Optimizing bulk segregant analysis of drug resistance using Plasmodium falciparum genetic crosses conducted in humanized mice

Background: Classical genetic crosses in malaria parasites involve isolation, genotyping, and phenotyping of multiple progeny parasites, which is time consuming and laborious. Bulk segregant analysis (BSA) offers a powerful and efficient alternative to identify loci underlying complex traits in the human malaria parasite, Plasmodium falciparum. Methods: We have used BSA, which combines genetic crosses using humanized mice with pooled sequencing of progeny populations to measure changes in allele frequency following selection with antimalarial drugs. We used dihydroartemisinin (DHA) drug selection in two genetic crosses (Mal31xKH004 and NF54xNHP1337). We specifically investigated how synchronization, cryopreservation, and the drug selection regimen of progeny pools impacted the success of BSA experiments. Findings: We detected a strong and repeatable quantitative trait locus (QTL) at chr13 kelch13 locus in both crosses, but did not detect QTLs at ferredoxin (fd), the apicoplast ribosomal protein S10 (arps10), multidrug resistance protein 2 (mdr2). QTLs were detected using synchronized, but not unsynchronized pools, consistent with the stage-specific action of DHA. We also successfully applied BSA to cryopreserved progeny pools. Interpretation: Our results provide proof-of-principal of the utility of BSA for rapid, robust genetic mapping of drug resistance loci. Use of cryopreserved progeny pools expands the utility of BSA because we can conduct experiments using archived progeny pools from previous genetic crosses. BSA provides a powerful approach that complements traditional QTL methods for investigating the genetic architecture of resistance to antimalarials, and to reveal new or accessory loci contributing to artemisinin resistance.

PENGGUNAAN KARTU PERSILANGAN GENETIKA TRIHIBRIDA SEBAGAI MEDIA PEMBELAJARAN BIOLOGI DAPAT MENINGKATKAN HASIL BELAJAR DAN AKTIVITAS SISWA KELAS XII MADRASAH ALIYAH NEGERI BINJAI

The purpose of this study was to increase student activity in Biology lessons. Based on research and data processing, it was found that the use of the Trihybrid Genetic Crosses card (three different traits) as a Biology learning medium, can improve the learning outcomes of class XII students of Madrasah Aliyah Negeri Binjai in the 2019/2020 school year in Biology lessons on Trihybrid Genetic Crosses (three different traits). ), this can be seen from the calculation of the correlation value in the first cycle between the pre-test to the first daily test of 0.827 with a t-test value of 8.333, and the second cycle between the post-test to the second daily test of 0.943 with a t-test value of 16.075. The use of the Trihybrid Genetic Cross card (three different traits) as a Biology learning medium, can increase the activity of class XII students of Madrasah Aliyah Negeri Binjai in the 2019/2020 school year in Biology lessons on Trihybrid Genetic Crosses (three different traits), this can be seen from the presence students during classroom action research (CAR), from 90% of the first cycle increased to 95% and 100% of the second cycle. Key words : Trihybrid Genetic Cross Card as Biology Learning Media, Learning Outcomes, Activities

The power and promise of genetic mapping from Plasmodium falciparum crosses utilizing human liver-chimeric mice

Genetic crosses are most powerful for linkage analysis when progeny numbers are high, parental alleles segregate evenly and numbers of inbred progeny are minimized. We previously developed a novel genetic crossing platform for the human malaria parasite Plasmodium falciparum, an obligately sexual, hermaphroditic protozoan, using mice carrying human hepatocytes (the human liver-chimeric FRG NOD huHep mouse) as the vertebrate host. We report on two genetic crosses—(1) an allopatric cross between a laboratory-adapted parasite (NF54) of African origin and a recently patient-derived Asian parasite, and (2) a sympatric cross between two recently patient-derived Asian parasites. We generated 144 unique recombinant clones from the two crosses, doubling the number of unique recombinant progeny generated in the previous 30 years. The allopatric African/Asian cross has minimal levels of inbreeding and extreme segregation distortion, while in the sympatric Asian cross, inbred progeny predominate and parental alleles segregate evenly. Using simulations, we demonstrate that these progeny provide the power to map small-effect mutations and epistatic interactions. The segregation distortion in the allopatric cross slightly erodes power to detect linkage in several genome regions. We greatly increase the power and the precision to map biomedically important traits with these new large progeny panels.

Somatic genetics analysis of sleep in adult mice

The molecular mechanisms of mammalian sleep regulation remain largely unknown. Classical forward and reverse mouse genetic approaches require germline mutations and, thus, are unwieldy to study the sleep functions of essential genes or redundant pathways. It is also costly and time-consuming to conduct large-scale electroencephalogram (EEG)/electromyogram (EMG)-based mouse sleep screening due to lengthy genetic crosses and labor-intensive surgeries. Here, we develop a highly efficient adult brain chimeric (ABC) expression/knockout (KO) platform and a highly accurate AI-augmented SleepV (video) system for high-througphut somatic genetics analysis of sleep in adult mice. This ABC platform involves intravenous administration of adeno-associated viruses (AAV) that bypass the blood brain barrier and transduce the majority of adult brain neurons. Constitutive or inducible ABC-expression of CREB and CRTC1 reduces both quantity and quality of non-rapid-eye-movement sleep (NREMS), whereas ABC-KO of CREB by AAV-mediated Cre/loxP recombination increases daily NREMS amount. Moreover, ABC-KO of exon 13 of Sik3 by AAV-Cre injection of Sik3-E13flox/flox adult mice phenocopies Sleepy (Sik3Slp/Slp) mice, which carry a germline splicing mutation resulting in skipping of exon 13 of Sik3. While both long and short isoforms of SLP kinase contribute to, ABC-KO of Slp allele by CRISPR/Cas9 rescues the hypersomnia of Sik3Slp/+ mice. Double ABC-KO of orexin/hypocretin receptors by CRISPR/Cas9 results in chocolate-induced narcolepsy episodes. We envision that these somatic genetics approaches should facilitate efficient and sophisticated studies of many brain-related cellular, physiological and behavioral processes in adult mice without genetic crosses.

Intraspecific Hybridization Of Normal Pigmented And Albino Clarias Gariepinus From Yola And Katsina under Hatchery Condition

The study examined the reproductive indices of albino and normal pigmented Clarias gariepinus fish from Katsina (KT) and Yola (YY), carried out under hatchery condition. The experiment compared the fecundity, testes, milt volume, percentage fertilization and hatchability of albino (AA) and normal pigmented (NN) Clarias gariepinus from Katsina (KT) and Yola (YY). Normal pigmented C. gariepinus from Katsina (KT) had the highest number of eggs (229,240), followed by YY (127,250) and the least was recorded in the Albino (AA) (124,750). The weights and lengths of the left and right lobes as well as the volumes of the milt were quantified . However, KT had the highest weight and length of the right testis (18g and 5.7cm respectively) while AA had the highest weight, length of the left testis and milt volume (30g, 11cm and 8.4ml respectively). The least length and weight of the testes was observed in YY. Meanwhile, KT and YY had milt volumes of 4.0ml and 4.7ml, respectively. The highest percentage fertilization and hatchability were recorded in KT × KT (98.7% and 98.5% respectively) among the purebred, while KT × AA had the highest percentage fertilization and hatchability (98.4% and 97.3% respectively) in the reciprocal hybrids. The percentage fertilization and hatchability among the genetic crosses showed significant differences (p<0.05). The results deduced the essentiality of fish hybridization. Furthermore, crossing of broodstocks from different regions have showcased the contingency of acquiring fish seed of improved reproductive potentials in the reciprocal hybrids in the aspects of fecundity, fertilization, hatchability rate, testis quality and faster growth. Keywords: Albino, Fecundity, Intra-specific Hybridization, Normal pigmented, Testis