DNA Bulks Research Articles

Multi-Population Analysis for Leaf and Neck Blast Reveals Novel Source of Neck Blast Resistance in Rice.

Rice blast is one of the most devastating biotic stresses that limits rice productivity. The North Eastern Hill (NEH) region of India is considered to be one of the primary centres of diversity for both rice and pathotypes of Magnaporthe grisea. Therefore, the present study was carried out to elucidate the genetic basis of leaf and neck blast resistance under Meghalaya conditions. A set of 80 diverse genotypes (natural population) and 2 F2 populations involving resistant parent, a wildtype landrace, LR 5 (Lal Jangali) and susceptible genotypes Sambha Mahsuri SUB 1 (SMS) and LR 26 (Chakhao Poireiton) were used for association analysis of reported major gene-linked markers with leaf and neck blast resistance to identify major effective genes under local conditions. Genotyping using twenty-five gene-specific markers across diverse genotypes and F2 progenies revealed genes Pi5 and Pi54 to be associated with leaf blast resistance in all three populations. Genes Pib and qPbm showed an association with neck blast resistance in both natural and LR 5 × SMS populations. Additionally, a set of 184 genome-wide polymorphic markers (SSRs and SNPs), when applied to F2-resistant and F2-susceptible DNA bulks derived from LR 5 × LR 26, suggested that Pi20(t) on chromosome 12 is one of the major genes imparting disease resistance. Markers snpOS318, RM1337 and RM7102 and RM247 and snpOS316 were associated with leaf blast and neck blast resistance, respectively. The genotypes, markers and genes will help in marker-assisted selection and development of varieties with durable resistance.

Development of female specific SCAR marker in spine gourd (Momordica dioica Roxb. Ex. Wild) and its validation

Momordica dioica is a dioecious vegetable crop belonging to the family Cucurbitaceae. Fifty RAPD primers were used to analyze the male and female DNA bulks consisting of six females and six male genotypes of Momordica dioica respectively and 23 of them have differentiated the reproductive DNA bulks. One of the primers, OPB07, amplified a female reproductive bulk specific fragment of 326 bp (OPB07326) and was also present exclusively in all female genotypes used to prepare the reproductive bulk. This unique fragment OPB07326 was cloned and sequenced and was developed into female specific SCAR marker MdFSOPB07255. This SCAR marker has also amplified the unique female-specific fragment of 255 bp in all the six female genotypes used for the preparation of the bulk. The existence of this female-specific fragment in M. dioca genome was also affirmed by southern hybridization of DIG-labeled probe of OPB07326 with EcoRI restricted profile of all the six female genotypes. Further, the MdFSOPB07255 marker was successfully employed to identify eight female genotypes out of ten M. dioica genotypes with unknown reproductive feature. Thus, MdFSOPB07255 could be utilized as an efficient and reliable DNA marker for early differentiation of reproductive type at the sapling stage.

Identification of salt stress-tolerant candidate genes in the BC2F2 population at the seedling stages of G. hirsutum and G. darwinii using NGS-based bulked segregant analysis.

Salinity is a major threat to the yield and productivity of cotton seedlings. In the present study, we developed a BC2F2 population of cotton plants from Gossypium darwinii (5-7) and Gossypium hirsutum (CCRI 12-4) salt-susceptible parents to identify salt-resistant candidate genes. The Illumina HiSeq™ strategy was used with bulked segregant analysis. Salt-resistant and salt-susceptible DNA bulks were pooled by using 30 plants from a BC2F2 population. Next-generation sequencing (NGS) technology was used for the sequencing of parents and both bulks. Four significant genomic regions were identified: the first genomic region was located on chromosome 18 (1.86 Mb), the second and third genomic regions were on chromosome 25 (1.06 Mb and 1.94 Mb, respectively), and the fourth was on chromosome 8 (1.41 Mb). The reads of bulk1 and bulk2 were aligned to the G. darwinii and G. hirsutum genomes, respectively, leading to the identification of 20,664,007 single-nucleotide polymorphisms (SNPs) and insertions/deletions (indels). After the screening, 6,573 polymorphic markers were obtained after filtration of the candidate regions. The SNP indices in resistant and susceptible bulks and Δ(SNP-index) values of resistant and susceptible bulks were measured. Based on the higher Δ(SNP-index) value, six effective polymorphic SNPs were selected in a different chromosome. Six effective SNPs were linked to five candidate genes in four genomic regions. Further validation of these five candidate genes was carried out using reverse transcription-quantitative polymerase chain reaction (RT-qPCR), resulting in an expression profile that showed two highly upregulated genes in the salt-tolerant species G. darwinii, i.e., Gohir.D05G367800 and Gohir.D12G239100; however, the opposite was shown in G. hirsutum, for which all genes, except one, showed partial expression. The results indicated that Gohir.D05G367800 and Gohir.D12G239100 may be salt-tolerant genes. We are confident that this study could be helpful for the cloning, transformation, and development of salt-resistant cotton varieties.

Towards the development of the PCR-based InDel markers associated with fruit skin quality traits in melon (Cucumis melo L.) using bulked segregant analysis

Melon's fruit skin quality is an agriculturally important and consumer-appealing trait. To identify molecular markers associated with fruit skin qualities, including reticulation and color, a total of 1200 Insertion/Deletion (InDel) markers randomly selected from the whole genome re-sequencing data of melon were combined with bulk segregant analysis to analyze a panel of melon genetic resources presenting reticulated- and smooth-skinned fruits as well as varying fruit skin colors. Four DNA bulks, including reticulated skin DNA bulk, smoothed skin DNA bulk, green skin DNA bulk, as well as white skin and light yellow skin DNA bulks, were developed. Four DNA pools were created by combining the DNA of 12 representative plants from each DNA bulk for polymorphism analysis, and 200 polymorphic InDel markers were selected. These polymorphic markers were used to characterize typical genetic bands within each DNA bulk. Two InDel markers, MC8-52 and MC4-7, were discovered to be related to fruit surface patterning (reticulated- and smooth-skinned fruits) and fruit color (green and white–light yellow fruits), respectively. These markers offer a high degree of detection accuracy. InDel marker MC8-52 on chromosome 8 achieved detection accuracies of 78.33% and 90.56%, whereas marker MC4-7 on chromosome 4 displayed detection accuracies of 92.92% and 83.35%, respectively. However, these markers were seen as a likely means of identifying strongly related markers for these traits. Hence, a high-resolution map of genomic regions carrying them is required for the development of highly linked markers for these traits.

Mapping of a soybean rust resistance in PI 594756 at the Rpp1 locus.

The online version contains supplementary material available at 10.1007/s11032-023-01358-4.

Genetic mapping and confirmation of the Rdm3 locus underlying resistance to southern stem canker in soybean

AbstractSouthern stem canker (SSC) in soybean [Glycine max (L.) Merrill], caused by the fungal pathogen Diaporthe aspalathi, is a major disease in the southern United States. It can cause up to 80% yield loss in severely infected fields. Plant introduction (PI) 398469, a Maturity Group VI soybean accession from South Korea, is one of six known sources of resistance to SSC. To determine the genetics of the resistance provided by PI 398469, F2:3 and F5‐derived recombinant inbred line (RIL) populations were developed from a cross with the susceptible breeding line G81‐2057. Both populations were phenotyped in the greenhouse using a modified toothpick method. The SSC‐resistant and susceptible DNA bulks consisting of 20 resistant and 18 susceptible families were created from the F2:3 population and genotyped using the Infinium SoySNP50K BeadChips for a bulked segregant analysis (BSA). Based on BSA results, 22 Kompetitive allele specific polymerase chain reaction (KASP) markers were designed to genotype the RIL population. Quantitative trait locus (QTL) analysis identified qRdm_14, on chromosome (Chr) 14 with an R2 of 62%. Linkage mapping in the F2:3 population confirmed the presence of a QTL on Chr 14. F5‐derived RILs derived from G81‐2057 × Crockett were phenotyped for SSC resistance and genotyped with the KASP markers to examine the relationship between Rdm3 from Crockett and qRdm_14 from PI 398469. Both PI 398469 and Crockett carried realleles at the Rdm3 locus for resistance to SSC. The QTL and KASP marker information could be used to screen breeding lines for resistance to SSC.

AAQSP increases mapping resolution of stable QTLs through applying NGS-BSA in multiple genetic backgrounds.

In this study, we present AAQSP as an extension of existing NGS-BSA applications for identifying stable QTLs at high resolution. GhPAP16 and GhIQD14 fine mapped on chromosome D09 of upland cotton are identified as important candidate genes for lint percentage (LP). Bulked segregant analysis combined with next generation sequencing (NGS-BSA) allows rapid identification of genome sequence differences responsible for phenotypic variation. The NGS-BSA approach applied to crops mainly depends on comparing two bulked DNA samples of individuals from an F2 population. Since some F2 individuals still maintain high heterozygosity, heterosis will exert complications in pursuing NGS-BSA in such populations. In addition, the genetic background influences the stability of gene expression in crops, so some QTLs mapped in one segregating population may not be widely applied in crop improvement. The AAQSP (Association Analysis of QTL-seq on Semi-homologous Populations) reported in our study combines the optimized scheme of constructing BSA bulks with NGS-BSA analysis in two (or more) different parental genetic backgrounds for isolating the stable QTLs. With application of AAQSP strategy and construction of a high-density linkage map, we have successfully identified a QTL significantly related to lint percentage (LP) in cultivated upland cotton, followed by map-based cloning to dissect two candidate genes, GhPAP16 and GhIQD14. This study demonstrated that AAQSP can efficiently identify stable QTLs for complex traits of interest, and thus accelerate the genetic improvement of upland cotton and other crop plants.

Bruchid Resistance Study through Bulked Segregant Analysis: Used as a Preliminary Step for Next-Generation Sequencing

Seed beetles, commonly known as bruchids, are serious insect pests of Indian pulses inflicting high damage during storage. Earlier, a bruchid resistant rice bean landrace (TNAU Red) from Manipur and a susceptible mung bean variety (VRMGg1) from Tamil Nadu were reported and their inter-specific cross was made in Tamil Nadu Agricultural University. In the present study, seeds from the segregating populations (recombinant inbred lines, RILs) of VRM (Gg)1 and TNAU Red were evaluated for bruchid Callosobruchus maculatus Fabricius resistance following standard methods under laboratory conditions. Bulked segregant analysis (BSA) is an efficient method for quick identification of molecular markers linked to any specific gene or genomic region. BSA significantly reduces the scale and cost by simplifying the procedure compared to conventional method of analyzing the entire population. BSA was carried out using 3 marker systems with an attempt to identify markers associated with the present trait of interest. Two DNA bulks namely resistant bulk (RB) and susceptible bulk (SB) were grouped by pooling equal amount of DNA from 5 each of resistant and susceptible RILs based on bruchid responses such as seed damage (%), mean developmental days (MDP) and adult emergence from seeds. Marker analysis carried out with 41 polymorphic SSRs showed skewed segregation distortion towards the susceptible parent. However, out of 9 RAPDs and 10 ISSRs, 2 RAPD (OPB08 and OPX04) and 1 ISSR (UBC810) primers amplified an allele at 200 - 300 and 300 - 400 bp, respectively, in resistant parent and resistant bulk but not in susceptible parent and susceptible bulk. The amplification of resistant parent allele in resistant bulk may indicate the presence of bruchid resistant gene(s) in the resistant RILs. This experiment was aimed at verifying the presence of bruchid resistant lines carrying resistant gene(s) prior to further association mapping or QTL mapping studies. Further, such type of study can be applied as a preliminary step in NGS technology.
 HIGHLIGHTS
 
 Bruchids are the most damaging storage insect in pulses and other legumes causing great economic losses
 Bulk segregant analysis (BSA) conducted using three marker systems in an attempt to identify markers associated with bruchid resistance
 Categorization of RILs based on bruchid responses such as seed damage (%), mean developmental days (MDP), adult emergence from seeds and scoring done
 Marker analysis using BSA useful for verification of the presence of bruchid resistant lines carrying resistant gene(s) prior to further advanced studies such as QTL mapping, NGS, etc.
 
 GRAPHICAL ABSTRACT

Two independent approaches converge to the cloning of a new Leptosphaeria maculans avirulence effector gene, AvrLmS-Lep2.

Brassica napus (oilseed rape, canola) seedling resistance to Leptosphaeria maculans, the causal agent of blackleg (stem canker) disease, follows a gene‐for‐gene relationship. The avirulence genes AvrLmS and AvrLep2 were described to be perceived by the resistance genes RlmS and LepR2, respectively, present in B. napus ‘Surpass 400’. Here we report cloning of AvrLmS and AvrLep2 using two independent methods. AvrLmS was cloned using combined in vitro crossing between avirulent and virulent isolates with sequencing of DNA bulks from avirulent or virulent progeny (bulked segregant sequencing). AvrLep2 was cloned using a biparental cross of avirulent and virulent L. maculans isolates and a classical map‐based cloning approach. Taking these two approaches independently, we found that AvrLmS and AvrLep2 are the same gene. Complementation of virulent isolates with this gene confirmed its role in inducing resistance on Surpass 400, Topas‐LepR2, and an RlmS‐line. The gene, renamed AvrLmS‐Lep2, encodes a small cysteine‐rich protein of unknown function with an N‐terminal secretory signal peptide, which is a common feature of the majority of effectors from extracellular fungal plant pathogens. The AvrLmS‐Lep2/LepR2 interaction phenotype was found to vary from a typical hypersensitive response through intermediate resistance sometimes towards susceptibility, depending on the inoculation conditions. AvrLmS‐Lep2 was nevertheless sufficient to significantly slow the systemic growth of the pathogen and reduce the stem lesion size on plant genotypes with LepR2, indicating the potential efficiency of this resistance to control the disease in the field.

QTL and candidate gene identification of the node of the first fruiting branch (NFFB) by QTL-seq in upland cotton (Gossypium hirsutum L.)

BackgroundThe node of the first fruiting branch (NFFB) is an important precocious trait in cotton. Many studies have been conducted on the localization of quantitative trait loci (QTLs) and genes related to fiber quality and yield, but there has been little attention to traits related to early maturity, especially the NFFB, in cotton.ResultsTo identify the QTL associated with the NFFB in cotton, a BC4F2 population comprising 278 individual plants was constructed. The parents and two DNA bulks for high and low NFFB were whole genome sequenced, and 243.8 Gb of clean nucleotide data were generated. A total of 449,302 polymorphic SNPs and 135,353 Indels between two bulks were identified for QTL-seq. Seventeen QTLs were detected and localized on 11 chromosomes in the cotton genome, among which two QTLs (qNFFB-Dt2–1 and qNFFB-Dt3–3) were located in hotspots. Two candidate genes (GhAPL and GhHDA5) related to the NFFB were identified using quantitative real-time PCR (qRT-PCR) and virus-induced gene silencing (VIGS) experiments in this study. Both genes exhibited higher expression levels in the early-maturing cotton material RIL182 during flower bud differentiation, and the silencing of GhAPL and GhHDA5 delayed the flowering time and increased the NFFB compared to those of VA plants in cotton.ConclusionsOur study preliminarily found that GhAPL and GhHDA5 are related to the early maturity in cotton. The findings provide a basis for the further functional verification of candidate genes related to the NFFB and contribute to the study of early maturity in cotton.

Mapping-by-sequencing the locus of EMS-induced mutation responsible for tufted-fuzzless seed phenotype in cotton.

Cotton fiber mutants are valuable resources for studying functions of altered genes and their roles in fiber development. The n4t is a recessive tufted-fuzzless seed mutant created through chemical mutagenesis with ethyl methanesulfonate. Genetic analysis indicated that the tufted-fuzzless phenotype is controlled by a single recessive locus. In this study, we developed an F2 population of 602 progeny plants and sequenced the genomes of the parents and two DNA bulks from F2 progenies showing the mutant phenotype. We identified DNA sequence variants between the tufted-fuzzless mutant and wild type by aligning the sequence reads to the reference TM-1 genome and designed subgenome-specific SNP markers. We mapped the n4t locus on chromosome D04 within a genomic interval of about 411kb. In this region, seven genes showed significant differential expression between the tufted-fuzzless mutant and wild type. Possible candidate genes are discussed in this study. The utilization of the n4t mutant along with other fiber mutants will facilitate our understanding of the molecular mechanisms of cotton fiber cell growth and development.

Development of polymorphic microsatellites for genetic studies of white scar oyster (Crassostrea belcheri) using paired-end shotgun sequencing.

White scar oyster Crassostrea belcheri is a commercially important bivalve species in Thailand. Appropriate genetic markers are needed for effective management to elevate its production efficiency. Type II microsatellites of C. belcheri were identified and characterized using an Illumina paired-end shotgun sequencing. A total of 14,743,710 reads were generated for which 198,849 reads containing microsatellites and 217,998 microsatellite loci were found. Twenty out of 60 microsatellite loci (33.33%) were polymorphic and these microsatellites were further tested against DNA bulks (N = 10 each) originating from 7 different geographic locations in Thai waters. Results indicated that newly developed microsatellites can be used for genetic diversity analysis of C. belcheri. Genotyping of C. belcheri collected from Surat Thani (Gulf of Thailand; N = 50) were performed. The number of alleles per locus ranged from 2 to 12 (average = 4.95). Observed and expected heterozygosities ranged from 0.0000 to 0.9400 (average = 0.3419) and 0.1139 to 0.8190 (average = 0.5844), respectively. Genome information and 20 newly isolated microsatellites will facilitate further studies in population genetics, stock management, and genetic improvement of C. belcheri in Thailand.

Mapping of a major QTL controlling plant height using a high-density genetic map and QTL-seq methods based on whole-genome resequencing in Brassica napus.

Plant height is a crucial element related to plant architecture that influences the seed yield of oilseed rape (Brassica napus L.). In this study, we isolated a natural B. napus mutant, namely a semi-dwarf mutant (sdw-e), which exhibits a 30% reduction in plant height compared with Zhongshuang 11-HP (ZS11-HP). Quantitative trait locus sequencing (QTL-seq) was conducted using two extreme DNA bulks in F2 populations in Wuchang-2017 derived from ZS11-HP × sdw-e to identify QTLs associated with plant height. The result suggested that two QTL intervals were located on chromosome A10. The F2 population consisting of 200 individuals in Yangluo-2018 derived from ZS11-HP × sdw-e was used to construct a high-density linkage map using whole-genome resequencing. The high-density linkage map harbored 4323 bin markers and covered a total distance of 2026.52 cM with an average marker interval of 0.47 cM. The major QTL for plant height named qPHA10 was identified on linkage group A10 by interval mapping and composite interval mapping methods. The major QTL qPHA10 was highly consistent with the QTL-seq results. And then, we integrated the variation sites and expression levels of genes in the major QTL interval to predict the candidate genes. Thus, the identified QTL and candidate genes could be used in marker-assisted selection for B. napus breeding in the future.

Deciphering the Genetic Diversity of Landraces With High-Throughput SNP Genotyping of DNA Bulks: Methodology and Application to the Maize 50k Array.

Genebanks harbor original landraces carrying many original favorable alleles for mitigating biotic and abiotic stresses. Their genetic diversity remains, however, poorly characterized due to their large within genetic diversity. We developed a high-throughput, cheap and labor saving DNA bulk approach based on single-nucleotide polymorphism (SNP) Illumina Infinium HD array to genotype landraces. Samples were gathered for each landrace by mixing equal weights from young leaves, from which DNA was extracted. We then estimated allelic frequencies in each DNA bulk based on fluorescent intensity ratio (FIR) between two alleles at each SNP using a two step-approach. We first tested either whether the DNA bulk was monomorphic or polymorphic according to the two FIR distributions of individuals homozygous for allele A or B, respectively. If the DNA bulk was polymorphic, we estimated its allelic frequency by using a predictive equation calibrated on FIR from DNA bulks with known allelic frequencies. Our approach: (i) gives accurate allelic frequency estimations that are highly reproducible across laboratories, (ii) protects against false detection of allele fixation within landraces. We estimated allelic frequencies of 23,412 SNPs in 156 landraces representing American and European maize diversity. Modified Roger’s genetic Distance between 156 landraces estimated from 23,412 SNPs and 17 simple sequence repeats using the same DNA bulks were highly correlated, suggesting that the ascertainment bias is low. Our approach is affordable, easy to implement and does not require specific bioinformatics support and laboratory equipment, and therefore should be highly relevant for large-scale characterization of genebanks for a wide range of species.

An EMS-induced mutation in a tetratricopeptide repeat-like superfamily protein gene (Ghir_A12G008870) on chromosome A12 is responsible for the liy short fiber phenotype in cotton.

The EMS-induced threonine/isoleucine substitution in a tetratricopeptide repeat-like superfamily protein encoded by gene Ghir_A12G008870 is responsible for the Ligon-lintless-y (liy) short fiber phenotype in cotton. A short fiber mutant Ligon-lintless-y was created through treating the seeds of the cotton line MD15 with ethyl methanesulfonate. Genetic analysis indicated that the short fiber phenotype is controlled by a single recessive locus designated liy. From F2 populations derived from crosses between the mutant and its wild type (WT), we selected 132 short fiber progeny (liy/liy) and made two DNA bulks. We sequenced these DNA bulks along with the two parents of the population. The liy locus was located on chromosome A12. Using multiple F2 populations and F3 progeny plants, we mapped the liy locus within a genomic region of 1.18Mb. In this region, there is only one gene, i.e., Ghir_A12G008870 encoding a tetratricopeptide repeat-like superfamily protein that has a non-synonymous mutation between the liy mutant and its WT. Analysis of a SNP marker representing this gene in the F2 and F3 progeny plants demonstrated its complete linkage with the liy short fiber phenotype. We further analyzed this SNP marker in a panel of 384 cotton varieties. The mutant allele is absent in all varieties analyzed. RNAseq and RT-qPCR analysis of the gene Ghir_A12G008870 during fiber development showed a significant expression difference between the liy mutant and its WT in developing fiber cells beginning at 12days post-anthesis. Virus-induced gene silencing of the gene Ghir_A12G008870 significantly reduced the fiber length of the WT cotton line MD15. Taken together, our results suggest that the gene Ghir_A12G008870 is involved in the cotton fiber cell elongation process and is a promising candidate gene responsible for the liy short fiber phenotype.

Mapping a Resistance Gene to Puccinia graminis f. sp. tritici in the Bread Wheat Cultivar 'Matlabas'.

Puccinia graminis f. sp. tritici race TTKSF+ was collected from the South African wheat cultivar 'Matlabas' in 2010. F2 and F3 populations derived from a Matlabas × Line 37-07 cross segregated for a single resistance gene to race TTKSF that is avirulent to Matlabas. In screening genomic DNA bulks of susceptible or resistant F2 plants with simple sequence repeat (SSR) markers, three chromosome arm 2BS markers and one multilocus marker amplified alleles present only in the resistant bulks and Matlabas. Additional 2B-specific SSR markers, incorporating markers spanning regions containing Sr9h, SrWLR, Sr28, and Sr47, were screened in the parental lines and mapped in the F2 population. Linkage and QTL mapping showed that the gene is located between Xbarc160 in the centromeric region and Xgwm47 on the long arm of chromosome 2B. When 2B-specific SNP markers were mapped, the area of interest was delimited to a 15.3 cM region on chromosome arm 2BL, with XIWA543-HRM and Xgwm47 as flanking loci. Matlabas, Webster, and related Sr9h lines all produced a similar, low infection type to race TTKSF, but were susceptible to race TTKSF+. Phenotypic data and allelic studies suggested that stem rust resistance in Matlabas was derived from an Sr9h source.

Identification of loci for single/double flower trait by combining genome‐wide association analysis and bulked segregant analysis in peach (Prunus persica)

AbstractBackgroundPeach is a typical perennial fruit crop that can be categorized both as an ornamental and edible crop. Double flower is an important appearance trait, in which breeders are pursuing in high‐quality ornamental peach breeding. The single/double flower character in peach is controlled by one gene, Dl, and the underlying genetic mechanism is unknown.ObjectiveIn this study, we explored the genetic basis of single/double flower trait in peach in a genome‐wide association study using 1 042 687 SNPs characterized in 201 accessions.MethodologyFour loci were identified to be significantly associated with the trait on chromosomes (Chr.) 1, 2 and 6. Besides, the bulked segregant analysis (BSA) was performed with two DNA bulks (single and double flower pool) from the F1 population plants of a cross 'Juhuatao' × 'Honggengansutao'.ResultsWe found a genomic region on Chr. 2 by the BSA, which overlapped the association peak on Chr. 2 in the GWAS—denoted as a region harbouring Dl. Finally, a candidate gene was identified by expression analysis of different plant tissues.ConclusionOur results provide insights into the genetic basis of flower shape and might facilitate marker‐assisted selection breeding for ornamental peach.

Identification of a locus for seed shattering in rice (Oryza sativa L.) by combining bulked segregant analysis with whole-genome sequencing

Seed shattering is an important trait related to the efficiency of harvesting seeds in crops. Although several genes have been identified to control seed shattering in rice (Oryza sativa L.), the genetic architecture underlying extensive variation of seed shattering is yet unclear. ‘Oonari’, a moderate-shattering indica rice cultivar, was developed from a mutant obtained through gamma-ray irradiation of an easy-shattering cultivar ‘Takanari’. The grain pedicel of ‘Oonari’ showed greater resistance to bending than that of ‘Takanari’, while there were no obvious differences in resistance to pulling and abscission zone formation. Investigation of the seed shattering of an F2 population from a cross between ‘Takanari’ and ‘Oonari’ indicated a single semi-dominant locus responsible for seed shattering, which was designated as Sh13. We used bulked DNA of F3 lines with the same shattering degree as that of ‘Oonari’ and DNAs of their parents for whole-genome sequencing, localizing Sh13 at the terminal region on the long arm of chromosome 2. In the candidate genomic region of ‘Oonari’, we identified a tandem duplicated segment containing a microRNA gene, osa-mir172d, whose wheat ortholog is involved in controlling grain threshability. Quantitative RT-PCR analysis indicated that the relative expression of osa-mir172d in ‘Oonari’ was twofold higher than that in ‘Takanari’. We suppose that the duplication of osa-mir172d be involved in reducing seed shattering in ‘Oonari’.

Genetic analysis and mapping of a striped rind gene (st3) in melon (Cucumis melo L.)

As an important commodity trait in melons, the rind is the most direct standard of evaluation for consumers. In this study, we obtained an F2 melon population derived from self-crossing an F1 generation from a cross between X010 (green rind with stripes) and M1-113 (white rind without stripes) and performed genetic analysis and mapping. Genetic analysis revealed that a single dominant gene (st3) controlled the striped rind trait in melons. According to bulked segregant analysis, two DNA bulks were constructed (20 striped and 20 solid-colored rind plants). Next-generation sequencing was used on two parental lines (X010 and M1-113) and two DNA bulks. The st3 gene was identified on chromosome 4 within 2.7 Mbp region. Fourty cleaved amplified polymorphic sequences (CAPS) markers were developed, and sixteen CAPS markers with polymorphisms on chromosome 4 were used for constructing a genetic linkage map, and identifying recombinants in a large F2 population with six new CAPS markers. The location of the st3 gene was narrowed to 172.8 kbp on chromosome 4 between markers M-4-28 (825,401 bp) and M-4-27 (998,261 bp). This study provides a theoretical basis for the fine mapping, cloning and functional characterization of st3.

BSA and molecular markers screening for salt stress tolerant mutant of Petunia obtained in in vitro culture

ABSTRACT: In this study, we performed BSA to identify genetic markers linked to salt tolerance. We tested the genetic diversity among four bulked DNA samples of EMS induced mutant clones and one bulked DNA sample of non-mutated clone of Petunia for salt tolerance in in vitro callus cultures using RAPD and ISSR markers. Out of the 36 RAPD and 16 ISSR primers identified, 25 and 13 were effectively used to amplify genomic DNA of all the five bulked samples, respectively. In total, 114 RAPD amplifications products were obtained, of which 28% were polymorphic and 2% were genotype-specific bands. Out of the 64 ISSR amplification products obtained, 51% were polymorphic and 1% was genotype-specific bands. Results of this study indicated the existence of two patterns of distorted segregation among the studied markers. The first one indicates the differences between non-mutated clones of Petunia and its putative mutants. The second one was observed only between putative mutants and putative mutants tested for salt tolerance in in vitro culture. Both RAPD and ISSR analysis successfully detected the association with changes induced by chemical mutagenesis and salinity. Furthermore, our results indicate that BSA method can be useful in the rapid detection of molecular markers for further marker-assisted selection.