Microsatellites to reveal genetic diversity and to distinguish four mangoes of Tegal District, Central Java, Indonesia

Hidayat A, Rahayu ES, Abdullah M, Retnoningsih A. 2021. Microsatellites to reveal genetic diversity and to distinguish four mangoes of Tegal District, Central Java, Indonesia. Biodiversitas 22: 3467-3473. The Wirasangka mango is Tegal District’s flora of identity, which requires recognition, and its existence deserves to be preserved. However, based on the morphology of the fruit, most of the people of Tegal still have difficulty distinguishing the wirasangka mango from other mangoes. Molecular markers are needed to ascertain the differences between these mango varieties. Microsatellite DNA is a repeating DNA of stable DNA markers with high repeatability and polymorphism. This study analyzes the level of genetic diversity and microsatellite alleles that can differentiate four mangoes from Tegal District, Central Java, Indonesia, i.e. wirasangka, tengkueh, golek, and okyong. Eleven accessions of six locations in Tegal District were analyzed using ten microsatellite loci. The microsatellite amplification result was separated using electrophoresis in 6% polyacrylamide gel and then visualized with silver dye. A total of 35 microsatellite alleles were found measuring 100-1000 bp, ranging from 1-7 alleles for each locus. The average polymorphism information content (PIC) of 0.54 indicates that genetic diversity is relatively high and informative. Therefore, the microsatellite alleles can be used to differentiate mango varieties. The specific allele characteristic of wirasangka mango accession is locus AJ63516, with an allele size of 600-700bp.

Applicability of human-specific STR systems, GlobalFiler™ PCR Amplification Kit, Investigator 24plex QS Kit, and PowerPlex® Fusion 6C in chimpanzee (Pan troglodytes)

Objectives Human identification systems based on STRs are widely used in human population genetics and forensic analysis. This study aimed to validate the cross-reactivity of three widely known human-specific STR identification systems i.e. GlobalFiler™ PCR Amplification Kit, Investigator 24plex QS Kit, and PowerPlex® Fusion 6C in chimpanzee. Results The present study revealed the successful amplification of 18 loci using GlobalFiler™ PCR Amplification Kit, 18 loci using Investigator 24plex QS Kit, and 20 loci using PowerPlex® Fusion 6C system. The marker Amelogenin (AMEL) showed differential allele size between male and female revealing the gender identity of chimpanzees and thus validates their application concerning forensic examination, population estimation, and genetic analysis.

FSHD1 Diagnosis in a Russian Population Using a qPCR-Based Approach

Facioscapulohumeral dystrophy (FSHD) is an autosomal dominant myodystrophy. Approximately 95% of cases of FSHD are caused by partial deletion of the D4Z4 macrosatellite tandem repeats on chromosome 4q35. The existing FSHD1 diagnostic methods are laborious and not widely used. Here, we present a comprehensive analysis of the currently used diagnostic methods (Southern blotting and molecular combing) against a new qPCR-based approach for FSHD1 diagnosis. We observed 93% concordance between the results obtained by the new qPCR-based approach, reference Southern blotting and molecular combing methods. Applying the qPCR-based approach in the studied population, we observed a prevalence (64.9%) of the permissive alleles in the range of 3–6 D4Z4 units for a group of patients, while in a group of carriers, the permissive alleles were mostly (84.6%) present in the range of 6–9 D4Z4 units. No prevalence of disease penetrance depending on gender was observed. The results confirmed the earlier established inverse correlation between permissive allele size and disease severity, disease penetrance. The results suggest the applicability of the qPCR-based approach for FSHD1 diagnosis and its robustness in a basic molecular genetics laboratory. To our knowledge, this is the first study of FSHD1 permissive allele distribution in a Russian population.

Applicability of Human-specific STR systems, GlobalFiler™ PCR Amplification Kit, Investigator 24plex QS Kit, and PowerPlex® Fusion 6C in Chimpanzee (Pan troglodytes)

Objectives The human identification systems based on STRs are widely used in human population genetics and forensic analysis. This study aimed to validate the cross-reactivity of three widely known Human-specific STR identification systems i.e. GlobalFiler™ PCR Amplification Kit, Investigator 24plex QS Kit, and PowerPlex® Fusion 6C in Chimpanzee. Result The present study revealed the successful amplification of 18 loci using GlobalFiler™ PCR Amplification Kit, 18 loci using Investigator 24plex QS Kit, and 20 loci using PowerPlex® Fusion 6C system. The marker Amelogenin (AMEL) showed differential allele size between male and female revealing the gender identity of Chimpanzees and thus validates their application concerning forensic examination, population estimation, and genetic analysis.

Approaches to Sequence the HTT CAG Repeat Expansion and Quantify Repeat Length Variation

Background: Huntington’s disease (HD) is an autosomal dominant neurodegenerative disorder caused by the expansion of the HTT CAG repeat. Affected individuals inherit ≥36 repeats and longer alleles cause earlier onset, greater disease severity and faster disease progression. The HTT CAG repeat is genetically unstable in the soma in a process that preferentially generates somatic expansions, the proportion of which is associated with disease onset, severity and progression. Somatic mosaicism of the HTT CAG repeat has traditionally been assessed by semi-quantitative PCR-electrophoresis approaches that have limitations (e.g., no information about sequence variants). Genotyping-by-sequencing could allow for some of these limitations to be overcome. Objective: To investigate the utility of PCR sequencing to genotype large (>50 CAGs) HD alleles and to quantify the associated somatic mosaicism. Methods: We have applied MiSeq and PacBio sequencing to PCR products of the HTT CAG repeat in transgenic R6/2 mice carrying ∼55, ∼110, ∼255 and ∼470 CAGs. For each of these alleles, we compared the repeat length distributions generated for different tissues at two ages. Results: We were able to sequence the CAG repeat full length in all samples. However, the repeat length distributions for samples with ∼470 CAGs were biased towards shorter repeat lengths. Conclusion: PCR sequencing can be used to sequence all the HD alleles considered, but this approach cannot be used to estimate modal allele size or quantify somatic expansions for alleles ⪢250 CAGs. We review the limitations of PCR sequencing and alternative approaches that may allow the quantification of somatic contractions and very large somatic expansions.

Development and validation in 500 female samples of a TP-PCR assay to identify AFF2 GCC expansions

The absence of FRAXE pathognomonic features hampers early recognition, delaying testing and molecular confirmation. Hence, our laboratory uses a multiplex PCR-based strategy to determine the allele size of both FRAXA and FRAXE in which around 20% of female samples are uninformative. The aim of this study is to develop a timely triplet-primed amplification (TP-PCR) screening strategy to size the AFF2 GCC repeat and accurately assess homozygosity in female samples. In order to achieve this, validation was performed in a cohort of 500 females with a previous uninformative FRAXE PCR result. Unlike the presence of AGG interspersion(s) in FRAXA triplet repeats, no interspersion was observed among the samples tested. Interestingly, the presence of a T>C SNP (rs868949662), contiguous to the GCC repetitive track, allows triplet primer binding in two additional GCCs, increasing the discrimination power of the TP-PCR assay in heterozygous and homozygous samples. Twelve alleles outside the normal range, eight intermediate and four premutated, were recognized, which seems relevant considering the rarity of the AFF2 expansions. Overall, this study confirmed the absence of interspersions in both normal and expanded FRAXE alleles, providing a strict, reproducible and low-cost homozygosity screening strategy. Furthermore, the occurrence of intermediate repeat sizes with unexpected frequency introduces new areas of clinical research in this cohort in understanding these less explored AFF2 repeat sizes and newly associated phenotypes.

Characterization of Varroa destructor Mites in Cuba Using Mitochondrial and Nuclear Markers

Varroa destructor has been present in Cuba since 1996, but without the use of acaricidal infestation rates remain at very low levels. The presence of Korean haplotype mites was described in 2007, but there is no information regarding the introgression of the less virulent Japanese haplotype that could account for a low pathogenicity of the mite. In this research, we carried out molecular characterization of Cuban Varroa mites through mitochondrial DNA and hypervariable nuclear loci. We applied an alternative RFLP technique and found that all the analyzed samples corresponded to Korean haplotypes. We analyzed the three STRs loci VD112, VD114 and VD016, previously described as highly variable and found new alleles in all of them, with an absolute allele size very different to those reported worldwide. We also detected genic and genotypic differentiation between samples from two nearby locations (P=0.08). We also tested a new RFLP method for mite haplotype discrimination with an intra-reaction positive control of digestion.

Molecular-genetic characteristics of Facioscapulohumeral muscular dystrophy in the Russian population

Миодистрофия Ландузи-Дежерина (МЛД) в 95% случаев вызвана частичной делецией массива макросателлитных повторов D4Z4 на одном из аллелей 4-й хромосомы в сцеплении с пермиссивным гаплотипом. В данной работе мы провели молекулярно-генетическую диагностику МЛД в группе российских пациентов с клиническими проявлениями МЛД и их фенотипически здоровых родственников. Полученные данные распределения патогенных аллелей по длине соответствуют ранее описанным зарубежным популяционным исследованиям. Кроме того, наличие здоровых носителей патогенного аллеля подтверждает, что для проявления заболевания требуется сочетания генетических, эпигенетических и средовых факторов. Facioscapulohumeral dystrophy (FSHD) is an autosomal dominant myodystrophy. In approximately 95% of cases FSHD is caused by partial deletion of the D4Z4 macrosatellite tandem repeat on chromosome 4q35 (FSHD1). Here we present results FSHD1 diagnostic of patients and unaffected relatives from Russian population. We demonstrate that FSHD1 permissive alleles are bigger in size for unaffected carriers, which indicate previously reported phenomenon of inverse correlation between permissive allele size and disease penetrance and expressivity. In addition, presence of patients and unaffected carriers from one family with the same permissive allele size demonstrate importance of non-genetic factors for disease development.

Use of local genetic ancestry to assess TOMM40-523′ and risk for Alzheimer disease

ObjectiveHere, we re-examine TOMM40-523′ as a race/ethnicity-specific risk modifier for late-onset Alzheimer disease (LOAD) with adjustment for local genomic ancestry (LGA) in Apolipoprotein E (APOE) ε4 haplotypes.MethodsThe TOMM40-523′ size was determined by fragment analysis and whole genome sequencing in homozygous APOE ε3 and APOE ε4 haplotypes of African (AF) or European (EUR) ancestry. The risk for LOAD was assessed within groups by allele size.ResultsThe TOMM40-523′ length did not modify risk for LOAD in APOE ε4 haplotypes with EUR or AF LGA. Increasing length of TOMM40-523′ was associated with a significantly reduced risk for LOAD in EUR APOE ε3 haplotypes.ConclusionsAdjustment for LGA confirms that TOMM40-523′ cannot explain the strong differential risk for LOAD between APOE ε4 with EUR and AF LGA. Our study does confirm previous reports that increasing allele length of the TOMM40-523′ repeat is associated with decreased risk for LOAD in carriers of homozygous APOE ε3 alleles and demonstrates that this effect is occurring in those individuals with the EUR LGA APOE ε3 allele haplotype.