Identification and Fine-Mapping of Clubroot (Plasmodiophora brassicae) Resistant QTL in Brassica rapa

European fodder turnips (Brassica rapa ssp. rapifera) were identified as sources of clubroot resistance (CR) and have been widely used in Brassica resistance breeding. An F2 population derived from a cross between a resistant turnip and a susceptible Chinese cabbage was used to determine the inheritance and locating the resistance Quantitative Trait Loci (QTLs). The parents showed to be very resistant/susceptible to the field isolates (pathotype 4) of clubroot from Henan in China. After inoculation, 27 very resistant or susceptible individuals were selected to construct bulks, respectively. Next-generation-sequencing-based Bulk Segregant Analysis Sequencing (BSA-Seq) was used and located resistance QTL on chromosome A03 (3.3–7.5 Mb) and A08 (0.01–6.5 Mb), named Bcr1 and Bcr2, respectively. Furthermore, an F3 population including 180 families derived from F2 individuals was phenotyped and used to verify and narrow candidate regions. Ten and seven Kompetitive Allele-Specific PCR (KASP) markers narrowed the target regions to 4.3–4.78 Mb (A03) and 0.02–0.79 Mb (A08), respectively. The phenotypic variation explained (PVE) of the two QTLs were 33.3% and 13.3% respectively. The two candidate regions contained 99 and 109 genes. In the A03 candidate region, there were three candidate R genes, namely Bra006630, Bra006631 and Bra006632. In the A08 candidate region, there were two candidate R genes, namely Bra030815 and Bra030846.

QTL Mapping for Pumpkin Fruit Traits Using a GBS-based High-density Genetic Map

Pumpkin is a popular vegetable crop and exhibits a broad diversity in fruit shape and size. Fruit-related traits are the decisive factors determining consumer acceptance and market value of pumpkin cultivar. As a result, deciphering the genetic basis of fruit-related traits is of great importance for pumpkin breeding. To address this problem, a F2 population was generated by two Cucurbita moschata inbred lines with contrasting fruit shapes and genotyping-by-sequencing approach were used to construct a high-density genetic map and localize the QTLs underlying the fruit-related traits in this study. The results showed that a high-quality genetic map was constructed for pumpkin, which comprised of 2413 bins and spanned a total length of 2252.10 cM with an average genetic distance of 0.94 cM. A total of 30 significant QTLs with moderate or small effects were identified for 7 fruit-related traits, including fruit length, fruit diameter, fruit shape index, fruit weight, fruit flesh thickness, seed cavity size, and total soluble solids content. Co-locations and close locations were observed between the QTLs underlying different traits, demonstrating that pleiotropic effect plays an important role in genetic control of fruit-related traits. The identified QTLs provide valuable information for further fine mapping of the related genes and pumpkin breeding programs with the aim of improving fruit quality.

Allelic Variation Analysis at the Vernalization Response and Photoperiod Genes in Russian Wheat Varieties Identified Two Novel Alleles of Vrn-B3

Heading time is an important agronomic trait affecting the adaptability and productivity of common wheat. In this study, 95 common wheat varieties from Russia and the late-maturing breeding line ‘Velut’ were tested for allelic diversity of genes having the strongest effect on heading. In this research, allelic variation at the Ppd-D1, Vrn-A1, Vrn-B1, Vrn-D1, and Vrn-B3 loci was tested. The Vrn-B1 and Vrn-B3 loci provided the largest contribution to genetic diversity. We found two novel allelic variants of the Vrn-B3 gene in the studied varieties. Ten varieties carried a 160 bp insertion in the promoter region, and the breeding line ‘Velut’ carried a 1617 bp insertion. These alleles were designated Vrn-B3e and Vrn-B3d, respectively. The analysis of the sequences showed the recent insertion of a retrotransposon homologous to the LTR retrotransposon (RLX_Hvul_Dacia_ RND-1) in the Vrn-B3d allele. Plants with the Vrn-B3e and the ‘Velut’ line with the Vrn-B3d allele headed later than the plants with the wild-type allele; among these plants, ‘Velut’ is the latest maturing wheat variety. Analysis of the gene expression of two groups of lines differing by the Vrn-B3 alleles (Vrn-B3d or vrn-B3) from the F2 population with ‘Velut’ as a parental line did not reveal a significant difference in the expression level between the groups. Additional research is required to study the reasons for the late maturation of the ‘Velut’ line. However, the studied wheat varieties could be used as a potential source of natural variation in genes controlling heading times.

Transcriptomic analyses implicate neuronal plasticity and chloride homeostasis in ivermectin resistance and recovery in a parasitic nematode

The antiparasitic drug ivermectin plays an essential role in human and animal health globally. However, ivermectin resistance is widespread in veterinary helminths and there are growing concerns of sub-optimal responses to treatment in related helminths of humans. Despite decades of research, the genetic mechanisms underlying ivermectin resistance are poorly understood in parasitic helminths. This reflects significant uncertainty regarding the mode of action of ivermectin in parasitic helminths, and the genetic complexity of these organisms; parasitic helminths have large, rapidly evolving genomes and differences in evolutionary history and genetic background can confound comparisons between resistant and susceptible populations. We undertook a controlled genetic cross of a multi-drug resistant and a susceptible reference isolate of Haemonchus contortus, an economically important gastrointestinal nematode of sheep, and ivermectin-selected the F2 population for comparison with an untreated F2 control. RNA-seq analyses of male and female adults of all populations identified high transcriptomic differentiation between parental isolates, which was significantly reduced in the F2, allowing differences associated specifically with ivermectin resistance to be identified. In all resistant populations, there was constitutive upregulation of a single gene, HCON_00155390:cky-1, a putative pharyngeal-expressed transcription factor, in a narrow locus on chromosome V previously shown to be under ivermectin selection. In addition, we detected sex-specific differences in gene expression between resistant and susceptible populations, including constitutive upregulation of a P-glycoprotein, HCON_00162780:pgp-11, in resistant males only. After ivermectin selection, we identified differential expression of genes with roles in neuronal function and chloride homeostasis, which is consistent with an adaptive response to ivermectin-induced hyperpolarisation of neuromuscular cells. Overall, we show the utility of a genetic cross to identify differences in gene expression that are specific to ivermectin selection and provide a framework to better understand ivermectin resistance and recovery in parasitic helminths.

Analysis of genetic variability in F2 interspecific hybrids of mung bean (Vigna radiata) using inter-retrotransposon amplified polymorphism marker system

Fatmawati Y, Setiawan AB, Purwantoro A, Respatie DW, Teo CH. 2021. Analysis of genetic variability in F2 interspecific hybrids of mung bean (Vigna radiata) using inter-retrotransposon amplified polymorphism marker system. Biodiversitas 22: 4880-4889. Mung bean (Vigna radiata L. Wilczek) categorized as one of the pivotal annual crops of Vigna genera is commonly cultivated in rotation with the cereal crops during the drought season. Conversely, to ameliorate its stunted productivity, the interspecific hybridization technique has been introduced between the mung bean and the common bean to promote genetic improvement with the breeding projects in Indonesia. However, since mung bean is a self-pollinated crop and has a narrow genetic base, the selection and improvement of a specific trait using marker-assisted selection is more challenging. Hence, a precautionary investigation is imperative to evaluate the progenies resulting from interspecific hybridization using an ideal marker. This study aimed to investigate the genetic variability of the F2 population of the interspecific mung bean hybrids using retrotransposon-based markers, particularly Inter-Retrotransposon Amplified Polymorphism (IRAP) markers. In this study, we identified retrotransposon from the mung bean genome and determined the Long Terminal Repeat (LTR) sequence using the LTR Finder. The IRAP primers were designed from a conserved region of the LTR sequence. One hundred of the F2 interspecific hybrids generated from the crossing between mung bean and common bean were successfully discriminated by IRAP markers. The IRAP marker showed high heterozygosity and moderate Polymorphic Information Content (PIC) values. The IRAP markers were able to detect genetic variability in the F2 progenies resulting from the interspecific hybridization. Cluster analysis showed that 100 of the F2 progenies were grouped into three clusters. This study demonstrated that retrotransposon-based markers can offer an effective approach for evaluating the segregation in the F2 population of intercross hybrids in the mung bean.

Identification and Validation of a QTL for Bacterial Leaf Streak Resistance in Rice (Oryza sativa L.) against Thai Xoc Strains

Rice is one of the most important food crops in the world and is of vital importance to many countries. Various diseases caused by fungi, bacteria and viruses constantly threaten rice plants and cause yield losses. Bacterial leaf streak disease (BLS) caused by Xanthomonas oryzae pv. oryzicola (Xoc) is one of the most devastating rice diseases. However, most modern rice varieties are susceptible to BLS. In this study, we applied the QTL-seq approach using an F2 population derived from the cross between IR62266 and Homcholasit (HSC) to rapidly identify the quantitative trait loci (QTL) that confers resistance to BLS caused by a Thai Xoc isolate, SP7-5. The results showed that a single genomic region at the beginning of chromosome 5 was highly associated with resistance to BLS. The gene xa5 was considered a potential candidate gene in this region since most associated single nucleotide polymorphisms (SNPs) were within this gene. A Kompetitive Allele-Specific PCR (KASP) marker was developed based on two consecutive functional SNPs in xa5 and validated in six F2 populations inoculated with another Thai Xoc isolate, 2NY2-2. The phenotypic variance explained by this marker (PVE) ranged from 59.04% to 70.84% in the six populations. These findings indicate that xa5 is a viable candidate gene for BLS resistance and may help in breeding programs for BLS resistance.

A Novel QTL for Resistance to Phytophthora Crown Rot in Squash

Phytophthora capsici Leonian causes significant yield losses in commercial squash (Cucurbita pepo) production worldwide. The deployment of resistant cultivars can complement integrated management practices for P. capsici, but resistant cultivars are currently unavailable for growers. Moderate resistance to Phytophthora crown rot in a selection of accession PI 181761 (C. pepo) (designated line #181761-36P) is controlled by three dominant genes (R4, R5 and R6). Introgression of these loci into elite germplasm through marker-assisted selection (MAS) can accelerate the release of new C. pepo cultivars resistant to crown rot, but these tools are currently unavailable. Here we describe the identification of a quantitative trait locus (QTL), molecular markers and candidate genes associated with crown rot resistance in #181761-36P. Five hundred and twenty-three SNP markers were genotyped in an F2 (n = 83) population derived from a cross between #181761-36P (R) and Table Queen (S) using targeted genotyping by sequencing. A linkage map (2068.96 cM) consisting of twenty-one linkage groups and an average density of 8.1 markers/cM was developed for the F2 population. The F2:3 families were phenotyped in the greenhouse with a virulent strain of P. capsica, using the spore-spray method. A single QTL (QtlPC-C13) was consistently detected on LG 13 (chromosome 13) across three experiments and explained 17.92–21.47% of phenotypic variation observed in the population. Nine candidate disease resistance gene homologs were found within the confidence interval of QtlPC-C13. Single nucleotide polymorphism (SNP) markers within these genes were converted into Kompetitive Allele Specific PCR (KASP) assays and tested for association with resistance in the F2 population. One SNP marker (C002686) was significantly associated with resistance to crown rot in the F2 population (p < 0.05). This marker is a potential target for MAS for crown rot resistance in C. pepo.

Genetic Mapping of the HLA1 Locus Causing Hybrid Lethality in Nicotiana Interspecific Hybrids

Hybrid lethality, a postzygotic mechanism of reproductive isolation, is a phenomenon that causes the death of F1 hybrid seedlings. Hybrid lethality is generally caused by the epistatic interaction of two or more loci. In the genus Nicotiana, N. debneyi has the dominant allele Hla1-1 at the HLA1 locus that causes hybrid lethality in F1 hybrid seedlings by interaction with N. tabacum allele(s). Here, we mapped the HLA1 locus using the F2 population segregating for the Hla1-1 allele derived from the interspecific cross between N. debneyi and N. fragrans. To map HLA1, several DNA markers including random amplified polymorphic DNA, amplified fragment length polymorphism, and simple sequence repeat markers, were used. Additionally, DNA markers were developed based on disease resistance gene homologs identified from the genome sequence of N. benthamiana. Linkage analysis revealed that HLA1 was located between two cleaved amplified polymorphic sequence markers Nb14-CAPS and NbRGH1-CAPS at a distance of 10.8 and 10.9 cM, respectively. The distance between these markers was equivalent to a 682 kb interval in the genome sequence of N. benthamiana.