Fecal DNA isolation and degradation in clam Cyclina sinensis: noninvasive DNA isolation for conservation and genetic assessment.

BACKGROUND: To avoid destructive sampling for conservation and genetic assessment, we isolated the DNA of clam Cyclina sinensis from their feces. DNA electrophoresis and PCR amplification were used to determine the quality of fecal DNA. And we analyzed the effects of different conditions on the degradation of feces and fecal DNA. RESULTS: The clear fecal DNA bands were detected by electrophoresis, and PCR amplification using clam fecal DNA as template was effective and reliable, suggesting that clam feces can be used as an ideal material for noninvasive DNA isolation. In addition, by analyzing the effects of different environmental temperatures and soaking times on the degradation of feces and fecal DNA, we found that the optimum temperature was 4 °C. In 15 days, the feces maintained good texture, and the quality of fecal DNA was good. At 28 °C, the feces degraded in 5 days, and the quality of fecal DNA was poor. CONCLUSIONS: The clam feces can be used as an ideal material for noninvasive DNA isolation. Moreover, the quality of fecal DNA is negatively correlated with environmental temperature and soaking time.

[Relationship between the choroid plexus cyst and the aneuploidy].

Objective: Using the method of the ultrasound and the noninvasive DNA to find the relationship between the choroid plexus cyst and the aneuploidy, and to provide the meaningful data for prenatal consultant. Methods: A total of 112 cases, that the gestational age were between 13 and 20 weeks, which were diagnosed with choroid plexus cyst in the Department of Gynecology and Obstetrics of the Second Hospital of Jilin University from January. 2016 to July. 2017 were tested by ultrasonography. They all accepted the noninvasive DNA. At the same time, a total of 100 normal fetuses were randomly involved in the control group by the combination of gestational age and the age of the pregnant woman, all of them had taken the non-invasive DNA examination and followed up until the birth. Those fetuses who combined with other malformations were induced labor in the two groups. If the results of noninvasive DNA indicated the high-risk, invasive examination for karyotype analysis were recommended. If the results were aneuploidy, they chose induced labor. The rest cases followed up until the birth. Results: Five cases of the 112 fetuses in the case group were found with obvious malformation (3 of them were found in the early trimester, 2 of them were found in the middle trimester). The numbers of high risk results of noninvasive DNA were 7, and 5 of them underwent the invasive karyotype analysis. When the aneuploidy had diagnosed definitely, induced labor had been taken. One case was found to be the aneuploidy in the control group, and took the induced labor. One case was diagnosed as right side aortic arch by ultrasound in the control group, with a good prognosis. The other cases were followed up until the birth with a good prognosis. Conclusions: (1)In the early trimester, the fetus with choroid plexus cyst has an increasing risk of aneuploidy. (2)When the maximum diameter of choroid plexus cyst is less than 1 cm and the cyst was single, most of them could disappear in the middle trimester, it has no effect on the fetus. (3)Noninvasive DNA test has a high accuracy, which can improve the positive rate of invasive examination.

[Relationship between sonographic markers and fetal chromosome abnormality during 16-18 weeks of pregnancy].

Objective: To analysis of fetal ultrasound soft index positive cases during 16-18 weeks of pregnancy, and to explore the relationship with chromosomal abnormalities in order to increase the positive rate of invasive prenatal diagnosis and reduce the rate of missed diagnosis. Methods: A total of 569 cases which were diagnosed with positive soft markers in the department of gynecology and obstetrics of the Second Hospital of Jilin University from Jan.2016 to Jan.2017 were studied by ultrasonography. Twenty-five cases were lost in follow-up and finally 544 cases were included as group A. Those fetuses who combined with other malformations were induced labor. Non-invasive DNA examination was recommended for continued pregnant women, and those pregnant women whose results were high risk underwent the amniotic cavity puncture. When the fetal aneuploidy was confirmed, they chose induced labor. We followed the rest of those patients until postnatal half year. Randomly selected 544 cases during 16-18 weeks of pregnancy without obvious abnormalities into group B, followed up to half a year after birth. Results: In group A, 7 of the 544 cases were combined with other severe malformation in the beginning, among the remaining 537 patients, 273 of them received non-invasive DNA examination. Ten cases were high risk results, all of them underwent the amniotic cavity puncture with the result of chromosome abnormality, and they chose induced labor. Six cases were found other malformation in the second trimester who chose induced labor, and the rest 521 cases followed until the fetuses was born after half year had a favorable prognosis. In group B, 1 cases of severe deformities and 1 cases of haploid fetuses were found in 544 fetuses. The incidence of haploid fetus in group A and group B were 1.8% and 0.2%, respectively, with statistically significant (P<0.05). The incidence of severe malformation in group A and group B were 2.3% and 0.2%, respectively, with statistically significant (P<0.05). Conclusions: During 16-18 weeks of pregnancy, sonographic markers may indicate an increased risk in fetal chromosomal abnormalities. The risk of serious malformation was increased in the fetuses with ultrasonic soft marker positive, but there was no specificity.

Reassessing the evolutionary history of ass-like equids: insights from patterns of genetic variation in contemporary extant populations.

All extant equid species are grouped in a single genus - Equus. Among those, ass-like equids have remained particularly unstudied and their phylogenetic relations were poorly understood, most probably because they inhabit extreme environments in remote geographic areas. To gain further insights into the evolutionary history of ass-like equids, we have used a non-invasive sampling approach to collect representative fecal samples of extant African and Asiatic ass-like equid populations across their distribution range and mitochondrial DNA (mtDNA) sequencing analyses to examine intraspecific genetic diversity and population structure, and to reconstruct phylogenetic relations among wild ass species/subspecies. Sequence analyses of 410 base pairs of the fast evolving mtDNA control region identified the Asiatic wild ass population of Kalamaili (China) as the one displaying the highest diversity among all wild ass populations. Phylogenetic analyses of complete cytochrome b sequences revealed that African and Asiatic wild asses shared a common ancestor approximately 2.3Mya and that diversification in both groups occurred much latter, probably driven by climatic events during the Pleistocene. Inferred genetic relationships among Asiatic wild ass species do not support E. kiang monophyly, highlighting the need of more extensive studies in order to clarify the taxonomic status of species/subspecies belonging to this branch of the Equus phylogeny. These results highlight the importance of re-assessing the evolutionary history of ass-like equid species, and urge to extend studies at the population level to efficiently design conservation and management actions for these threatened species.

Efficient isolation method for high-quality genomic DNA from cicada exuviae.

In recent years, animal ethics issues have led researchers to explore nondestructive methods to access materials for genetic studies. Cicada exuviae are among those materials because they are cast skins that individuals left after molt and are easily collected. In this study, we aim to identify the most efficient extraction method to obtain high quantity and quality of DNA from cicada exuviae. We compared relative DNA yield and purity of six extraction protocols, including both manual protocols and available commercial kits, extracting from four different exoskeleton parts. Furthermore, amplification and sequencing of genomic DNA were evaluated in terms of availability of sequencing sequence at the expected genomic size. Both the choice of protocol and exuvia part significantly affected DNA yield and purity. Only samples that were extracted using the PowerSoil DNA Isolation kit generated gel bands of expected size as well as successful sequencing results. The failed attempts to extract DNA using other protocols could be partially explained by a low DNA yield from cicada exuviae and partly by contamination with humic acids that exist in the soil where cicada nymphs reside before emergence, as shown by spectroscopic measurements. Genomic DNA extracted from cicada exuviae could provide valuable information for species identification, allowing the investigation of genetic diversity across consecutive broods, or spatiotemporal variation among various populations. Consequently, we hope to provide a simple method to acquire pure genomic DNA applicable for multiple research purposes.

Blood meal analysis of tabanid fly after it biting the rare Sumatran rhinoceros.

OBJECTIVE: To demonstrate a noninvasive large mammalian genetic sampling method using blood meal obtained from a tabanid fly. METHODS: Blood meal was recovered from the abdomen of an engorged tabanid fly (Haematopota sp.) which was captured immediately after biting a Sumatran rhino in captivity. The blood was applied on to a Whatman FTA(®) blood card. Subsequent laboratory work was conducted to extract, amplify and sequence the DNA from the sample. Validation was done by sampling the hair follicles and blood samples from the rhinoceros and subjecting it to the same laboratory process. RESULTS: BLAST search and constructed phylogenetic trees confirmed the blood meal samples were indeed from the rhino. CONCLUSIONS: This method could be used in the field application to noninvasively collect genetic samples. Collection of tabanids and other haematophagous arthropods (e.g. mosquitoes and ticks) and other blood-sucking parasites (e.g. leeches and worms) could also provide information on vector-borne diseases.

Blood meal analysis of tabanid fly after it biting the rare Sumatran rhinoceros

<p><b>OBJECTIVE</b>To demonstrate a noninvasive large mammalian genetic sampling method using blood meal obtained from a tabanid fly.</p><p><b>METHODS</b>Blood meal was recovered from the abdomen of an engorged tabanid fly (Haematopota sp.) which was captured immediately after biting a Sumatran rhino in captivity. The blood was applied on to a Whatman FTA(®) blood card. Subsequent laboratory work was conducted to extract, amplify and sequence the DNA from the sample. Validation was done by sampling the hair follicles and blood samples from the rhinoceros and subjecting it to the same laboratory process.</p><p><b>RESULTS</b>BLAST search and constructed phylogenetic trees confirmed the blood meal samples were indeed from the rhino.</p><p><b>CONCLUSIONS</b>This method could be used in the field application to noninvasively collect genetic samples. Collection of tabanids and other haematophagous arthropods (e.g. mosquitoes and ticks) and other blood-sucking parasites (e.g. leeches and worms) could also provide information on vector-borne diseases.</p>