Single Recessive Gene Research Articles

Mapping a mutation causing pale yellow petals in Brassica rapa.

Petal color is an important trait for both ornamental purposes and also for attracting pollinators. Here, we report a mutation of Brassica rapa R-o-18 with pale yellow petals that we retrieved from an EMS population and named whiter shade of pale ( wsp ). Phenotypic segregation ratio of an F2 mapping population indicates the phenotype is controlled by a single recessive gene. Mapping data from the whole genome sequencing coupled with allele frequency analysis suggests the mutation is located in a ~2 Mbp interval on chromosome 2. The interval contains a putative esterase/lipase/thioesterase protein previously demonstrated to account for floral color in B. rapa . We demonstrate that wsp carries a G to A missense mutation causing an aspartate to asparagine substitution within the putative lysophospholipid acyltransferase domain.

Characterization of a barley (Hordeum vulgare L.) mutant with multiple stem nodes and spikes and dwarf (msnsd) and fine-mapping of its causal gene.

Multiple nodes and dwarf mutants in barley are a valuable resource for identifying genes that control shoot branching, vegetative growth and development. In this study, physiological, microscopic and genetic analysis were conducted to characterize and fine-map the underling gene of a barley mutant with Multiple Stem Nodes and Spikes and Dwarf (msnsd), which was selected from EMS- and 60Co-treated barley cv. Edamai 934. The msnsd mutant had more stem nodes, lower plant height and a shorter plastochron than Edamai 934. Moreover, the mutant had two or more spikes on each tiller. Microscopic analysis showed that the dwarf phenotype of msnsd resulted from reduced cell lengths and cell numbers in the stem. Further physiological analysis showed that msnsd was GA3-deficient, with its plant height increasing after external GA3 application. Genetic analysis revealed that a single recessive nuclear gene, namely, HvMSNSD, controlled the msnsd phenotype. Using a segregating population derived from Harrington and the msnsd mutant, HvMSNSD was fine-mapped on chromosome 5H in a 200 kb interval using bulked segregant analysis (BSA) coupled with RNA-sequencing (BSR-seq), with a C-T substitution in the exon of HvTCP25 co-segregating with the msnsd phenotype. RNA-seq analysis showed that a gene encoding gibberellin 2-oxidase 8, a negative regulator of GA biosynthesis, was upregulated in the msnsd mutant. Several known genes related to inflorescence development that were also upregulated and enriched in the msnsd mutant. Collectively, we propose that HvMSNSD regulates the plastochron and morphology of reproductive organs, likely by coordinating GA homeostasis and changed expression of floral development related genes in barley. This study offers valuable insights into the molecular regulation of barley plant architecture and inflorescence development.

OsChlC1, a Novel Gene Encoding Magnesium-Chelating Enzyme, Affects the Content of Chlorophyll in Rice

Leaf-color mutants in rice (Oryza sativa L.) are excellent models for studying chlorophyll biosynthesis and chloroplast development. In this study, a yellow-green-leaf mutant generated by 60Co irradiation, ygl9311, was isolated: it displayed a yellow-green leaf phenotype during the complete growth cycle. Compared with the wild type, the photosynthetic pigment contents of leaves in ygl9311 were significantly reduced, and chloroplast development was retarded. Genetic analysis indicated that the ygl9311 phenotype was controlled by a single recessive nuclear gene. Map-based cloning and transcriptome sequencing analysis suggested that the candidate gene was OsChlC1 (BGIOSGA012976), which encodes a Mg-chelatase I subunit. The results of CRISPR/Cas9 system and RNAi knockout tests show that mutation of OsChlC1 could reproduce the phenotype of yellow-green leaves of the mutant ygl9311. In conclusion, the novel rice leaf-color gene OsChlC1 affects the content of chlorophyll in rice, showing a relatively conserved function in indica and japonica rice cultivars.

Inheritance of dwarfism and narrow lobed-leaf in two rapeseed (Brassica napus L.) mutant lines

There is a need for dwarf and narrow lobed-leaves rapeseed cultivars to reduce transpiration under drought prone areas. A dwarf mutant line ‘H2M-1’ and a mutant with reduced lobed-leaf ‘H2M-2’ were developed. To exploit these mutated traits properly in an effective breeding program, one should understand their mode of inheritance. There are conflicting findings for plant dwarfism and limited studies for leaf size in mutant genetic backgrounds. Therefore, the objective of this study was to investigate the inheritance of dwarfism and narrow lobed-leaf mutated traits. Plants of the wild-type variety ‘INRA-CZH2’ were reciprocally crossed with plants of the line ‘H2M-1’ and plants of the line ‘H2M-2’. A genetic study was conducted by analyzing segregation of mutated traits in F1, F2 and BC1F1 generations. The results revealed that two recessive genes with dominant epistasis action controlled the heredity of plant height in the dwarf line, whereas only a single recessive gene is involved in determining reduced lobed-leaf in the line H2M-2. Thus, there is a possibility to easily and quickly transfer these characters into rapeseed breeding germplasm or varieties towards the development of suitable cultivars for areas marked by increasing drought stress.

Novel Bisexual Flower Control Gene Regulates Sex Differentiation in Melon (Cucumis melo L.).

The sex-control system involves several mechanisms in melon. The present study identified a novel bisexual flower control gene from the hermaphroditic melon germplasm, different from the previously recognized one. Genetic analysis showed that a single recessive gene in the newly identified locus b controlled the bisexual flower phenotype in melons. We generated 1431 F2 segregating individuals for genetic mapping of locus b, which was delimited to a 47.94 kb region. Six candidate genes were identified in the delimited interval, and candidate No. 4 encoding melon CPR5 protein was selected as the suitable one for locus b and was denoted CmCPR5. CPR5 reportedly interacted with ethylene receptor ETR1 to regulate ethylene signal transduction. Moreover, the ethephon assays showed that the parental lines (unisexual line and bisexual line) had contrasting expression patterns of CmCPR5. The BiFC and LCI assays also confirmed that CmCPR5 interacted with CmETR1 in 0426 but not in Y101. However, crossover tests showed that CmETR1 functioned normally in both parental lines, suggesting CPR5 malfunction in Y101. This study proposed a corollary mechanism of bisexual flower regulation during stamen primordium development in which the inhibition of stamen primordia development was prevented by the malfunctioning CmCPR5, resulting in bisexual flowers.

Brittle culm 3, encoding a cellulose synthase subunit 5, is required for cell wall biosynthesis in barley (Hordeum vulgare L.).

The cell wall plays an important role in plant mechanical strength. Cellulose is the major component of plant cell walls and provides the most abundant renewable biomass resource for biofuels on earth. Mutational analysis showed that cellulose synthase (CESA) genes are critical in cell wall biosynthesis in cereal crops like rice. However, their role has not been fully elucidated in barley. In this study, we isolated a brittle culm mutant brittle culm 3 (bc3) derived from Yangnongpi 5 ethyl methanesulfonate (EMS) mutagenesis in barley. The bc3 mutants exhibited reduced mechanical strength of the culms due to impaired thickening of the sclerenchyma cell wall and reduced cellulose and hemicellulose content in the culms. Genetic analysis and map-based cloning revealed that the bc3 mutant was controlled by a single recessive gene and harbored a point mutation in the HvCESA5 gene, generating a premature stop codon near the N-terminal of the protein. Quantitative real-time PCR (qRT-PCR) analysis showed that the HvCESA5 gene is predominantly expressed in the culms and co-expressed with HvCESA4 and HvCESA8, consistent with the brittle culm phenotype of the bc3 mutant. These results indicate that the truncated HvCESA5 affects cell wall biosynthesis leading to a brittle culm phenotype. Our findings provide evidence for the important role of HvCESA5 in cell wall biosynthesis pathway and could be a potential target to modify cell wall in barley.

A 2-bp deletion in the protein kinase domain region of the ERECTA-like receptor kinase gene in cucumber results in short internode phenotype

Cucumber varieties with shortend internodes require less space than regular vining varieties, thus have great significance for germplasm improvement. Here, we found a novel spontaneous cucumber mutant si107 that exhibited short intenodes (si), smaller leaves, fruits, and seeds. The decrease in longitudinal cell length led to the shortened internodes of si107. The genetic analysis revealed a single recessive gene si-2 that was responsible for the mutation. Through multiple lines of evidence, we demonstrated that CsSI is the possible candidate gene for si-2, which encodes an ERECTA leucine-rich repeat receptor-like kinase. The shortened internode in si107 is attributed to a 2-bp deletion in the protein kinase domain region of this gene. The expression of CsSI was higher in the internodes, petioles, and fruit peels of si107 than in the wild type (WT). The transcriptome analysis between the si107 mutant and WT indicated that differentially expressed genes were significantly enriched in the plant hormone signal transduction pathway, in which auxin signal genes comprised the largest group, and all were downregulated in si107. Phytohormone quantitation confirmed that endogenous auxin levels in the stems of si107 were decreased. Our results provide new insights into the molecular mechanisms underlying the internode length control in cucumber.

Identification of a Leafy Head Formation Related Gene in Chinese Cabbage (Brassica rapa L. ssp. pekinensis)

Leafy head formation is one of the most important characteristics of Chinese cabbage, and the process is regulated by a series of genes and environmental factors. In this study, a non-heading short leaf mutant slm was identified from an ethyl methane sulfonate mutagenesis (EMS) population of the heading Chinese cabbage line FT. The most significant phenotypic characteristics of slm was shortening leaves and increasing leaf numbers, which led to failure to form a leafy head. Genetic analysis showed that a single recessive gene Brslm was responsible for the mutant phenotype. Mutmap analysis suggested that Brslm was located on chromosome A07, and four candidate genes were predicted. KASP analysis demonstrated that BraA07g039390.3C was the target gene of the candidates. BraA07g039390.3C is a homologous to Arabidopsis CLV1 encoding receptor kinase with an extracellular leucine-rich domain. Sequencing analysis revealed that a single SNP from G to A occurred in 904th nucleotide of Brclv1, which resulted in the change of the 302nd amino acid from Asp to Asn. The SNP was co-segregated with the mutant phenotype in F2 individuals and located on the conserved domains. These results indicated that BrCLV1 was the mutant gene for slm which led to shortening leaves and increasing leaf numbers, disrupting the leafy heading formation in FT. These findings contribute to revealing the BrCLV1 function in leafy head formation in Chinese cabbage.

A splice site mutation in the FvePHP gene is associated with leaf development and flowering time in woodland strawberry.

Leaves and flowers are crucial for the growth and development of higher plants. In this study we identified a mutant with narrow leaflets and early flowering (nlef) in an ethyl methanesulfonate-mutagenized population of woodland strawberry (Fragaria vesca) and aimed to identify the candidate gene. Genetic analysis revealed that a single recessive gene, nlef, controlled the mutant phenotype. We found that FvH4_1g25470, which encodes a putative DNA polymerase α with a polymerase and histidinol phosphatase domain (PHP), might be the candidate gene, using bulked segregant analysis with whole-genome sequencing, molecular markers, and cloning analyses. A splice donor site mutation (C to T) at the 5' end of the second intron led to an erroneous splice event that reduced the expression level of the full-length transcript of FvePHP in mutant plants. FvePHP was localized in the nucleus and was highly expressed in leaves. Silencing of FvePHP using the virus-induced gene silencing method resulted in partial developmental defects in strawberry leaves. Overexpression of the FvePHP gene can largely restore the mutant phenotype. The expression levels of FveSEP1, FveSEP3, FveAP1, FveFUL, and FveFT were higher in the mutants than those in 'Yellow Wonder' plants, probably contributing to the early flowering phenotype in mutant plants. Our results indicate that mutation in FvePHP is associated with multiple developmental pathways. These results aid in understanding the role of DNA polymerase in strawberry development.

Volatile Compounds Governed by Single Recessive Gene Impart Aroma in Sponge Gourd (Luffa cylindrica L. Roem).

As a vegetable crop, sponge gourd is widely consumed worldwide due to its health promoting and nutraceutical value. This study describes genetics of an aromatic genotype VRSG-7-17 and deciphers the genetic control and volatile compound composition of sponge gourd. To study the inheritance of this trait, a cross was made between aromatic light-green-fruited VRSG-7-17 and non-aromatic dark-green-fruited VRSG-194 genotypes. The F1s were found to be non-aromatic and have a green fruit colour. Chi-square (χ2) analysis of backcross and F2 population segregating for aroma suggested that the inheritance of aroma in VRSG-7-17 is governed by a single recessive gene in a simple Mendelian fashion. The SPME-GC/MS analysis of the volatile compounds suggested that the compounds responsible for Basmati rice-like aroma were mainly hexanal, 1-octen-3-ol, 3-octanone and limonene. The aroma persists in the cooked VRSG-7-17 fruits, that did not lose fragrance traits at high temperatures. The inheritance of fruit colour was found to be controlled by a single gene with incomplete dominance. The segregation analysis showed that the aroma and fruit colour were not linked, and they segregated independently. The findings will lead to understanding the inheritance of the aromatic compounds in the sponge gourd and may be utilised in the breeding programmes for developing improved aromatic varieties.

A Major Recessive Gene Associated with Anthracnose (Colletotrichum capsici) Resistance in Chilli Pepper

Chilli anthracnose caused by Colletotrichum sp., is an important disease in Bangladesh and many other Asian countries. Recently the local Bangladeshi genotype ‘Comilla-2’ was found resistant to C. capsici. Inheritance of resistant to C. capsici was analyzed in segregating populations derived from a cross of ‘BARI chilli-1’ x ‘Comilla-2’. BARI chilli-1 as susceptible and Comilla-2 as resistant parent was used in the study. Detached matured green fruits were inoculated using the microinjection method. Disease response was evaluated using disease incidence and over all lesion diameter at 8 days after inoculation. The disease reaction of F1 plants in case of disease incidence and overall lesion diameter were clearly skewed to the susceptible parent, with average values of 46.70% and 13.2 mm, respectively. The distribution of disease incidence in the F2 population was skewed toward the susceptible parent and the distribution of overall lesion diameter in the F2 population showed a similar trend. Based on the scale of resistance and susceptibility, less than 25.0% disease incidence or less than 9.0 mm overall lesion diameter were evaluated as resistance. In the cross ‘BARI chilli-1’ x ‘Comilla-2’, the segregation ratios of resistance and susceptibility scored by disease incidence and overall lesion diameter in the F2 , BCr and BCs populations and chi-squared test significantly fitted one recessive gene model i.e. 1:3 Mendelian model. The result indicates that the resistance of ‘Comilla-2’ to C. capsici is controlled by a single recessive gene.

Characterization of Glossy Spike Mutants and Identification of Candidate Genes Regulating Cuticular Wax Synthesis in Barley (Hordeum vulgare L.).

Cuticular waxes comprise the hydrophobic layer that protects crops against nonstomatal water loss and biotic and abiotic stresses. Expanding on our current knowledge of the genes that are involved in cuticular wax biosynthesis and regulation plays an important role in dissecting the processes of cuticular wax metabolism. In this study, we identified the Cer-GN1 barley (Hordeum vulgare L.) mutant that is generated by ethyl methanesulfonate mutagenesis with a glossy spike phenotype that is controlled by a single recessive nuclear gene. A physiological analysis showed that the total cuticular wax loads of Cer-GN1 were one-third that of the progenitor wild-type (WT), and its water loss rate was significantly accelerated (p < 0.05). In addition, Cer-GN1 was defective in the glume's cuticle according to the toluidine blue dye test, and it was deficient in the tubule-shaped crystals which were observed on the glume surfaces by scanning electron microscopy. Using metabolomics and transcriptomics, we investigated the impacts of cuticular wax composition and waxy regulatory genes on the loss of the glaucous wax in the spikes of Cer-GN1. Among the differential metabolites, we found that 16-hydroxyhexadecanoic acid, which is one of the predominant C16 and C18 fatty acid-derived cutin monomers, was significantly downregulated in Cer-GN1 when it was compared to that of WT. We identified two novel genes that are located on chromosome 4H and are downregulated in Cer-GN1 (HvMSTRG.29184 and HvMSTRG.29185) that encode long-chain fatty acid omega-monooxygenase CYP704B1, which regulates the conversion of C16 palmitic acid to 16-hydroxyhexadecanoic acid. A quantitative real-time PCR revealed that the expression levels of HvMSTRG.29184 and HvMSTRG.29185 were downregulated at 1, 4, 8, 12, and 16 days after the heading stage in Cer-GN1 when it was compared to those of WT. These results suggested that HvMSTRG.29184 and HvMSTRG.29185 have CYP704B1 activity, which could regulate the conversion of C16 palmitic acid to 16-hydroxyhexadecanoic acid in barley. Their downregulation in Cer-GN1 reduced the synthesis of the cuticular wax components and ultimately caused the loss of the glaucous wax in the spikes. It is necessary to verify whether HvMSTRG.29184 and HvMSTRG.29185 truly encode a CYP704B1 that regulates the conversion of C16 palmitic acid to 16-hydroxyhexadecanoic acid in barley.

Fine Mapping and Candidate-Gene Analysis of an open glume multi-pistil 3 (mp3) in Rice (Oryza sativa L.)

The rice mutant mp3 was derived from an indica–japonica cross between Rejing35 and XieqingzaoB, producing an inconstant number of pistils ranging from one to four pistils in a floret at heading stage, which also developed an open-glume with one or two seeds and twin seedlings at mature and seedling stage. Several altered characteristics, including filling grain panicle–1 (62.90), grain-setting rate (60.48%) and grain yield plant–1 (13.42 g), decreased but an increase in 1000-grain weight (36.87 g) was observed. Genetic analysis revealed that the mp3 mutant phenotype was controlled by a single recessive gene. Using a chromosome walking strategy in the F2 population of 02428/mp3, the mp3 gene was fine mapped between L3-135 and RM7576, with a physical distance of 30.617 kb on rice chromosome 3. Four candidate genes were found in this region referred to the rice genome annotations. LOC_Os03g11614/OsMADS1 corresponded with the mutant mp3 phenotype. Sequencing showed no sequence alterations in the coding and promoter sequence of the LOC_Os03g11614/OsMADS1 of mp3. The mp3 gene may be an allelic gene with three previously reported genes but controlled different mutant floral organ phenotypes in rice. Therefore, this mp3 gene provided a novel perspective on the biological function of OsMADS1 in the development of rice floral organ.

Mapping and functional verification of leaf yellowing genes in watermelon during whole growth period.

Increasing light energy utilization efficiency is an effective way to increase yield and improve quality of watermelon. Leaf is the main place for photosynthesis, and the color of leaf is directly related to the change of photosynthesis. In addition, leaf yellowing can be used as a marker trait to play an important role in watermelon hybrid breeding and improve seed breeding. It can not only be used to eliminate hybrids at seedling stage, but also be used to determine seed purity. In this study, transcriptome analysis was first carried out using the whole growth period leaf yellowing watermelon mutant w-yl and inbred line ZK, and identified 2,471 differentially expressed genes (DEGs) in the comparison group w-yl-vs-ZK. Among the top 20 terms of the gene ontology (GO) enrichment pathway, 17 terms were related to photosynthesis. KEGG pathway enrichment analysis showed that the most abundant pathway was photosynthesis-antenna proteins. The F2 population was constructed by conventional hybridization with the inbred line ZK. Genetic analysis showed that leaf yellowing of the mutant was controlled by a single recessive gene. The leaf yellowing gene of watermelon located between Ind14,179,011 and InD16,396,362 on chromosome 2 by using indel-specific PCR markers, with a region of 2.217 Mb. In the interval, it was found that five genes may have gene fragment deletion in w-yl, among which Cla97C02G036010, Cla97C02G036030, Cla97C02G036040, Cla97C02G036050 were the whole fragment loss, and Cla97C02G0360 was the C-terminal partial base loss. Gene function verification results showed that Cla97C02G036040, Cla97C02G036050 and Cla97C02G036060 may be the key factors leading to yellowing of w-yl leaves.

Map-based cloning and transcriptome analysis of the more-tiller and small-grain mutant in rice.

A G to T nucleotide substitution of OsTSG2 led to more tillers and smaller grains in rice by participating in phytohormone signal transduction and starch and sucrose metabolism. Rice is one of the most important food crops worldwide. Grain size and tiller number are the most important factors determining rice yield. The more-tiller and small-grain 2 (tsg2) mutant in rice, developed by ethyl methanesulfonate (EMS) mutagenesis, has smaller grains, more tillers, and a higher yield per plant relative to the wild-type (WT). Based on the genetic analysis, the tsg2 traits were conferred by a single recessive nuclear gene located on the long arm of chromosome 2. After fine-mapping the OsTSG2 locus, a G to T nucleotide substitution was identified, which resulted in an A to S mutation in a highly conserved domain of the growth-regulation factor protein. The single-strand conformation polymorphism (SSCP) marker was developed based on the SNP associated with the phenotypic segregation of traits. The functional complementation of OsTSG2 from the tsg2 mutant to the WT led to an increase in grain size and weight. The differentially expressed genes (DEGs) identified by RNA sequencing were involved in phytohormone signal transduction and starch and sucrose metabolism. Enzyme-linked immunosorbent assay (ELISA) analysis detected variation in the indole acetic acid (IAA) and jasmonic acid (JA) content in the tsg2 inflorescence, while the cellular organization, degree of chalkiness, gel consistency, amylose content, and alkaline spreading value were affected in the tsg2 grains. The findings elucidated the regulatory mechanisms of the tsg2 traits. This mutant could be used in marker-assisted breeding for high-yield and good-quality rice.

Molecular Characterizations of the er1 Alleles Conferring Resistance to Erysiphe pisi in Three Chinese Pea (Pisum sativum L.) Landraces.

Powdery mildew caused by Erysiphe pisi DC. is a major disease affecting pea worldwide. This study aimed to confirm the resistance genes contained in three powdery mildew-resistant Chinese pea landraces (Suoshadabaiwan, Dabaiwandou, and Guiwan 1) and to develop the functional markers of the novel resistance genes. The resistance genes were identified by genetic mapping and PsMLO1 gene sequence identification. To confirm the inheritance of powdery mildew resistance in the three Landraces, the susceptible cultivars Bawan 6, Longwan 1, and Chengwan 8 were crossed with Suoshadabaiwan, Dabaiwandou, and Guiwan 1 to produce F1, F2, and F2:3 populations, respectively. All F1 plants were susceptible to E. pisi, and phenotypic segregation patterns in all the F2 and F2:3 populations fit the 3:1 (susceptible: resistant) and 1:2:1 (susceptible homozygotes: heterozygotes: resistant homozygotes) ratios, respectively, indicating powdery mildew resistance in the three Landraces were controlled by a single recessive gene, respectively. The analysis of er1-linked markers and genetic mapping in the F2 populations suggested that the recessive resistance genes in three landraces could be er1 alleles. The cDNA sequences of 10 homologous PsMLO1 cDNA clones from the contrasting parents were obtained. A known er1 allele, er1-4, was identified in Suoshadabaiwan. Two novel er1 alleles were identified in Dabaiwandou and Guiwan 1, which were designated as er1-13 and er1-14, respectively. Both novel alleles were characterized with a 1-bp deletion (T) in positions 32 (exon 1) and 277 (exon 3), respectively, which caused a frame-shift mutation to result in premature termination of translation of PsMLO1 protein. The co-dominant functional markers specific for er1-13 and er1-14, KASPar-er1-13, and KASPar-er1-14 were developed and effectively validated in populations and pea germplasms. Here, two novel er1 alleles were characterized and their functional markers were validated. These results provide powerful tools for marker-assisted selection in pea breeding.

Ubiquitin-Specific Protease 2 (OsUBP2) Negatively Regulates Cell Death and Disease Resistance in Rice.

Lesion mimic mutants (LMMs) are great materials for studying programmed cell death and immune mechanisms in plants. Various mechanisms are involved in the phenotypes of different LMMs, but few studies have explored the mechanisms linking deubiquitination and LMMs in rice (Oryza sativa). Here, we identified a rice LMM, rust spots rice (rsr1), resulting from the mutation of a single recessive gene. This LMM has spontaneous reddish-brown spots on its leaves, and displays enhanced resistance to both fungal leaf blast (caused by Magnaporthe oryzae) and bacterial blight (caused by Xanthomonas oryzae pv. oryzae). Map-based cloning showed that the mutated gene in rsr1 encodes a Ubiquitin-Specific Protease 2 (OsUBP2). The mutation of OsUBP2 was shown to result in reactive oxygen species (ROS) accumulation, chloroplast structural defects, and programmed cell death, while the overexpression of OsUBP2 weakened rice resistance to leaf blast. OsUBP2 is therefore a negative regulator of immune processes and ROS production. OsUBP2 has deubiquitinating enzyme activity in vitro, and the enzyme active site includes a cysteine at the 234th residue. The ubiquitinated proteomics data of rsr1 and WT provide some possible target protein candidates for OsUBP2.

Identification of the powdery mildew resistance gene in wheat breeding line Yannong 99102-06188 via bulked segregant exome capture sequencing.

Powdery mildew of wheat (Triticum aestivum), caused by Blumeria graminis f.sp. tritici (Bgt), is a destructive disease that seriously threatens the yield and quality of its host. Identifying resistance genes is the most attractive and effective strategy for developing disease-resistant cultivars and controlling this disease. In this study, a wheat breeding line Yannong 99102-06188 (YN99102), an elite derivative line from the same breeding process as the famous wheat cultivar Yannong 999, showed high resistance to powdery mildew at the whole growth stages. Genetic analysis was carried out using Bgt isolate E09 and a population of YN99102 crossed with a susceptible parent Jinhe 13–205 (JH13–205). The result indicated that a single recessive gene, tentatively designated pmYN99102, conferred seedling resistance to the Bgt isolate E09. Using bulked segregant exome capture sequencing (BSE-Seq), pmYN99102 was physically located to a ~33.7 Mb (691.0–724.7 Mb) interval on the chromosome arm 2BL, and this interval was further locked in a 1.5 cM genetic interval using molecular markers, which was aligned to a 9.0 Mb physical interval (699.2–708.2 Mb). Based on the analysis of physical location, origin, resistant spectrum, and inherited pattern, pmYN99102 differed from those of the reported powdery mildew (Pm) resistance genes on 2BL, suggesting pmYN99102 is most likely a new Pm gene/allele in the targeted interval. To transfer pmYN99102 to different genetic backgrounds using marker-assisted selection (MAS), 18 closely linked markers were tested for their availability in different genetic backgrounds for MAS, and all markers expect for YTU103-97 can be used in MAS for tracking pmYN99102 when it transferred into those susceptible cultivars.

Identification of single major QTL and candidate gene(s) governing hull-less seed trait in pumpkin.

The hull-less pumpkin (Cucurbita pepo) seed does not require de-hulling before use for human consumption, as a result highly preferred by the oil, nut, and baking industries. In hull-less seeds, a single recessive gene is responsible for the absence of outer thick seed coat layers; however, the genomic region and gene(s) controlling the trait are unclear to date. In this study, four crosses attempted to derive F2 and backcross populations confirmed the single recessive gene inheritance of hull-less seed trait in populations adapted to the sub-tropical climate. The candidate genomic region for hull-less seed trait was identified through the BSA-QTLseq approach using bulks of F2:3 progenies from a cross of HP111 (hulled) and HLP36 (hull-less). A novel genomic region on chromosome 12 ranging from 1.80 to 3.86 Mb was associated with the hull-less seed trait. The re-sequencing data identified a total of 396 SNPs within this region and eight were successfully converted into polymorphic KASP assays. The genotyping of segregating F2 (n = 160) with polymorphic KASP assays resulted in a 40.3 cM partial linkage map and identified Cp_3430407 (10 cM) and Cp_3498687 (16.1 cM) as flanking markers for hull-less locus (Cphl-1). These flanking markers correspond to the 68.28 kb region in the reference genome, and the marker, Cp_3430407 successfully predicted the genotype in 93.33% of the C. pepo hull-less germplasm lines, thus can be used for marker-assisted selection in parents polymorphic for the hull-less seed trait. The Cphl-1-linked genomic region (2.06 Mb) encompasses a total of 182 genes, including secondary cell wall and lignin biosynthesis-related transcriptional factors viz., “NAC” (Cp4.1LG12g04350) and “MYB” (Cp4.1LG12g03120). These genes were differentially expressed in the seeds of hulled and hull-less genotypes, and therefore could be the potential candidate genes governing the hull-less seed trait in pumpkin.

Identification and characterization of tsyl1, a thermosensitive chlorophyll-deficient mutant in rice (Oryza sativa)

Chloroplast development and chlorophyll biosynthesis are affected by temperature. However, the underlying molecular mechanism of this phenomenon remains elusive. Here, we isolated and characterized a thermosensitive yellow-green leaf mutant named tsyl1 (thermosensitive yellow leaf 1) from an ethylmethylsulfone (EMS)-mutagenized pool of rice. The mutant exhibits a yellow-green leaf phenotype and decreased leaf chlorophyll contents throughout development. At the mature stage of the tsyl1 mutant, the plant height, tiller number, number of spikelets per panicle and 1000 seed weight were decreased significantly compared to those of wild-type plants, but the seed setting rate and panicle length were not. The mutant phenotype was controlled by a single recessive nuclear gene on the short arm of rice chromosome 11. Map-based cloning of TSYL1, followed by a complementation experiment, showed a G base deletion at the coding region of LOC_Os11g05552, leading to the yellow-green phenotype. The TSYL1 gene encodes a signal recognition particle 54 kDa (SRP54) protein that is conserved in all organisms. The expression of tsyl1 was induced by high temperature. Furthermore, the expression of chlorophyll biosynthesis- and chloroplast development-related genes was influenced in tsyl1 at different temperatures. These results indicated that the TSYL1 gene plays a key role in chlorophyll biosynthesis and is affected by temperature at the transcriptional level.