conspecific divergence and was considered as barcode gap. The maximum divergence among the samples of T. progeneius and T. putitora was 0.8% that was much below the barcode gap, indicating them being synonymous. The query sequences of N. hexastichus invariably formed a discrete and a congeneric clade with the database sequences and maintained the interspecific divergence that supported its distinct species status. Notably, N. hexastichus was encountered in a single site and seemed to be under threat. CONCLUSION: This study substantiated the identification of N. hexastichus to be a true species, and tentatively regarded T. progeneius to be a synonym of T. putitora. It would guide the conservationists to initiate priority conservation of N. hexastichus and T. putitora.