InDel Markers Research Articles

Development and validation of PCR marker array for molecular selection towards spring, vernalization-independent and winter, vernalization-responsive ecotypes of white lupin (Lupinus albus L.)

White lupin (Lupinus albus L.) is an ancient grain legume that is still undergoing improvement of domestication traits, including vernalization-responsiveness, providing frost tolerance and preventing winter flowering in autumn-sowing agriculture, and vernalization-independence, conferring drought escape by rapid flowering in spring-sowing. A recent genome-wide association study highlighted several loci significantly associated with the most contrasting phenotypes, including deletions in the promoter of the FLOWERING LOCUS T homolog, LalbFTc1, and some DArT-seq/silicoDArT loci. The present study aimed to develop and validate a versatile PCR marker array enabling molecular selection of spring- and winter-type white lupin ecotypes. Candidate DArT-seq and silicoDArT loci were transformed into cleaved amplified polymorphic sequence (CAPS) or derived CAPS markers. Developed markers, together with those previously published for LalbFTc1 INDELs and quantitative trait loci from linkage maps, were implemented for screening of white lupin germplasm panel subjected to 2-year phenotyping of phenology traits. Three DArT-seq, two silicoDArT and seven LalbFTc1 INDEL markers were positively validated, constituting a convenient PCR-based marker assay for rapid and accurate reselection of white lupin germplasm towards early flowering and thermoneutrality or late flowering and vernalization-responsiveness, as well as for tracking high genetic and phenotypic diversity within white lupin landraces, revealed in the present study.

Integrated Genetic Diversity and Multi-Omics Analysis of Colour Formation in Safflower.

Safflower (Carthamus tinctorius L.) is a medicinal and edible cash crop that is widely cultivated worldwide. However, the genetic diversity of safflower germplasm resources and the reasons for the variations in safflower flower colour remain unclear. In this study, we used a combination of agronomic traits and Indel markers to assess the genetic diversity of 614 safflower germplasm resources. The results showed that most of the evaluated agronomic traits had high variability. The mean values of the Shannon's information index (I) and polymorphism information content (PIC) in 50 pairs of Indel markers were 0.551 and 0.296, respectively. The population structure, neighbour-joining phylogeny, and principal coordinate analyses classified all genotypes into four subgroups, and 214 safflower core germplasms were constructed. Multiple analyses of genetic diversity parameters, range conformity, and the percentage of variance difference showed that the core germplasm did not differ significantly and could represent the original germplasm better. Transcriptome and metabolome analyses revealed that flavonoid synthesis-related genes, including CHS, F3H, ANS, and BZ1, were differentially expressed in different coloured safflowers. Most significantly, different genes and metabolite compounds in white safflowers were enriched upstream from the phenylpropanoid metabolic pathway to the production of naringenin, whereas those in red safflowers were concentrated in the downstream pathway from eriodictyol. Meanwhile, the preliminary quantification of anthocyanins and carotenoids extracted from red, orange, and white types of safflower showed that the level of both anthocyanins and carotenoids were highest in red types. This work provides new insights into the formation of different safflower flower colours and in the conservation and management of safflower germplasm.

Identification of Starmerella aleppica f.a., sp. nov. and large indels in the rRNA cistron that split the Starmerella genus.

Yeast strains representing a novel asexual ascomycetous species were isolated from seven Malva sylvestris flowers. Sequencing of the chromosomal regions coding for the D1/D2 domains of the large subunit ribosomal RNA, the ITS1-5.8S-ITS2 segments and parts of the gene coding for the small subunit ribosomal RNA showed that the isolates were conspecific. Comparative analysis of these sequences and the corresponding sequences of the type strains of ascomycetous yeasts revealed that the strains represent a hitherto undescribed species belonging to the sensu stricto subclade of the genus Starmerella. The new species is osmotolerant and can develop invasive pseudohyphae, but does not form spores. For the new species, the name Starmerella aleppica f.a. (forma asexualis) is proposed. The holotype, preserved in a metabolically inactive state, is CBS 12960T (extype cultures: 2-1361 and CCY 90-2-1, NCAIM Y.02123). The GenBank accession numbers of barcode sequences are JX515983 (D1/D2 domain), JX515985 (ITS1-5.8S-ITS2 and partial 18S rRNA gene), PQ613837 (TEF1 partial sequence) and PQ613838 (RPB2 partial sequence). MycoBank: MB855459. The analysis of the D1/D2 and internal transcribed spacer (ITS) sequences of the type strains of species of the genus identified multiple multinucleotide indels that can be used as taxonomic markers (InDel markers). The indel patterns of the subclades are very different and homogeneous within the subclades. This result reinforces the idea raised, but also refuted, in previous studies that the Starmerella subclades may represent different genera.

Allelic divergence and heritable interrelationship studies for yield and submergence tolerance in rice (Oryza sativa L.)

Global low-lying rice cultivation faces a serious threat of flash floods, exacerbated by climate change. Significant progress has been made by plant breeders to introgress Sub1 locus into elite rice background through marker-assisted breeding approaches. The F2 population derived from the ADT 36 × #91-27 (NIL of CO 43 Sub1) was used to identify high-yielding lines that are homozygous for the Sub1 locus and Pi54 gene. INDEL markers, ART 5 and Pi 54, were used for genotyping the Sub1 locus and Pi54 gene, respectively. Out of 83 F2 plants, 21 plants are homozygotes and 45 plants are heterozygotes for the Sub1 locus. Whereas 21 plants are homozygotes and 46 are heterozygotes for the Pi54 gene. Four plants were identified to be homozygotes for the Sub1 locus and Pi54 gene. Genetic analysis of the F2 plants identified that productive tillers (number/plant) and the filled grains (number/panicle) exerted the highest positive effect on the single plant yield and also had the highest heritability. Also, these traits have a significant positive correlation per se. These traits, particularly productive tillers (number/plant), can potentially be used in subsequent generations to select high-yielding submergence tolerance and blast resistant lines of ADT 36 × #91-27 (NIL of CO 43 Sub1).

Development of Intragenic InDel Markers From RNA‐Seq Data for Map‐Based Cloning and Marker‐Assisted Selection in Maize (Zea mays L.)

ABSTRACTMaize is one of the most extensively grown and produced crops in the world. Functional molecular markers (FMM) are essential tools for enhancing the efficiency of both breeding and basic research. The transcriptome obtained from high‐throughput sequencing is a valuable resource for developing FMM. In this study, we precisely identified high‐density and highly polymorphic RNA‐derived InDel markers from 368 high‐quality, high read depth maize RNA‐seq datasets using stringent criteria. The nonredundant marker dataset comprised 13,904 InDel markers with a Polymorphism Information Content (PIC) ≥ 0.5 and a principal allelic difference (length difference between the most and second most frequent alleles) ≥ 3 bp, covering 7749 genes. Subsequently, the polymorphism of InDel markers was verified experimentally. Fifty InDel markers were randomly selected, and their average PIC values in 20 maize inbred lines selected from 368 RNA‐seq samples and 20 newly selected maize inbred lines were 0.53 and 0.54, with range intervals of 0.18 to 0.77 and 0.22 to 0.74, respectively. The high density and polymorphism of RNA‐derived InDel markers identified in this study make them effective tools for maize gene cloning, genetic diversity studies, genome‐wide association analysis, and marker‐assisted selection.

Assessment of Parental Polymorphism between Swarna / Oryza rufipogon IC309814 Derived Mapping Population

Detection of molecular markers with parental polymorphism is a pre-requisite for QTL mapping and further identification of genomic regions. PCR based InDel markers tend to have a greater degree to show polymorphism among the parental genotypes for any specific cross. Polymorphic studies were carried out between the two parents Swarna (cultivated rice) and Oryza rufipogon IC309814 (Wild rice) using 192 randomly selected InDel markers and 40 gene specific markers for Iron and Zinc covering the entire 12 chromosomes. Results revealed that InDel markers were polymorphic between both the parents and highest percentage of polymorphism observed on chromosome 10 (75%) followed by chromosome 5 (60%) and chromosome 9 (55.6%) and lowest polymorphism percentage was shown in chromosome 3 (8.3%) with an overall 22.4 % polymorphism percentage among all the markers used in this study. Detected polymorphic (SSR) markers were for genotyping the mapping population derived from the cross between Swarna cultivar and Oryza rufipogon to map Quantitative Trait Loci (QTLs) associated with yield and grain quality traits.

Fine-Mapping of Tomato male sterile-24 Locus and Marker Development for ms-24 and Its Alleles, ms-10, ms-35, and ms-36

Male sterility is an essential trait for minimizing costs and ensuring seed purity in the production of tomato hybrid seeds. However, its application in commercial breeding faces challenges such as instability, low stigma exsertion rates, and the lack of molecular markers to facilitate the efficient backcrossing of male sterile loci into elite tomato lines. This study characterized the male sterile-24 (ms-24) mutant, which demonstrated exserted stigmas and complete male sterility. Our histological examination revealed that ms-24 anthers displayed dysfunctional development of the tetrads and tapetum. The ms-24 locus was finely mapped to a 149 kb interval containing 22 putative genes. Among these, Solyc02g079810, also known as MS-10, encodes a basic helix–loop–helix (bHLH) transcription factor essential for tapetum and pollen development. A sequencing analysis revealed an approximately 4.9 kb retrotransposon insertion in the first exon of the MS-10 gene in ms-24. Allelism tests confirmed that ms-24 was allelic to ms-10, which is also allelic to ms-35 and ms-36. The same retrotransposon insertion was also identified in the ms-10 mutant, and a similar insertion of retrotransposon was detected in the second exon of the MS-10 gene in the ms-36 mutant. Based on these retrotransposon sequences, codominant insertion–deletion (InDel) markers, MS-24I and MS-35I, were developed for the precise identification of the ms-24, ms-10, ms-35, and ms-36 alleles. These findings establish a foundational basis for subsequent investigations into the molecular mechanisms underlying male sterility and enhance the selection process of male sterile lines in tomato hybrid breeding programs.

Map-based cloning revealed BhAPRR2 gene regulating the black peel formation of mature fruit in wax gourd (Benincasa hispida).

Map-based cloning revealed BhAPRR2, encoding a two-component response-regulating protein that regulates the black peel formation of mature fruit in wax gourd. Wax gourd is an economically significant vegetable crop, and peel color is a crucial agronomic trait that influences its commercial value. Although genes controlling light green or white peel have been cloned in wax gourd, the genetic basis and molecular mechanism underlying black peel remain unclear. Here, we confirmed that the peel color of wax gourd is a qualitative trait governed by single gene, with black being dominant over green. Through bulked segregant analysis sequencing (BSA-seq) and map-based cloning, we identified Bh.pf3chr5g483 as the candidate gene. This gene encodes a two-component response-regulating protein and is homologous to APRR2, referred to as BhAPRR2. Compared to P170, the BhAPRR2 in YD1 exhibits multiple mutations in both its coding and promoter regions. Notably, the mutations in the coding region do not affect its nuclear localization or transcriptional activation activity. However, the mutations in the promoter region substantially increase its expression in the peel of YD1, potentially contributing to the black peel phenotype observed in this variety. Furthermore, we developed an insertion/deletion (InDel) marker based on a 93-base pair (bp) insertion/deletion mutation in the promoter region of BhAPRR2, which achieved up to 95.8% phenotypic accuracy in a natural population comprising 165 wax gourd germplasms. In summary, our findings suggest that mutations in the promoter region of BhAPRR2 may contribute to the development of black peel in wax gourd. This discovery provides new insights into the molecular and genetic mechanisms underlying peel color diversity and offers a valuable molecular marker for wax gourd breeding efforts.

Mapping of resistance genes to powdery mildew based on DNA re-sequencing and bulk segregant analysis in Capsicum.

Powdery mildew caused by Leveillula taurica adversely affects the development and growth of pepper plants. However, there have been few reports on the fine mapping and quantitative trait locus (QTLs) gene cloning of resistance genes to powdery mildew in pepper. Herein, an F2 segregating population was constructed using the high resistance material "NuMex Suave Red" and the extremely susceptible material "c89" for bulked segregant analysis and DNA re-sequencing (BSA-seq). Molecular markers were used to achieve fine mapping, followed by expression verification. A major QTL located on chromosome 5 (Chr5, 7.20-11.75Mb) that is associated with resistance to powdery mildew in pepper was mapped using BSA-seq. A narrow interval of 64.86kb encompassing five genes was refined using InDel and KSAP molecular markers developed from the QTL region. Among them, the expression of the ubiquitin-conjugating enzyme E2 gene, Capana05g000392, was significantly upregulated in multiple resistant materials. In addition, there was a single nucleotide polymorphism (SNP) of A/G in the 241st position of the CDS sequence of Capana05g000392, which in turn leads to an amino acid polymorphism of M/V between susceptible parent and resistant parent. Overall, these results indicate that the Capana05g000392 gene may serve as a robust potential factor against powdery mildew in pepper. These findings further elucidate the genetic mechanism of resistance to powdery mildew in pepper and facilitate molecular marker-assisted breeding.

Identification of SNPs and INDELS associated with duck egg quality traits through a genome-wide association analysis

Egg quality traits are economically important in the poultry industry. To explore the genetic architecture and identify potential candidate genes, a genome-wide association study (GWAS) was performed for 13 egg quality traits using data from whole-genome sequencing of 299 Longyan Shan-ma female ducks, including 12 quantitative traits and one qualitative trait, eggshell color (ESC; white, light green, green). From estimation of pedigree genetic parameters, heritability (h2) ranged from 0.022 to 0.996 for the 12 quantitative traits, with the highest h2 (0.996) for eggshell color a* value (ESCA) and the lowest h2 (0.022) for egg yolk percentage relative to EW. A total of 8,874 single nucleotide polymorphism (SNP)-based significant associations (1.0 × 10-6) and 247 insertion-deletion (indel)-based significant associations (1.00 × 10-5) were identified, including 5,980 SNPs and 159 indel markers. From 5,924 SNPs and 143 indels associated with ESC traits, 181 potential candidate genes were identified, and most significant SNPs and indels (P < 1.0 × 10-20) were located at 1.86 Mb (44.29-46.15 Mb) on chromosome 4. The top SNP (chr4:45325309:C>A; P = 7.97 × 10-43) and the top indel (chr4:45299595:delTTCCACTCCAC; P = 4.20 × 10-36) for the ESC a* value were within two known ESC candidate genes; ATP-binding cassette subfamily G member 2 (ABCG2) and protein kinase cGMP-dependent 2 (PRKG2). Of 56 SNPs and 16 indels associated with other egg quality traits, 46 potential candidate genes were identified including synapse differentiation-inducing 1-like (SYNDIG1L) for EW, and core histone macro-H2A.1 (LOC101795967) and neurogenin 1 (NEUROG1) for egg shape index; and four genes including collagen type VI alpha 3 chain (COL6A3), lysine demethylase 7A (KDM7A), LOC101802169, and sperm-associated antigen 16 (SPAG16) for egg yolk weight and the percentage of yolk to total egg weight. Of the 46 genes, the molecular functions of 22 are related to protein binding, indicating important roles in the formation of egg quality traits. Our findings provide new insight into the genetic basis of egg quality traits in ducks.

A single-base deletion in exon 2 of Hd1 delineates monogenic recessive photoperiod insensitivity in aromatic Joha rice: a novel allele for seasonal adaptability

BackgroundAssam's aromatic Joha rice is a unique rice class famous for its aroma, taste, and nutritional benefits, which fetch high market prices in domestic and international markets. Joha landraces are inherently poor yielders due to their strong aroma and predominantly photoperiod sensitivity. Hybridization involving non-aromatic HYVs improves yield with concomitant loss of quality. In this context, mutation breeding, a sustainable approach where genetic mutations are induced to create desirable traits, often provides useful allelic variation in specific morpho-agronomic traits. The present study delves into the genetic characterization of a photoperiod-insensitive mutant. As part of our mutation breeding programme, this mutant was isolated from a gamma ray-induced M2 population of a Joha rice landrace, Kon Joha.ResultsThe mutant was unique, and a single recessive gene conditions the induced photoperiod insensitivity. Mutant gene tagging involved 402 SSR and InDel markers, and later polymorphic markers were used for bulk segregant analysis (BSA) in the F2 population of ‘mutant × Kalijeera (distant parent)’. BSA revealed an association between the SSR marker RM527 and this mutant trait. This marker is present on chromosome 6 of the rice genome. Using chromosome 6-specific SSR markers in polymorphic screening and BSA revealed another associated marker, RM19725, for the mutant trait. The genomic interval between RM527 and RM19725 harbors a photoperiod-insensitive gene, Hd1, on chromosome 6. Cloning and sequencing of Hd1 genomic fragments from the parents and mutants revealed a single-base deletion in exon 2, leading to a frameshift mutation in the Hd1 protein. This mutation in exon 2 leads to severe structural abnormalities in the CCT domain of the Hd1 protein that is critical for the interaction of the repressing complex with conserved response elements in the florigen gene under long-day conditions, thereby causing photoperiod insensitivity.ConclusionsThe mutant's pleasant aroma and other quality characteristics, comparable to those of the parent cultivar, hold significant promise. They expand its potential use in a structured breeding programme aimed at developing high-value aromatic Joha rice. This rice, resilient to winter- and summer-growing environments and with broad seasonal adaptability, could revolutionize the rice market. The practical value of our research is underscored by this exciting possibility.

The Universal Set of 99 InDel Markers for Human Identification

The aim of this work was to select InDel markers sufficient for human identification and to create a routine method for their genotyping. We analyzed the allele distribution of all known InDels in European, East Asian, South Asian, African, and American populations and selected markers whose minor allele frequency, MAF, was ≥ 0.30. Thus, a set of 99 polymorphisms was formed with an average MAF for all five populations of 0.397 and a distance between markers of ≥3 million bp. The expected values of combined random match probability differ very little between the five superpopulations and are in the range of 1.44 × 10−41–3.68 × 10−41. For the genotyping of this set of markers, we have developed a method that includes single-step multiplex PCR followed by hybridization on a biochip. The average amplicon length was 72 bp. Aiming to verify the usefulness of 99 biallelic InDels in genetic identification and kinship testing, two phylogenetically distant populations were studied: Russian and Ecuadorian. For the Russian population, the combined random match probability was 2.09 × 10−40, and the combined power of exclusion, CPE, was 0.999999989. For the Ecuadorian population, the combined random match probability was 1.02 × 10−40, and the combined power of exclusion was 0.999999978. The proposed method is intended to become an alternative to human identification based on differences in the length of STR loci.

Complete chloroplast genomes of three Polygala species and indel marker development for identification of authentic polygalae radix (Polygala tenuifolia).

In Korea, only Polygala tenuifolia is registered as Polygalae Radix in the pharmacopoeia, while in China, both P. tenuifolia and P. sibirica are used equally. Accurate identification of herbal medicines is crucial for their safety and efficacy, but commercial products are typically sold in dried form, making morphological distinction difficult. Therefore, a quick and accurate method to distinguish P. tenuifolia is necessary for proper utilization of medicinal herb. We aimed to identify specific molecular markers for P. tenuifolia to avoid confusion regarding its pharmacological efficacy and to evaluate the classification of Polygala using plastid phylogenetic data. We analyzed the sequences of three species distributed in Korea, P. tenuifolia, P. japonica, and P. sibirica, and assembled their chloroplast genome sequences. Comparative analysis revealed regions of local divergence, and six molecular markers were developed from these hotspots. Additionally, a phylogenetic tree was constructed to determine the phylogenetic positions of the three Polygala species. The marker successfully identified the three Polygala species, and all commercial products and breeding lines tested were confirmed to be P. tenuifolia and recognized as authentic. Phylogenetic analysis revealed that P. tenuifolia forms a distinct cluster from P. sibirica and P. japonica. We determined the chloroplast genomes of the three Polygala species and performed phylogenetic tree analysis and marker development. Indel markers were developed to identify the critical herbal species, P. tenuifolia. This comprehensive study of the Polygala chloroplast genome provides useful information for P. tenuifolia identification.

Development of Novel InDel Markers for Breeding Applications in Pomegranate (Punica granatum L.)

ABSTRACTThe present study aims to enrich the genetic marker resources that are amenable for future trait mapping and quality improvement programmes in pomegranate. We analysed 140 well‐characterized genes that belonged to seven important gene families involved in the growth and development of pomegranate. We retrieved 140 representative genes for seven gene families from four draft genomes of pomegranate, cvs. Tunisia, Dabenzi, Taishanhong and Bhagawa, using BLASTn homology search. A total of 245 InDel primers were designed based on the comparison among four pomegranate genomes for the representative gene sequences, focusing on a size difference of at least 8 base pairs (bp). One hundred forty‐eight (60.41%) markers were in silico verified through e‐PCR on the Tunisia genome and further confirmed on the other three genomes, and we identified 107 polymorphic markers with an average PIC value of 0.62. By using 148 InDel markers physically mapped on the genome, we found that Tunisia had the highest syntenic relations with the Bhagawa (100%), Dabenzi (97.97%) and Taishanhong (97.30%) genomes. Experimental validation of 54 InDel primers on six pomegranate genotypes identified a set of 37 (71.15%) polymorphic markers, 24 of which had PIC values of ≥ 0.48. An investigation on genetic diversity and phenotype correlations in 16 pomegranate genotypes using 16 InDel markers demonstrated the utility of these markers. The developed InDel markers are an excellent resource for diverse applications in pomegranate including genetic diversity, trait mapping, gene discovery and gene editing.

An INDEL genomic approach to explore population diversity of phytoplankton

BackgroundAlthough metabarcoding and metagenomic approaches have generated large datasets on worldwide phytoplankton species diversity, the intraspecific genetic diversity underlying the genetic adaptation of marine phytoplankton to specific environmental niches remains largely unexplored. This is mainly due to the lack of biological resources and tools for monitoring the dynamics of this diversity in space and time.ResultsTo gain insight into population diversity, a novel method based on INDEL markers was developed on Bathycoccus prasinos (Mamiellophyceae), an abundant and cosmopolitan species with strong seasonal patterns. Long read sequencing was first used to characterize structural variants among the genomes of six B. prasinos strains sampled from geographically distinct regions in the world ocean. Markers derived from identified insertions/deletions were validated by PCR then used to genotype 55 B. prasinos strains isolated during the winter bloom 2018–2019 in the bay of Banyuls-sur-Mer (Mediterranean Sea, France). This led to their classification into eight multi-loci genotypes and the sequencing of strains representative of local diversity, further improving the available genetic diversity of B. prasinos. Finally, selected markers were directly tracked on environmental DNA sampled during 3 successive blooms from 2018 to 2021, showcasing a fast and cost-effective approach to follow local population dynamics.ConclusionsThis method, which involves (i) pre-identifying the genetic diversity of B. prasinos in environmental samples by PCR, (ii) isolating cells from selected environmental samples and (iii) identifying genotypes representative of B. prasinos diversity for sequencing, can be used to comprehensively describe the diversity and population dynamics not only in B. prasinos but also potentially in other generalist phytoplankton species.

Fine mapping and identification of the downy mildew resistance gene BoDMR2 in Cabbage (Brassica oleracea L. var. capitata).

Cabbage (Brassica oleracea L. var. capitata) is an important crop within the Brassica oleracea species and is extensively cultivated worldwide. In recent years, outbreaks of downy mildew caused by Hyaloperonospora parasitica have resulted in substantial losses in cabbage production. Despite this, there have been limited studies on genes associated with resistance to downy mildew in cabbage. This study identified sister lines exhibiting significant differences in disease resistance and susceptibility. Using bulked segregant analysis followed by sequencing (BSA-seq) and linkage analysis, the cabbage resistance locus BoDMR2 was accurately mapped to an approximately 300kb interval on chromosome 7. Among the candidate genes identified, several single nucleotide polymorphisms (SNPs) and a 3-bp insertion were found within the conserved domain of the Bo7g117810 gene, encoding a leucine-rich repeat domain protein, in susceptible genotypes. Additionally, real-time quantitative polymerase chain reaction (RT‒qPCR) analysis revealed that the expression level of Bo7g117810 in resistant specimens was 2.5-fold higher than that in susceptible specimens. An insertion‒deletion (InDel) marker was designed based on the identified insertion in susceptible materials, facilitating the identification and selection of downy mildew-resistant cabbage cultivars. This study identifies Bo7g117810 as a potential candidate gene associated with adult-stage resistance to downy mildew in cabbage, supported by observed differences in gene sequence and expression levels. Furthermore, the development of an InDel marker I1-3, based on its mutation, provides valuable resources for breeding resistant cabbage cultivars.

Genome-Wide Association Studies (GWAS) and Transcriptome Analysis Reveal Male Heterogametic Sex-Determining Regions and Candidate Genes in Northern Snakeheads (Channa argus).

The Northern snakehead (Channa argus) is a significant economic aquaculture species in China. Exhibiting sexual dimorphism in the growth rate between females and males, mono-sex breeding holds substantial value for aquaculture. This study employed GWAS and transcriptome analysis were applied to identify sex determination genomic regions and develop sex-specific markers. A total of 270 single-nucleotide polymorphisms (SNPs) and 31 insertion-deletions (InDels) were identified as being sexually dimorphic through GWAS and fixation index (Fst) scanning. Based on GWAS results, two sex-specific InDel markers were developed, effectively distinguishing genetic sex for XX females, XY males, and YY super-males via (polymerase chain reaction) PCR amplification. A major genomic segment of approximately 115 kb on chromosome 3 (Chr 03) was identified as the sex-determination region. A comparative transcriptome analysis of gonads for three sexes identified 158 overlapping differentially expressed genes (DEGs). Additionally, three sex-related candidate genes were identified near the sex determination region, including id2, sox11, and rnf144a. Further studies are required to elucidate the functions of these genes. Overall, two sex-specific InDel markers support a male heterogametic XX/XY sex-determination system in Northern snakeheads and three candidate genes offer new insights into sex determination and the evolution of sex chromosomes in teleost fish.

Co-localization of quantitative trait loci for pod and kernel traits and development of molecular marker for kernel weight on chromosome Arahy05 in peanut (Arachis hypogaea L.)

Key messageStable QTL for pod and kernel traits were co-localized on chromosome Arahy05, and an INDEL marker at 106,411,957 on Arahy05 was developed and validated to be useful for marker-assisted selection of kernel weight.Pod and kernel traits, such as hundred pod weight (HPW), and hundred kernel weight (HKW), along with pod and kernel sizes, are pivotal determinants of yield in peanut breeding programs. This study sought to identify quantitative trait loci (QTL) that are associated with these pod and kernel traits in peanuts. To achieve this, a recombinant inbred line (RIL) population, was derived from a cross between Yuhua15, a cultivar known for its high yield, and a germplasm accession W1202. The investigation uncovered stable and major QTL that are significantly associated with both pod and kernel weight and were consistently co-localized on chromosomes Arahy05 and Arahy08. Furthermore, an INDEL marker was identified and characterized in the QTL interval on Arahy05. An extensive re-sequencing analysis comprising 395 germplasm accessions led to the discovery of two principal haplotypes within a 500-kb window flanking the aforementioned INDEL marker. The haplotypes exhibited a significant correlation with the HKW in our diverse panel of germplasm accessions. Notably, the 170 accessions harboring the haplotype associated with an increased HKW primarily represented botanical varieties, specifically Arachis hypogaea var. hypogaea and A. hypogaea var. hirsuta. On the other hand, the 137 accessions associated with the alternative haplotype, which corresponded to a reduced HKW, were predominately identified as belonging to botanical varieties within A. hypogaea subsp. fastigiata. The INDEL marker located on Arahy05, which demonstrates close linkage to the pod and kernel traits, would be an efficient approach for marker-assisted selection (MAS) of pod and kernel weight in breeding programs.

Allelic Diversity and Development of Breeder-Friendly Marker Specific to floury2 Gene Regulating the Accumulation of α-Zeins and Essential Amino Acids in Maize Kernel.

Maize zeins lack essential amino acids, such as methionine, lysine, and tryptophan. The floury2 (fl2) mutation reduces zein synthesis and increases methionine and lysine content in kernels. In this study, fl2 gene (1612bp) was sequenced in eight wild-type and two mutant inbreds and detected 218 SNPs and 18 InDels. Transversion of C to T at 343bp position caused the substitution of alanine by valine in the fl2 mutant. A PCR-based marker (FL-SNP-CT) was developed, which distinguished the favorable mutant fl2 allele (T) from the wild-type (C) Fl2 allele. Gene-based diversity analysis using seven gene-based InDel markers grouped 48 inbred lines into three major clusters, with an average genetic dissimilarity coefficient of 0.534. The average major allele frequency, gene diversity, heterozygosity, and polymorphism information content of the InDel markers were 0.701, 0.392, 0.039, and 0.318, respectively. Haplotype analysis revealed 29 haplotypes of fl2 gene among these 48 inbreds. Amino acid substitution (Ala-Val) at the signal peptide cleavage site produced unprocessed 24-kDa mutant protein instead of 22-kDa zein found in normal genotype. Eight paralogues of fl2 detected in the study showed variation in exon lengths (616-1170bp) and translation lengths (135-267 amino acids). Orthologue analysis among 15 accessions of Sorghum bicolor and two accessions of Saccharum spontaneum revealed a single exon in fl2 gene, ranging from 267 to 810bp. The study elucidated the molecular basis of fl2 mutation and reported a breeder-friendly marker for molecular breeding programs. This is the first study to characterize fl2 gene in a set of subtropically adapted inbreds.

In vivo tetraploid induction of mono-embryonic citrus genotypes by colchicine treatment

Polyploid holds great significance in citrus breeding, to produce dwarf tetraploid rootstocks with wide adaptability and triploid scion varieties bearing seedless fruits. In citrus, tetraploids have been generated through somatic hybridization and autonomous chromosome doubling of nucellar embryos. However, most tetraploids were recovered from poly-embryonic (apomictic) citrus, while tetraploids of mono-embryonic (non-apomictic) citrus were seldomly reported. Herein, we developed an in vivo tetraploid induction method by colchicine treatment, which is especially high-efficient in mono-embryonic citrus. The epicotyl of citrus seedling was decapitated and incubated in dark for different time durations (0–15 d) to induce callus formation on the epicotyl wound. The callus on wound was then immersed in different concentrations (0.0125 %-0.100 %) of colchicine for 2 h, enabling regeneration of tetraploid shoots. We achieved high tetraploid induction rate (20.69 %) but low mixoploid induction rate (1.72 %), when 0.025 % colchicine was applied on wound cultured in dark for 9 d By using this method, 147 tetraploids were recovered from mono-embryonic mandarin, tangerine, sweet orange, pummelo and citron. Our method exhibited high tetraploid induction efficiency (14.28 %-20.69 %), low rate of mixoploid (1.72 %-5.30 %), short recovery period (45 d), and no need of tissue culture. The majority (75 %-100 %) of the recovered tetraploids maintained stable ploidy during subsequent growth, and the mono-embryonic nature of the tetraploids was identified by InDel marker analysis. The mono-embryonic tetraploids obtained herein enriched polyploid germplasm pool for citrus breeding.