Kompetitive Allele Specific PCR Research Articles

Genetic mapping of regions associated with root system architecture in rice using MutMap QTL-seq

The root system architecture is an important complex trait in rice. With changing climatic conditions and soil nutrient deficiencies, there is an immediate need to breed nutrient-use-efficient rice varieties with robust root system architectural (RSA) traits. To map the genomic regions associated with crucial component traits of RSA viz. root length and root volume, a biparental F2 mapping population was developed using TI-128, an Ethyl Methane Sulphonate (EMS) mutant of a mega variety BPT-5204 having high root length (RL) and root volume (RV) with wild type BPT-5204. Extreme bulks having high RL and RV and low RL and RV were the whole genome re-sequenced along with parents. Genetic mapping using the MutMap QTL-Seq approach elucidated two genomic intervals on Chr.12 (3.14–3.74 Mb, 18.11–20.85 Mb), and on Chr.2 (23.18–23.68 Mb) as potential regions associated with both RL and RV. The Kompetitive Allele Specific PCR (KASP) assays for SNPs with delta SNP index near 1 were associated with higher RL and RV in the panel of sixty-two genotypes varying in root length and volume. The KASP_SNPs viz. Chr12_S4 (C→T; Chr12:3243938), located in the 3’ UTR region of LOC_Os12g06670 encoding a protein kinase domain-containing protein and Chr2_S6 (C→T; Chr2:23181622) present upstream in the regulator of chromosomal condensation protein LOC_Os2g38350. Validation of these genes using qRT-PCR and in-silico studies using various online tools and databases revealed higher expression in TI-128 as compared to BPT- 5204 at the seedling and panicle initiation stages implying the functional role in enhancing RL and RV.

Cloning and functional verification of the male sterile gene BrQRT3 in Chinese cabbage

Chinese cabbage is a cross-pollinated crop with significant heterosis, and male sterile lines are an important way to produce hybrid seeds. In this study, a male sterile mutant msm0795 was identified in an EMS-mutagenized population of Chinese cabbage. Cytological observations revealed that the microspores failed to separate after the tetrad stage, and thus developed into abnormal pollen grains, resulting in anther abortion. MutMap combined with Kompetitive Allele Specific PCR genotyping showed that BraA01g011280.3.5 C was identified as the candidate gene, which encodes polygalacturonase QRT3 and plays a direct role in the degradation of pollen mother cell wall during microspore development, named BrQRT3. Subcellular localization and expression analyses demonstrated that BrQRT3 was localized in the cell membrane and was ubiquitously expressed in roots, stems, leaves, flower buds, and flowers, but the expression of BrQRT3 was gradually suppressed with the anther development. Ectopic expression confirmed that over-expression of BrQRT3 in qrt3 background Arabidopsis mutant can rescue the pollen defects caused by loss of AtQRT3 function. It is the first time to achieve a male sterile mutant caused by the mutation of BrQRT3 in Chinese cabbage. These findings contribute to elucidate the mechanism of BrQRT3 in regulating stamen development of Chinese cabbage.

Fine mapping of QYrsv.swust-1BL for resistance to stripe rust in durum wheat Svevo.

Stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is a serious disease that affects wheat worldwide. There is a great need to develop cultivars with combinations of all-stage resistance (ASR) and adult-plant resistance (APR) genes for sustainable control of the disease. QYrsv.swust-1BL in the Italian durum wheat (Triticum turgidum ssp. durum) cultivar Svevo is effective against Pst races in China and Israel, and the gene has been previously mapped to the long arm of chromosome 1B. The gene is flanked by SNP (single nucleotide polymorphism) markers IWB5732 and IWB4839 (0.75 cM). In the present study, we used high-density 660K SNP array genotyping and the phenotypes of 137 recombinant inbred lines (RILs) to fine map the QYrsv.swust-1BL locus within a 1.066 Mb region in durum wheat Svevo (RefSeq Rel. 1.0) on chromosome arm 1BL. The identified 1.066 Mb region overlaps with a previously described map of Yr29/QYr.ucw-1BL, a stripe rust APR gene. Twenty-five candidate genes for QYrsv.swut-1BL were identified through comparing polymorphic genes within the 1.066 Mb region in the resistant cultivar. SNP markers were selected and converted to Kompetitive allele-specific polymerase chain reaction (KASP) markers. Five KASP markers based on SNP were validated in a F2 and F2:3 breeding population, providing further compelling evidence for the significant effects of QYrsv.swut-1BL. These markers should be useful in marker-assisted selection for incorporating Yr29/QYrsv.swust-1BL into new durum and common wheat cultivars for resistance to stripe rust.

Identification and molecular mapping of resistance genes for adult-plant resistance to stripe rust in spring wheat germplasm line PI660076

Wheat is one of the major food crops worldwide. Stripe rust can cause a great loss of wheat yield, especially when the disease is prevalent. Chemical control not only causes the loss of resistance to stripe rust but also has a serious impact on the human body and environment. Therefore, the most economical measure to control wheat stripe rust is to cultivate resistant varieties. Rapid variation of stripe rust races often leads to rapid "loss" of resistance to stripe rust disease; therefore, breeders and researchers have to continuously explore new stripe rust resistance genes to provide new resistance sources for the rapid variation of stripe rust races. Previous studies have confirmed that PI660076, a spring wheat line, showed stripe rust resistance under natural conditions at the adult stage, which has great value in breeding programs. In this study, a recombinant inbred lines (RIL) population was constructed by crossing the wheat line PI660076 with the stripe rust-susceptible line AvS. Genotyping of the population was performed using a wheat 15 K SNP array. Three QTLs were identified using phenotypic data over four years across three environments. The resistance type of each QTL was determined by inoculating the RIL lines with single and homozygous QTL during the seedling and adult stages under controlled conditions. The all-stage resistance (ASR) QTL, QYr076.jaas-2A (flanked by SNP marker AX-11048464 and Kompetitive Allele-Specific PCR (KASP) marker KASP_4940) explained 7.13–16.58% and 6.95–7.25% of infection type (IT) and disease severity (DS), respectively. The adult-plant resistance (APR) resistance QTL, QYr076.jaas-4D.1 (flanked by KASP marker KASP_0795 and SNP marker AX-111567243,) explained 6.85–12.70% and 7.94–17.26% of IT and DS, respectively. The APR resistance QTL, QYr076.jaas-4D.2 flanked by KASP markers KASP_9130 and KASP_6535, explained 7.97–39.19% and 8.77–20.55% of the phenotypic variation in IT and DS, respectively. All the three QTLs are likely to be new. The obtained results lay a foundation for further utilization of the stripe rust-resistant line PI660076, as well as for fine mapping and molecular marker-assisted selection breeding.Graphical

Molecular characterization of QTL for grain zinc and iron concentrations in wheat landrace Chinese Spring.

Three stable QTL for grain zinc concentration were identified in wheat landrace Chinese Spring. Favorable alleles were more frequent in landraces than in modern wheat cultivars. Wheat is a major source of dietary energy for the growing world population. Developing cultivars with enriched zinc and iron can potentially alleviate human micronutrient deficiency. In this study, a recombinant inbred line (RIL) population with 245 lines derived from cross Zhou 8425B/Chinese Spring was used to detect quantitative trait loci (QTL) for grain zinc concentration (GZnC) and grain iron concentration (GFeC) across four environments. Three stable QTL for GZnC with all favorable alleles from Chinese Spring were identified on chromosomes 3BL, 5AL, and 5BL. These QTL explaining maxima of 8.7%, 5.8%, and 7.1% of phenotypic variances were validated in 125 resequenced wheat accessions encompassing both landraces and modern cultivars using six kompetitive allele specific PCR (KASP) assays. The frequencies of favorable alleles for QGZnCzc.caas-3BL, QGZnCzc.caas-5AL and QGZnCzc.caas-5BL were higher in landraces (90.4%, 68.0%, and 100.0%, respectively) compared to modern cultivars (45.9%, 35.4%, and 40.9%), suggesting they were not selected in breeding programs. Candidate gene association studies on GZnC in the cultivar panel further delimited the QTL into 8.5Mb, 4.1Mb, and 47.8Mb regions containing 46, 4, and 199 candidate genes, respectively. The 5BL QTL located in a region where recombination was suppressed. Two stable and three less stable QTL for GFeC with favorable alleles also from Chinese Spring were identified on chromosomes 4BS (Rht-B1a), 4DS (Rht-D1a), 1DS, 3AS, and 6DS. This study sheds light on the genetic basis of GZnC and GFeC in Chinese Spring and provides useful molecular markers for wheat biofortification.

Genomic-wide analysis reveals seven in absentia genes regulating grain development in wheat (Triticum aestivum L.).

Seven in absentia proteins, which contain a conserved SINA domain, are involved in regulating various aspects of wheat (Triticum aestivum L.) growth and development, especially in response to environmental stresses. However, it is unclear whether TaSINA family members are involved in regulating grain development until now. In this study, the expression pattern, genomic polymorphism, and relationship with grain-related traits were analyzed for all TaSINA members. Most of the TaSINA genes identified showed higher expression levels in young wheat spikes or grains than other organs. The genomic polymorphism analysis revealed that at least 62 TaSINA genes had different haplotypes, where the haplotypes of five genes were significantly correlated with grain-related traits. Kompetitive allele-specific PCR markers were developed to confirm the single nucleotide polymorphisms in TaSINA101 and TaSINA109 among the five selected genes in a set of 292 wheat accessions. The TaSINA101-Hap II and TaSINA109-Hap II haplotypes had higher grain weight and width compared to TaSINA101-Hap I and TaSINA109-Hap I in at least three environments, respectively. The qRT-PCR assays revealed that TaSINA101 was highly expressed in the palea shell, seed coat, and embryo in young wheat grains. The TaSINA101 protein was unevenly distributed in the nucleus when transiently expressed in the protoplast of wheat. Three homozygous TaSINA101 transgenic lines in rice (Oryza sativa L.) showed higher grain weight and size compared to the wild type. These findings provide valuable insight into the biological function and elite haplotype of TaSINA family genes in wheat grain development at a genomic-wide level.

Identification and validation of two major QTL for grain number per spike on chromosomes 2B and 2D in bread wheat (Triticum aestivum L.).

Major QTL for grain number per spike were identified on chromosomes 2B and 2D. Haplotypes and candidate genes of QGns.cib-2B.1 were analyzed. Grain number per spike (GNS) is one of the main components of wheat yield. Genetic dissection of their regulatory factors is essential to improve the yield potential. In present study, a recombinant inbred line population comprising 180 lines developed from the cross between a high GNS line W7268 and a cultivar Chuanyu12 was employed to identify quantitative trait loci (QTL) associated with GNS across six environments. Two major QTL, QGns.cib-2B.1 and QGns.cib-2D.1, were detected in at least four environments with the phenotypic variations of 12.99-27.07% and 8.50-13.79%, respectively. And significant interactions were observed between the two major QTL. In addition, QGns.cib-2B.1 is a QTL cluster for GNS, grain number per spikelet and fertile tiller number, and they were validated in different genetic backgrounds using Kompetitive Allele Specific PCR (KASP) markers. QGns.cib-2B.1 showed pleotropic effects on other yield-related traits including plant height, spike length, and spikelet number per spike, but did not significantly affect thousand grain weight which suggested that it might be potentially applicable in breeding program. Comparison analysis suggested that QGns.cib-2B.1 might be a novel QTL. Furthermore, haplotype analysis of QGns.cib-2B.1 indicated that it is a hot spot of artificial selection during wheat improvement. Based on the expression patterns, gene annotation, orthologs analysis and sequence variations, the candidate genes of QGns.cib-2B.1 were predicted. Collectively, the major QTL and KASP markers reported here provided a wealth of information for the genetic basis of GNS and grain yield improvement.

Pyramiding effects of favorable haplotypes of loci on major fiber yield and quality traits in Upland Cotton (Gossypium hirsutum L.)

Enhancing both fiber yield and quality is crucial in cotton breeding programs. Typically, a negative correlation exists between fiber yield and quality, and the strategy to decouple this relationship remains obscure. In this study, we generated three multi-parent intercross populations, encompassing 2,852 F2:3 lines, which represent fiber yield potential (FY), fiber quality potential (FQ), and a combination of both potentials (FYQ), derived from sixteen diverse Upland cotton cultivars. We utilized twenty-four kompetitive allele specific PCR (KASP) molecular markers, based on previously identified loci associated with fiber quality (FL5/FS1, FL3/FS2, and FL2) and fiber yield (BW1, BW2, and LP1), for genotyping purposes. The pyramiding of favorable haplotypes from three fiber quality-associated loci resulted in an increase exceeding 10% in fiber length (FL5+FL3+FL2+LP1) and in an increase exceeding 17% in fiber strength (FL5+FL3+FL2). Notably, our findings suggest that the combination of FL3+FL2+BW1+LP1 alleles has the potential to disrupt the negative correlation between fiber yield and quality. Lines harboring these alleles simultaneously exhibited an elevated lint percentage (LP), as well as enhanced fiber length and quality. This study offers robust molecular markers for the efficient selection of superior progeny with desired phenotypes in the early stages of cotton breeding. Furthermore, the allelic combination that concurrently improves fiber quality and yield sheds light on for further exploration the molecular basis of breaking unfavorable linkages in crop breeding.

Characterization of Quantitative Trait Loci for Leaf Rust Resistance in the Uzbekistani Wheat Landrace Teremai Bugdai.

Leaf rust, caused by Puccinia triticina, is a major cause of wheat yield losses globally, and novel leaf rust resistance genes are needed to enhance wheat leaf rust resistance. Teremai Bugdai is a landrace from Uzebekistan that is highly resistant to many races of P. triticina in the United States. To unravel leaf rust resistance loci in Teremai Bugdai, a recombinant inbred line (RIL) population of Teremai Bugdai × TAM 110 was evaluated for response to P. triticina race Pt54-1 (TNBGJ) and genotyped using single nucleotide polymorphism (SNP) markers generated by genotyping-by-sequencing (GBS). Quantitative trait loci (QTL) analysis using 5,130 high-quality GBS-SNPs revealed three QTLs, QLr-Stars-2DS, QLr-Stars-6BL, and QLr.Stars-7BL, for leaf rust resistance in two experiments. QLr-Stars-2DS, which is either a new Lr2 allele or a new resistance locus, was delimited to an ∼19.47-Mb interval between 46.4 and 65.9 Mb on 2DS and explained 31.3 and 33.2% of the phenotypic variance in the two experiments. QLr-Stars-6BL was mapped in an ∼84.0-kb interval between 719.48 and 719.56 Mb on 6BL, accounting for 33 to 36.8% of the phenotypic variance in two experiments. QLr.Stars-7BL was placed in a 350-kb interval between 762.41 and 762.76 Mb on 7BL and explained 4.4 to 5.3% of the phenotypic variance. Nine GBS-SNPs flanking these QTLs were converted to kompetitive allele specific PCR (KASP) markers, and these markers can be used to facilitate their introgression into locally adapted wheat lines.

Three Novel er1 Alleles and Their Functional Markers for Breeding Resistance to Powdery Mildew (Erysiphe pisi) in Pea.

Powdery mildew caused by Erysiphe pisi DC is a global notorious disease on peas. Deploying resistance pea cultivars is the most efficient and environmentally friendly method for disease control. This study focuses on revealing the resistance genes in three pea germplasms and developing their functional markers for resistance breeding. The identification of resistance genes involved genetic mapping and the sequencing of the pea mildew resistance locus O homolog PsMLO1 gene. To confirm the heredity of three resistant germplasms, they were crossed with susceptible cultivars to generate F1, F2, and F2:3 populations. The F1 generation exhibited susceptibility to E. pisi, whereas the segregation patterns in subsequent generations adhered to the 3:1 (susceptible: resistant) and 1:2:1 (susceptible homozygotes: heterozygotes: resistant homozygotes) ratios, indicating that powdery mildew resistance was governed by a single recessive gene in each germplasm. Analysis of er1-linked markers and genetic mapping suggested that the resistance genes could be er1 alleles in these germplasms. The multiple clone sequencing results of the three homologous PsMLO1 genes showed they were novel er1 alleles, named er1-15, er1-16, and er1-17. The er1-15 and er1-16 were caused by 1-bp deletion at position 335 (A) and 429 (T) in exon 3, respectively, whereas er1-17 was caused by a 1-bp insertion at position 248 in exon 3, causing a frame-shift mutation and premature termination of PsMLO1 protein translation. Their respective functional markers, kompetitive allele-specific PCR (KASP)-er1-15, KASP-er1-16, and KASP-er1-17, were successfully developed and validated in respective mapping populations and pea germplasms. These results provide valuable tools for pea breeding resistance to E. pisi.

Characterization of a new greenbug resistance gene Gb9 in a synthetic hexaploid wheat.

Greenbug [Schizaphis graminum (Rondani)] is a serious insect pest that not only damages cereal crops, but also transmits several destructive viruses. The emergence of new greenbug biotypes in the field makes it urgent to identify novel greenbug resistance genes in wheat. CWI 76364 (PI 703397), a synthetic hexaploid wheat (SHW) line, exhibits greenbug resistance. Evaluation of an F2:3 population from cross OK 14319 × CWI 76364 indicated that a dominant gene, designated Gb9, conditions greenbug resistance in CWI 76364. Selective genotyping of a subset of F2 plants with contrasting phenotypes by genotyping-by-sequencing identified 25 SNPs closely linked to Gb9 on chromosome arm 7DL. Ten of these SNPs were converted to Kompetitive allele-specific polymerase chain reaction (KASP) markers for genotyping the entire F2 population. Genetic analysis delimited Gb9 to a 0.6-Mb interval flanked by KASP markers located at 599,835,668bp (Stars-KASP872) and 600,471,081bp (Stars-KASP881) on 7DL. Gb9 was 0.5cM distal to Stars-KASP872 and 0.5cM proximal to Stars-KASP881. Allelism tests indicated that Gb9 is a new greenbug resistance gene which confers resistance to greenbug biotypes C, E, H, I, and TX1. TX1 is one of the most widely virulent biotypes and has overcome most known wheat greenbug resistance genes. The introgression of Gb9 into locally adapted wheat cultivars is of economic importance, and the KASP markers developed in this study can be used to tag Gb9 in cultivar development.

Genetic mapping reveals a candidate gene CmoFL1 controlling fruit length in pumpkin.

Fruit length (FL) is an important economical trait that affects fruit yield and appearance. Pumpkin (Cucurbita moschata Duch) contains a wealth genetic variation in fruit length. However, the natural variation underlying differences in pumpkin fruit length remains unclear. In this study, we constructed a F2 segregate population using KG1 producing long fruit and MBF producing short fruit as parents to identify the candidate gene for fruit length. By bulked segregant analysis (BSA-seq) and Kompetitive Allele-Specific PCR (KASP) approach of fine mapping, we obtained a 50.77 kb candidate region on chromosome 14 associated with the fruit length. Then, based on sequence variation, gene expression and promoter activity analyses, we identified a candidate gene (CmoFL1) encoding E3 ubiquitin ligase in this region may account for the variation of fruit length. One SNP variation in promoter of CmoFL1 changed the GT1CONSENSUS, and DUAL-LUC assay revealed that this variation significantly affected the promoter activity of CmoFL1. RNA-seq analysis indicated that CmoFL1 might associated with the cell division process and negatively regulate fruit length. Collectively, our work identifies an important allelic affecting fruit length, and provides a target gene manipulating fruit length in future pumpkin breeding.

Fine mapping and candidate gene analysis of CRA8.1.6, which confers clubroot resistance in turnip (Brassica rapa ssp. rapa).

Clubroot disease poses a significant threat to Brassica crops, necessitating ongoing updates on resistance gene sources. In F2 segregants of the clubroot-resistant inbred line BrT18-6-4-3 and susceptible DH line Y510, the genetic analysis identified a single dominant gene responsible for clubroot resistance. Through bulk segregant sequencing analysis and kompetitive allele-specific polymerase chain reaction assays, CRA8.1.6 was mapped within 110 kb (12,255-12,365 Mb) between markers L-CR11 and L-CR12 on chromosome A08. We identified B raA08g015220.3.5C as the candidate gene of CRA8.1.6. Upon comparison with the sequence of disease-resistant material BrT18-6-4-3, we found 249 single-nucleotide polymorphisms, seven insertions, six deletions, and a long terminal repeat (LTR) retrotransposon (5,310 bp) at 909 bp of the first intron. However, the LTR retrotransposon was absent in the coding sequence of the susceptible DH line Y510. Given the presence of a non-functional LTR insertion in other materials, it showed that the LTR insertion might not be associated with susceptibility. Sequence alignment analysis revealed that the fourth exon of the susceptible line harbored two deletions and an insertion, resulting in a frameshift mutation at 8,551 bp, leading to translation termination at the leucine-rich repeat domain's C-terminal in susceptible material. Sequence alignment of the CDS revealed a 99.4% similarity to Crr1a, which indicate that CRA8.1.6 is likely an allele of the Crr1a gene. Two functional markers, CRA08-InDel and CRA08-KASP1, have been developed for marker-assisted selection in CR turnip cultivars. Our findings could facilitate the development of clubroot-resistance turnip cultivars through marker-assisted selection.

Development of Novel KASP Markers for Improved Germination in Deep-Sown Direct Seeded Rice

BackgroundThe lack of stable-high yielding and direct-seeded adapted varieties with better germination ability from deeper soil depth and availability of molecular markers are major limitation in achieving the maximum yield potential of rice under water and resource limited conditions. Development of high-throughput and trait-linked markers are of great interest in genomics-assisted breeding. The aim of present study was to develop and validate novel KASP (Kompetitive Allele-Specific PCR) markers associated with traits improving germination and seedling vigor of deep sown direct seeded rice (DSR).ResultsOut of 58 designed KASP assays, four KASP assays did not show any polymorphism in any of the eleven genetic backgrounds considered in the present study. The 54 polymorphic KASP assays were then validated for their robustness and reliability on the F1s plants developed from eight different crosses considered in the present study. The third next validation was carried out on 256 F3:F4 and 713 BC3F2:3 progenies. Finally, the reliability of the KASP assays was accessed on a set of random 50 samples from F3:F4 and 80–100 samples from BC3F2:3 progenies using the 10 random markers. From the 54 polymorphic KASP, based on the false positive rate, false negative rate, KASP utility in different genetic backgrounds and significant differences in the phenotypic values of the positive (desirable) and negative (undesirable) traits, a total of 12 KASP assays have been selected. These 12 KASP include 5 KASP on chromosome 3, 1 on chromosome 4, 3 on chromosome 7 and 3 on chromosome 8. The two SNPs lying in the exon regions of LOC_Os04g34290 and LOC_Os08g32100 led to non-synonymous mutations indicating a possible deleterious effect of the SNP variants on the protein structure.ConclusionThe present research work will provide trait-linked KASP assays, improved breeding material possessing favourable alleles and breeding material in form of expected pre-direct-seeded adapted rice varieties. The marker can be utilized in introgression program during pyramiding of valuable QTLs/genes providing adaptation to rice under DSR. The functional studies of the genes LOC_Os04g34290 and LOC_Os08g32100 possessing two validated SNPs may provide valuable information about these genes.

BrBCAT1 mutation resulted in deficiency of epicuticular wax crystal in Chinese cabbage.

BrBCAT1 encoding a branched-chain amino acid aminotransferase was responsible for the glossy trait, which was verified by allelic mutants in Chinese cabbage. The glossy characteristic, thanks to the epicuticular wax crystal deficiency, is an excellent commodity character for leafy vegetables. Herein, two allelic glossy green mutants, wdm11 and wdm12, were isolated from an ethyl methane sulfonate (EMS)-mutagenized population of Chinese cabbage, and the mutant phenotype was recessive inherited. Cryo-SEM detected that epicuticular wax crystal in the mutant leaves was virtually absent. MutMap and Kompetitive allele-specific PCR analyses demonstrated that BraA06g006950.3C (BrBCAT1), homologous to AtBCAT1, encoding a branched-chain amino acid aminotransferase was the candidate gene. A SNP (G to A) on the fourth exon of BrBCAT1 in wdm11 caused the 233rd amino acid to change from glycine (G) to aspartic acid (D). A SNP (G to A) on the second exon of BrBCAT1 in wdm12 led to the 112th amino acid change from glycine (G) to arginine (R). Both of the allelic mutants had genetic structural variation in the candidate gene, which indicated that the mutant phenotype was triggered by the BrBCAT1 mutation. The expression levels of BrBCAT1 and genes related to fatty acid chain extension were decreased significantly in the mutant compared to the wild-type, which might result in epicuticular wax crystal deficiency in the mutants. Our findings proved that the mutation of BrBCAT1 induced the glossy phenotype and provided a valuable gene resource for commodity character improvement in Chinese cabbage.

Development of near homozygous lines for diploid hybrid TPS breeding in potatoes

Diploid inbred-based F1 hybrid True Potato Seed (DHTPS) breeding is a novel technique to transform potato breeding and cultivation across the globe. Significant efforts are being made to identify elite diploids, dihaploids and develop diploid inbred lines for heterosis exploitation in potatoes. Self-incompatibility is the first obstacle for developing inbred lines in diploid potatoes, which necessitates the introgression of a dominant S locus inhibitor gene (Sli) for switching self-incompatibility to self-compatibility. We evaluated a set of 357 diploid clones in different selfing generations for self-compatibility and degree of homozygosity using Kompetitive Allele Specific PCR (KASP) Single Nucleotide Polymorphism (SNP) markers. A subset of 10 KASP markers of the Sli candidate region on chromosome 12 showed an association with the phenotype for self-compatibility. The results revealed that the selected 10 KASP markers for the Sli gene genotype could be deployed for high throughput rapid screening of self-compatibility in diploid populations and to identify new sources of self-compatibility. The homozygosity assessed through 99 KASP markers distributed across all the chromosomes of the potato genome was 20–78 % in founder diploid clones, while different selfing generations, i.e., S0, S1, S2 and S3 observed 36.1–80.4, 56.9–82.8, 59.5–85.4 and 73.7–87.8 % average homozygosity, respectively. The diploid plants with ∼80 % homozygosity were also observed in the first selfing generation, which inferred that homozygosity assessment in the early generations itself could identify the best plants with high homozygosity to speed up the generation of diploid inbred lines.

QTL Mapping for Agronomic Important Traits in Well-Adapted Wheat Cultivars

Wheat (Triticum aestivum L.) is one of the most important food crops worldwide and provides the staple food for 40% of the world’s population. Increasing wheat production has become an important goal to ensure global food security. The grain yield of wheat is a complex trait that is usually influenced by multiple agronomically important traits. Thus, the genetic dissection and discovery of quantitative trait loci (QTL) of wheat-yield-related traits are very important to develop high-yield cultivars to improve wheat production. To analyze the genetic basis and discover genes controlling important agronomic traits in wheat, a recombinant inbred lines (RILs) population consisting of 180 RILs derived from a cross between Xinong822 (XN822) and Yannong999 (YN999), two well-adapted cultivars, was used to map QTL for plant height (PH), spike number per spike (SNS), spike length (SL), grain number per spike (GNS), spike number per plant (SN), 1000- grain weight (TGW), grain length (GL), grain width (GW), length/width of grain (GL/GW), perimeter of grain (Peri), and surface area of grains (Sur) in three environments. A total of 64 QTL were detected and distributed on all wheat chromosomes except 3A and 5A. The identified QTL individually explained 2.24–38.24% of the phenotypic variation, with LOD scores ranging from 2.5 to 29. Nine of these QTL were detected in multiple environments, and seven QTL were associated with more than one trait. Additionally, Kompetitive Allele Specific PCR (KASP) assays for five major QTL QSns-1A.2 (PVE = 6.82), QPh-2D.1 (PVE = 37.81), QSl-2D (PVE = 38.24), QTgw-4B (PVE = 8.78), and QGns-4D (PVE = 13.54) were developed and validated in the population. The identified QTL and linked markers are highly valuable in improving wheat yield through marker-assisted breeding, and the large-effect QTL can be fine-mapped for further QTL cloning of yield-related traits in wheat.

KASP genotyping reveals disease resistance and yield enhancement in swarna introgression lines

Deployment of marker-assisted selection (MAS) in the molecular breeding programmes has transformed the crop breeding over the last decade, bringing speed and precision to the breeding programmes. The experiment was conducted during rainy (kharif) seasons of 2019, 2020, and 2021 at ICAR-Indian Institute of Rice Research, Rajendranagar, Hyderabad, Telangana for validation of the Kompetitive Allele Specific PCR (KASP) SNP markers for routine marker- assisted selection through genotyping and phenotyping of a set of breeding lines segregating for grain number, yield and resistance to bacterial blight and blast diseases. Initially, 150 introgression lines of Swarna possessing bacterial leaf blight (Xa21), blast (Pi54) and yield enhancing gene (Gn1a) at ICF4 generation were genotyped using trait specific KASP markers, and a set of 22 plants was identified to be homozygous for all the three genes, 68 plants were found positive for two genes possessing either Xa21 + Gn1a and Pi54 + Gn1a, rest were having different single gene. Simultaneously, plants which were triple gene positives were also subjected for stringent phenotypic screening for the targeted stresses, viz. bacterial blight and blast disease resistance and further evaluated for grain number and other key agronomic traits. All the three-gene positive plants exhibited high level of resistance to bacterial blight and blast diseases in comparison with positive checks and displayed higher grain number and yield than the recurrent parent, Swarna. The efficiency of SNP marker system provided by Intertek is accurate, cost effective and accurate with phenotype.

KASP genotyping reveals disease resistance and yield enhancement in swarna introgression lines

Deployment of marker-assisted selection (MAS) in the molecular breeding programmes has transformed the crop breeding over the last decade, bringing speed and precision to the breeding programmes. The experiment was conducted during rainy (kharif) seasons of 2019, 2020, and 2021 at ICAR-Indian Institute of Rice Research, Rajendranagar, Hyderabad, Telangana for validation of the Kompetitive Allele Specific PCR (KASP) SNP markers for routine marker- assisted selection through genotyping and phenotyping of a set of breeding lines segregating for grain number, yield and resistance to bacterial blight and blast diseases. Initially, 150 introgression lines of Swarna possessing bacterial leaf blight (Xa21), blast (Pi54) and yield enhancing gene (Gn1a) at ICF4 generation were genotyped using trait specific KASP markers, and a set of 22 plants was identified to be homozygous for all the three genes, 68 plants were found positive for two genes possessing either Xa21 + Gn1a and Pi54 + Gn1a, rest were having different single gene. Simultaneously, plants which were triple gene positives were also subjected for stringent phenotypic screening for the targeted stresses, viz. bacterial blight and blast disease resistance and further evaluated for grain number and other key agronomic traits. All the three-gene positive plants exhibited high level of resistance to bacterial blight and blast diseases in comparison with positive checks and displayed higher grain number and yield than the recurrent parent, Swarna. The efficiency of SNP marker system provided by Intertek is accurate, cost effective and accurate with phenotype.

Promoter variations of ClERF1 gene determines flesh firmness in watermelon

BackgroundFlesh firmness is a critical factor that influences fruit storability, shelf-life and consumer’s preference as well. However, less is known about the key genetic factors that are associated with flesh firmness in fresh fruits like watermelon.ResultsIn this study, through bulk segregant analysis (BSA-seq), we identified a quantitative trait locus (QTL) that influenced variations in flesh firmness among recombinant inbred lines (RIL) developed from cross between the Citrullus mucosospermus accession ZJU152 with hard-flesh and Citrullus lanatus accession ZJU163 with soft-flesh. Fine mapping and sequence variations analyses revealed that ethylene-responsive factor 1 (ClERF1) was the most likely candidate gene for watermelon flesh firmness. Furthermore, several variations existed in the promoter region between ClERF1 of two parents, and significantly higher expressions of ClERF1 were found in hard-flesh ZJU152 compared with soft-flesh ZJU163 at key developmental stages. DUAL-LUC and GUS assays suggested much stronger promoter activity in ZJU152 over ZJU163. In addition, the kompetitive allele-specific PCR (KASP) genotyping datasets of RIL populations and germplasm accessions further supported ClERF1 as a possible candidate gene for fruit flesh firmness variability and the hard-flesh genotype might only exist in wild species C. mucosospermus. Through yeast one-hybrid (Y1H) and dual luciferase assay, we found that ClERF1 could directly bind to the promoters of auxin-responsive protein (ClAux/IAA) and exostosin family protein (ClEXT) and positively regulated their expressions influencing fruit ripening and cell wall biosynthesis.ConclusionsOur results indicate that ClERF1 encoding an ethylene-responsive factor 1 is associated with flesh firmness in watermelon and provide mechanistic insight into the regulation of flesh firmness, and the ClERF1 gene is potentially applicable to the molecular improvement of fruit-flesh firmness by design breeding.