Molecular Breeding

Correction to: Genetic analysis and gene mapping of the orange flower trait in Chinese cabbage ( Brassica rapa L.) The original article unfortunately contains an incorrect supplementary materials.

QTL mapping in Vigna radiata × Vigna umbellata population uncovers major genomic regions associated with bruchid resistance Abstract Mungbean (Vigna radiata), genus Vigna, is an economically important legume crop that is valued for its protein-rich dry seeds. However, bruchid infestation causes severe threat to seed storage in terms of deterioration in quantity along with nutritional quality. A new resistance source was found in Vigna umbellata, a species that is cross compatible with mungbean. Thus, the objective of this study is to identify the quantitative trait locus (QTL) controlling the bruchid resistance in RIL (recombinant inbred line) population developed from a hybridization between VRM (Gg) 1 (V. radiata) and TNAU RED (V. umbellata). The RIL population was screened for bruchid resistance using the following traits, viz. per cent of seed damage (SD), the total developmental period (TDP) and per cent of adult emergence (AE) in 2017 and 2018. The QTL analysis of these traits using a genetic map composed of 538 single nucleotide polymorphism (SNP) markers covering 11 chromosomes detected 12 QTLs in over the 2 years. Among them, the QTLs on chromosomes 05 and 08, designated qSD05 and qAE08, respectively, were stably detected in both years. qSD05 exhibited large effects in both years and mapped to 1.58-Mb genomic region of the mungbean reference genome. Genome mining of this QTL region identified the likely candidate genes involved in bruchid resistance. The outcomes and QTLs found in this study may provide useful information for fine mapping, marker-assisted selection (MAS), gene cloning and breeding for the resistance to bruchids.

Molecular cytogenetic characterization of a novel wheat– Psathyrostachys huashanica Keng 5Ns (5D) disomic substitution line with stripe rust resistance Abstract Psathyrostachys huashanica Keng (2n = 2x = 14, NsNs) is a valuable genetic resource for improved wheat breeding, because of its high fecundity, vigorous growth, and resistance to diseases. In this study, a novel wheat–P. huashanica 5Ns (5D) disomic substitution line DH66 was isolated from the F6 progeny of the heptaploid hybrid H8911 (2n = 7x = 49, AABBDDNs) and Triticum durum line Trs-372. Mitotic and meiotic observations showed that the chromosome karyotype of DH66 was 2n = 42 = 21II. Genomic in situ hybridization indicated that DH66 contained 40 wheat chromosomes and two P. huashanica chromosomes, which paired stably and were transmitted to the offspring. Fluorescence in situ hybridization showed that chromosome 5D was absent from DH66. Analysis using a 15K wheat chip demonstrated that the genotype of DH66 had strong matches with P. huashanica in terms of many single-nucleotide polymorphism (SNP) loci on the 5D chromosome, but few with line 7182 at the same SNPs. Verification using markers confirmed that a pair of wheat 5D chromosomes in DH66 were substituted by a pair of P. huashanica5Ns chromosomes, and thus DH66 was characterized as a wheat–P. huashanica 5Ns (5D) disomic substitution line. Agronomic trait evaluations showed that, compared with its wheat parents, DH66 exhibited significant improvements, with reduced plant height and dough stability time, and superior resistance to stripe rust in the adult stage. Therefore, the novel cytogenetically stable substitution line DH66 can be used in wheat disease-resistant and high-quality breeding programs.

Fine mapping and identification of the rice blast-resistance locus Pi-kf2(t) as a new member of the Pi2/Pi9 multigene family Abstract The identification and utilization of broad-spectrum resistance genes is the most effective and economical strategy of controlling rice blast disease. Kangfeng B (KFB), an elite Chinese rice cultivar, has been shown to exhibit broad-spectrum resistance to 53 isolates of Magnaporthe oryzae, the rice blast causative agent, from different regions of China. In this study, we identified a dominant blast-resistance gene at the Pi2/Pi9 locus from cultivar KFB, through genetic analysis and physical mapping. Allele-specific marker-based assessment revealed that Pi2, Pi9, and Piz-t are not the blast-resistance genes in KFB. By combining bulked segregation analysis (BSA) and recessive class analysis (RCA), the blast-resistance gene was fine-mapped to an approximately 249-kb interval between markers InDel-22 and Rm7213 on chromosome 6. Three bacterial artificial chromosome (BAC) clones spanning the region were identified. This region contains 19 predicted genes, including 7 nucleotide binding site–leucine-rich repeat (NBS-LRR) genes at the Pi2/Pi9 locus in japonica cv. Nipponbare genome. Further sequence comparison of the four functional NBS-LRR genes revealed that NBS-LRR2 and NBS-LRR4, as evidenced by their allelic/orthologous to Pi9 or Pi2, had significant differences of 9 to 43 and 14 to 48 amino acids in KFB, respectively, unlike the other known Pi2/Pi9alleles, suggesting that KFB carries a hitherto undocumented member of the Pi2/Pi9 multigene family. It was tentatively designated as Pi-kf2(t). Our results provide essential information for the isolation of the Pi-kf2(t) gene and will facilitate both map-based cloning and marker-assisted selection of this gene for rice blast-resistance breeding.

Limited yield advantage of early-season rice hybrids over inbreds in middle-lower reaches of the Yangtze River Abstract Early-season rice plays a significant role in double rice-cropping system in China to improve the grain yield per unit of area. But recently, the stagnation of grain yield increase has been witnessed for early-season rice varieties in middle-lower reaches of the Yangtze River, particular in the national rice regional adaption trials of southern China. Further, in the early cropping season, hybrid rice did not always perform better than inbred rice in grain production. It is relevant to question whether yield ceilings have been reached in early-season indica rice before a new breakthrough on the germplasm exploitation. Field experiments were conducted to evaluate the yield performance of five inbreds and their 12 derived hybrid rice varieties in 2017 and 2018 in Hangzhou, China. Limited yield advantage of early-season hybrids over inbreds was observed, commonly less than 5.00% and averaged as 0.62% in 2017 and 3.20% in 2018. The hybrids always produced more panicles per plant but less grains per panicle with similar growing periods compared with the inbreds. Then, the genetic distance of the hybrid parents was surveyed using 48 selected molecular markers, showing low polymorphisms. Further, they were genotyped for four critical quantitative trait loci for both yield traits and heading date and three ones only for yield traits. So did 60 major early-season indica rice varieties at the four pleiotropic loci. Evidence was shown for the high uniform of their haplotype combinations across the tested materials, which could lead to early heading and low yield potential. Our work revealed the low genetic diversity, or called genetic bottleneck, for early-season hybrids to further improve grain yield potential. Moreover, along with the popularization of low-input requirements, the inbred rice varieties of cheap seed price might be a more suitable alternative for this cropping season in middle-lower reaches of the Yangtze River.

Construction of the first high-density genetic map and QTL mapping for photosynthetic traits in Lycium barbarum L. Abstract Photosynthesis is essential for plant development as well as crop yield. QTL mapping was conducted for photosynthetic traits such as net photosynthetic rate (PN), stomatal conductance (Cond), inner-cellular carbon dioxide (Ci), transpiration rate (Trmmol), limiting value of the stoma (Ls), and water use efficiency (WUE). A high-density genetic map covering 964.03 cM was developed based on a hybrid population of the Goji (Lycium barbarum L.). The genetic map consisted of 23,967 markers with an average distance of 0.040 cM between two adjacent markers. Twenty-nine and three quantitative trait loci (QTLs) for photosynthetic traits and trunk diameter (TD), respectively, were detected, of which 8 QTLs, including 3 for PN, 2 for Cond, 1 for Trmmol, 1 for Ci, and 1 for Ls, can be detected in at least 2-year measurements (from 2017 and 2018, as well as the averaged data from 2017 and 2018, which was regarded as a 3rd year, named 1718). Among these measurements, qPN1 was detected in all 3 years and considered a stable QTL whereas qLs1 and qLs2 with the highest phenotypic variance explained (PVE%) 32.012 and 17.965 were detected as major QTLs. PN, Cond, Ci, Trmmol, Ls, and WUE showed significant correlations with each other except for Cond and WUE whereas PN and Cond were significantly correlated with TD (P < 0.05). These findings indicate that PNand Cond are critical factors for the growth of plants and QTLs contribute to PN, Cond, and TD, which could provide an improved way to enhance the breeding of the Goji in relation to its growth rate.

Association mapping seed kernel oil content in upland cotton using genome-wide SSRs and SNPs Abstract Cotton is a major fiber and oilseed crop; however, few studies have been focused on oil content in cotton. To dissect the genetic basis of seed oil content (SOC), genome-wide association study was performed by using SOC phenotypic data of 503 upland cotton inbred accessions in eight environments and two sets of genotypic data including 179 simple sequence repeats (SSRs) and 11,975 single-nucleotide polymorphism (SNPs) from our previous study. SOC for a given genotype was fairly stable which was reflected in a broad-sense heritability of H2 = 96.6%. Using the mixed linear model (K + Q), 16 significant SSRs (P < 0.01) and 26 significant SNPs (P < 1/11,975), were detected cross BLUP values and at least one individual environments. Based on the LD block analysis, eight SOC-associated QTL on chromosome 6, 10, 12, 13, 15, 17, and 24 covering more than one significant markers were identified, two QTL of them on chromosome 10 and 13 could be matched between SSR and SNP markers. The confidence interval of candidate genes in each QTL ranged from 0.14 to 1.50 Mb, and 12 to 70 genes were identified in these QTL. According to the gene function annotation and gene FPKM value of the 20~35 days post anther (DPA) cotton ovules, key genes involved in lipid metabolism and transport; tricarboxylic acid cycle which might affect seed oil content were found near the leading markers. These results will facilitate the molecular marker assisted breeding of cotton oil content.

DLT/OsGRAS-32, regulating leaf width and thickness by controlling cell number in Oryza sativa Abstract The improvement of rice leaf morphology is an important goal of rice plant breeding. Studying its molecular development mechanism will facilitate the breeding of a maximally efficient plant type. To further study the developmental mechanism of rice leaves, a dwarf broad-leaf mutant, g44, was obtained by ethyl methanesulfonate mutagenesis of the rice restorer line Jinhui10, and genetic analysis indicated that the trait was controlled by a pair of recessive nuclear gene. The locus was fine mapped on chromosome 6 with 16-kb physical distance. Sequencing revealed that an A-T base substitution occurred in the mutant, resulting in the early termination of LOC_Os06g03710/DLTtranslation in g44 and yielding only one residual peptide containing 558 amino acids. The phenotypic and sequencing results of the complementary transgenic plants and the allelic mutant g44-1 further confirmed this result. Mutant plants grown under field conditions showed dwarfing, a leaf colour of dark green, leaf widening, and grain enlargement. Paraffin section and cryosection analysis showed that the increase in the number of large vascular bundles and small vascular bundles and the increase in the spacing between adjacent small vascular bundles were the main reasons for the broadening of mutant leaves. In addition, the increase in thickness of the mesophyll cell layer may be the main reason for the increase in leaf colour darkness and net photosynthetic rate. The qPCR results preliminarily predicted that the increase in the number of mutant cells was caused by an enhanced cell division ability, and the DLT/G44 gene was proposed to participate in the regulation of cell division by suppressing the expression of cyclin-related genes.

Identification and validation of SNP markers associated with Wz -mediated Phytophthora nicotianae resistance in Nicotiana tabacum L. Abstract A chromosome segment designated as Wz found to positively affect resistance of to black shank disease caused by Phytophthora nicotianae was previously introgressed from Nicotiana rustica into cultivated tobacco (N. tabacum). DNA markers associated with this region can aid in efforts to pyramid multiple mechanisms of black shank resistance into single new tobacco cultivars. In order to identify DNA markers associated with Wz, we generated RNA-seq data from roots of nearly isogenic lines (NILs) differing for the presence/absence of the alien genomic region. Analysis of SNPs, INDELs, and differentially expressed genes that distinguished the NILs suggested that the N. rustica Wzintrogression replaced an approximately 65 Mb region of N. tabacum chromosome 19. The utility of a subset of derived Kompetitive Allele Specific PCR (KASP) and Cleaved Amplified Polymorphic Sequence (CAPS) markers for predicting the presence of Wz was verified using segregating populations and F3:4 families that were evaluated for field black shank resistance. The new markers are useful for overall efforts to develop tobacco cultivars containing novel gene combinations affecting soil-borne pathogen resistance.

Overexpressing Sesamum indicum L.’s DGAT1 increases the seed oil content of transgenic soybean Abstract Soybean (Glycine max) is an important oilseed crop that provides ~30% of the vegetable oil used for food, feed and industrial applications. Genetic engineering can produce precise changes in plants in a short period of time and complements conventional plant breeding methods. However, soybean has the lower transformation efficiency and more genotypic limitations than other species, which makes it more difficult to transform. In this study, we introduced diacylglycerolacyl transferase (DGAT) isolated from sesame (Sesamum indicum L.) into the soybean cultivar Dongnong 47, using Agrobacterium-mediated transformation to produce high-oil transgenic lines that are more suitable for breeding. After a 2-year single-location field trial, the transgenic soybean overexpressing SiDGAT1 had increase seed oil content, by an average of over 1.0 percentage point, compared with wild-type plants. Additionally, the transgenic plants expressing SiDGAT1 had significantly reduced protein and soluble sugar contents in mature seeds. SiDGAT1-overexpressing soybean exhibited an altered fatty acid composition, with increases in palmitic (C16:0) and linoleic (C18:2) acid contents and decreases in stearic (C18:0) and oleic (18:1) acid contents, but no major yield change. Thus, engineering the SiDGAT1 enzyme is an effective strategy to improve the oil content and value of soybean.