Validation of molecular markers linked to Cercospora leaf spot disease resistance in mungbean (Vigna radiata [L.] Wilczek)

Cercospora leaf spot (CLS) resistance is a highly desirable trait for mungbean (Vigna radiata [L.] Wilczek) production in Thailand. ‘V4718’ is a vital resistance source that shows high and stable resistance to CLS disease. A previous study identified a major quantitative trait locus (QTL) (qCLSC72V18-1) controlling CLS resistance and found the marker (I16274) that was located closest to the resistance gene by using F2:9 and F2:10 recombinant inbred line populations derived through a cross between ‘V4718’ and the susceptible variety ‘Chai Nat 72’ (‘CN72’). Here, we evaluated three newly reported simple sequence repeat (SSR) markers and one InDel marker together with six previously identified markers that were linked to qCLSC72V18-1 to further identify the markers that were located close to this QTL. By performing bulk segregant analysis on two validation populations, we found that two SSR markers (Vr6gCLS037 and Vr6gCLS133) and one InDel marker (VrTAF5_indel) were putatively associated with CLS resistance. Of these markers, only the VrTAF5_indel marker showed a significant association with the CLS resistance gene with a logarithm of odds score > 3 across the phenotypic data for 2016 and 2018. QTL analysis with inclusive composite interval mapping revealed that the VrTAF5_indel marker was integrated into the genetic map with other previously identified markers. The I16274 and VrTAF5_indel markers flanking the QTL of interest accounted for 41.56%-60.38% of the phenotypic variation with genetic distances of 4.0 and 5.0 cM from the resistance gene, respectively. Both markers together permitted only 0.40% recombination with the CLS resistance gene in marker-assisted selection and thus could be useful in future breeding efforts for CLS resistance in mungbean.

Detection of Candidate Gene LsACOS5 and Development of InDel Marker for Male Sterility by DdRAD-Seq and Whole-Genome Sequencing in Lettuce (Lactuca Sativa L.)

A new breeding method of F1 hybrid using male sterility would open an exciting frontier in lettuce breeding, a self-pollinating crop. Male sterility is a crucial trait in F1 hybrid breeding. It is essential to map the causative gene for using male sterility. The ms-S, male-sterile gene of ‘CGN17397’, was mapped to LG8 by double-digest restriction site-associated DNA sequencing (ddRAD-seq) and narrowed down between two markers using two F2 populations. This region spans approximately 10.16 Mb, where 94 genes were annotated according to the lettuce reference genome sequence (version8 from crisphead cultivar ‘Salinas’). The whole-genome sequencing of the male-sterile and fertile lines of ‘CGN17397’ revealed that only one gene differed in the area of Lsat_1_v5_gn_8_148221.1, a homolog of Arabidopsis acyl-CoA synthetase5 (AtACOS5), and was deleted in the male-sterile lines. It was reported that AtACOS5 was needed for pollen wall formation and that the null mutants of AtACOS5 were entirely male sterility. Thus, I concluded that Lsat_1_v5_gn_8_148221.1 designated as LsACOS5 was a biologically plausible candidate gene for the ms-S locus. By using the structural polymorphism of LsACOS5, an insertion/deletion (InDel) marker was developed to select the male-sterile trait. The results obtained here provide valuable information for the genic male-sterility in lettuce.

Marker-Assisted Evaluation of Two Powdery Mildew Resistance Candidate Genes in Korean Cucumber Inbred Lines

Two genes, CsLRR-RPK2 (CsGy5G015660) and CsaMLO8 (Csa5G623470), have been considered as powdery mildew (PM) resistance genes in cucumbers. In this study, we evaluated the involvement of the alleles of these two genes in PM resistance in 100 commercial Korean cucumber inbred lines. To achieve this, we developed cleaved amplified polymorphic sequences (CAPS) and InDel markers from CsLRR-RPK2 and CsaMLO8. Genotyping analysis indicated that the CsLRR-RPK2-CAPS marker showed a stronger correlation with the PM-resistant phenotype, with an 84% consistency compared to the CsaMLO8-InDel marker. The use of the CsaMLO8-InDel marker showed a 70% consistency between phenotype and genotype results. It was proposed that the CsLRR-RPK2-CAPS marker successfully eliminated PM-susceptible inbred lines, since both genotype and phenotype results were 100% identical. Furthermore, the present study revealed that the introduction of one of these alleles is probably enough to confer PM resistance in cucumbers. However, seven PM-resistant inbred lines harbored either CsaMLO8 or CsLRR-RPK2 alleles, indicating that there is another PM-resistant resource(s) besides CsaMLO8- and CsLRR-RPK2–originated resistance in the commercial Korean inbred lines. Our results provide reliable evidence confirming two PM-resistant candidate genes for the detection of PM resistance resources in cucumber inbred lines.

A SmelAAT Acyltransferase Variant Causes a Major Difference in Eggplant (Solanum melongena L.) Peel Anthocyanin Composition

Eggplant berries are rich in anthocyanins like delphinidin-3-rutinoside (D3R) and nasunin (NAS), which are accumulated at high amounts in the peel. NAS is derived by D3R through acylation and glycosylation steps. The presence of D3R or NAS is usually associated with black-purple or lilac fruit coloration of the most cultivated varieties, respectively. Building on QTL mapping position, a candidate gene approach was used to investigate the involvement of a BAHD anthocyanin acyltransferase (SmelAAT) in determining anthocyanin type. The cDNA sequence comparison revealed the presence of a single-base deletion in D3R-type line ‘305E40’ (305E40_aat) with respect to the NAS-type reference line ‘67/3’. This is predicted to cause a frame shift mutation, leading to a loss of SmelAAT function and, thus, D3R retention. RT-qPCR analyses confirmed SmelAAT and 305E40_aat expression during berry maturation. In D3R-type lines, ‘305E40’ and ‘DR2’ overexpressing the functional SmelAAT allele from ‘67/3’, the transcript levels of the transgene, correlated with the accumulation of NAS in fruit peel. Furthermore, it was also found a higher expression of the transcript for glucosyltransferase Smel5GT1, putatively involved with SmelAAT in the last steps of anthocyanin decoration. Finally, an indel marker matching with anthocyanin type in the ‘305E40’ × ’67/3’ segregating population was developed and validated in a wide number of accessions, proving its usefulness for breeding purposes.

PCR-Based InDel Marker Associated with Powdery Mildew-Resistant MR-1

Powdery mildew (PM) is a fungal disease occurring in both field and greenhouse conditions worldwide. It infects many plant species and reduces both the productivity and quality of crops. Melon (Cucumis melo L.) is an economically important crop. In order to develop a molecular marker that can be used more conveniently in the development of PM-resistant melon using MR-1 melon resources, the previously reported cleaved amplified polymorphic sequence (CAPS) marker was improved with a length polymorphism PCR marker. Two cleaved CAPS markers—BSA12-LI3ECORI and BSA12-LI4HINFI—associated with BPm12.1, a major quantitative trait locus (QTL) corresponding to the PM resistance of MR-1, have been reported. In this study, we found that in the BSA12-LI3ECORI CAPS marker specifically, a 41 bp deletion was present in the PCR DNA region of the MR-1 melon genome. A new marker capable of distinguishing polymerase chain reaction (PCR) length polymorphism was produced using insertion-deletion (InDel) information in this region. This PCR-based InDel marker distinguished the genotypes of PM-resistant MR-1, PM-susceptible Top Mark, and their F1 progeny. These results suggest that this InDel marker could be used to develop PM-resistant melon varieties based on MR-1.

Exploring the Distribution of Blast Resistance Alleles at the Pi2/9 Locus in Major Rice-Producing Areas of China by a Novel Indel Marker

Rice blast disease caused by the fungus Magnaporthe oryzae damages cereal crops and poses a high risk to rice production around the world. Currently, planting cultivars with resistance (R) genes is still the most environment-friendly approach to control this disease. Effective identification of R genes existing in diverse rice cultivars is important for understanding the distribution of R genes and predicting their contribution to resistance against blast isolates in regional breeding. Here, we developed a new insertion/deletion (InDel) marker, Pigm/2/9InDel, that can differentiate the cloned R genes (Pigm, Pi9, and Pi2/Piz-t) at the Pi2/9 locus. Pigm/2/9InDel combined with the marker Pi2-LRR for Pi2 was applied to determine the distribution of these four R genes among 905 rice varieties, most of which were collected from the major rice-producing regions in China. In brief, nine Pigm-containing varieties from Fujian and Guangdong provinces were identified. All of the 62 Pi2-containing varieties were collected from Guangdong, and 60 varieties containing Piz-t were from seven provinces. However, Pi9 was not found in any of the Chinese varieties. The newly identified varieties carrying the Pi2/9 alleles were further subjected to inoculation tests with regional blast isolates and field trials. Our results indicate that Pigm and Pi2 alleles have been introgressed for blast resistance breeding mainly in the Fujian and Guangdong region, and Pi9 is a valuable blast resistance resource to be introduced into China.

Identification of Vibrio cholerae Strains of the «Haitian» Group by PCR Based on INDEL-Typing

The aim of the work was to find a genetic INDEL-marker of the Haitian group of Vibrio cholerae strains, what allow carrying out their identification by means PCR.Materials and methods. For searching INDEL-markers we used the data from GenBank database on complete genomic sequences of V. cholerae strains El Тor isolated in different continents in different years. For the analysis we used the author's software written in the Java programming language. The NextGIS system was used for mapping.Results and discussion. We found that the deletion of 8 nucleotides in the gene VCA1095 located on a small chromosome and encoding chemotaxis protein СheA is a characteristic genetic feature of the «Haitian» group strains. Primers have been developed to detect this deletion in PCR.Conclusion. The method of detection of strains of cholera vibrions «Haitian group» on the basis of INDELmarkers was developed and the distribution of such strains in the world before and after 2010 was shown.

Development of Molecular Marker Linked with Bacterial Fruit Blotch Resistance in Melon (Cucumis melo L.)

Bacterial fruit blotch (BFB) causes losses in melon marketable yield. However, until now, there has been no information about the genetic loci responsible for resistance to the disease or their pattern of inheritance. We determined the inheritance pattern of BFB resistance from a segregating population of 491 F2 individuals raised by crossing BFB-resistant (PI 353814) and susceptible (PI 614596) parental accessions. All F1 plants were resistant to Acidovorax citrulli strain KACC18782, and F2 plants segregated with a 3:1 ratio for resistant and susceptible phenotypes, respectively, in a seedling bioassay experiment, indicating that BFB resistance is controlled by a monogenic dominant gene. In an investigation of 57 putative disease-resistance related genes across the melon genome, only the MELO3C022157 gene (encoding TIR-NBS-LRR domain), showing polymorphism between resistant and susceptible parents, revealed as a good candidate for further investigation. Cloning, sequencing and quantitative RT-PCR expression of the polymorphic gene MELO3C022157 located on chromosome 9 revealed multiple insertion/deletions (InDels) and single nucleotide polymorphisms (SNPs), of which the SNP A2035T in the second exon of the gene caused loss of the LRR domain and truncated protein in the susceptible accession. The InDel marker MB157-2, based on the large (504 bp) insertion in the first intron of the susceptible accession, was able to distinguish resistant and susceptible accessions among 491 F2 and 22 landraces/inbred accessions with 98.17% and 100% detection accuracy, respectively. This novel PCR-based, co-dominant InDel marker represents a practical tool for marker-assisted breeding aimed at developing BFB-resistant melon accessions.