Functional analysis of <Emphasis Type="Italic">PI-</Emphasis>like gene in relation to flower development from bamboo (<Emphasis Type="Italic">Bambusa oldhamii</Emphasis>)

Bamboo flowering owns many unique characteristics and remains a mystery. To investigate the molecular mechanisms underlying flower development in bamboo, a petal-identity gene was identified as a PISTILLATA homologue named BoPI from Bambusa oldhamii (bamboo family). Expression analysis showed that BoPI was highly expressed in flower organs and gradually increased during flower development stage, suggesting that BoPI played an important role in flower development. Ectopic expression of BoPI in Arabidopsis caused conversion of sepals to petals. 35S::BoPI fully rescued the defective petal formation in the pi-1 mutant. BoPI could interact with BoAP3 protein in vitro. These results suggested that BoPI regulated flower development of bamboo in a similar way with PI. Besides flower organs, BoPI was also expressed in leaf and branch, which revealed that BoPI may involve in leaf and branch development. Similar to other MIKC-type gene, BoPI contained the C-terminal sequence but its function was controversial. Ectopic expression of the C-terminal deletion construct (BoPI- ΔC) in Arabidopsis converted sepals to petals; BoPI- ΔC interacted with BoAP3 on yeast two-hybrid assay, just like the full-length construct. The result implied that the C-terminal sequence may not be absolutely required for organ identity function in the context of BoPI.     

Natural genetic variation in <Emphasis Type="Italic">Brassica</Emphasis> homologs of <Emphasis Type="Italic">FLOWERING LOCUS T</Emphasis> and characterization of its expression domains

FLOWERING LOCUS T (FT), a major effect gene, regulates flowering time in Arabidopsis. We analyzed evolutionary changes distinguishing two FT homeologous loci in B. rapa, described genetic variation in homologs isolated and reported expression pattern of FT in B. juncea. Synteny analysis confirmed presence of two FT genomic copies in B. rapa ssp. pekinensis and resolved pre-existing anomalies regarding copy number in “AA” genome. Synteny analysis of B. rapa homeologous regions CR1 (129 kb) and CR2 (232 kb) revealed differential gene fractionation and wide-spread re-arrangements. Seven genomic DNA (gDNA) variants (2.1–2.2 kb) and 10 complementary DNA (cDNA) variants (528 bp) were isolated from 6 Brassica species. The gDNA variants shared 72–99 % similarity within Brassica and 58–60 % between Arabidopsis and Brassica. FT cDNA variants shared 92–100 % similarity within Brassica and 87 % between Arabidopsis and Brassica. Phylogenetic analysis of FT gDNA, cDNA and protein sequences revealed two major clades, differentiating homologs derived from species containing shared “BB” and “CC” genomes. Phylogram based on Brassica FT gDNA differentiated homeologs derived from AA-LF (Least fractioned) and AA-MF1 (Moderately fractioned) sub-genomes. Analysis of FT expression pattern in B. juncea revealed increasing levels correlating with attainment of physiological maturity; highest levels were detected in older leaves implying conservation in spatio-temporal expression pattern vis-à-vis Arabidopsis. In conclusion, our study reveals that polyploidy in Brassicas resulted in expansion of FT gene copies with homologs charting independent evolutionary course through accumulation of mutations. However, expression domains of FT remained conserved across Brassicaceae to preserve the critical function of FT in controlling flowering time.     

Functional analysis of an <Emphasis Type="Italic">APETALA1</Emphasis>-like MADS box gene from <Emphasis Type="Italic">Eustoma grandiflorum</Emphasis> in regulating floral transition and formation

An Eustoma grandiflorum APETALA1 (EgAP1) gene showing high homology to the SQUA subfamily of MADS-box genes was isolated and characterized. EgAP1, containing a conserved euAP1 motif at the C-terminus, showed high sequence identity to Antirrhinum majus SQUAMOSA in the SQUA subfamily. EgAP1 mRNA was detected in the leaf and expressed significantly higher in young flower buds than in mature flower buds. In flowers, EgAP1 mRNA was strongly detected in sepal, weakly detected in petal and was absent in stamen and carpel. Transgenic Arabidopsis plants ectopically expressing EgAP1 flowered early and produced terminal flowers. In addition, the conversion of petals into stamen-like structures was also observed in 35S::EgAP1 flowers. 35S::EgAP1 was able to complement the ap1 flower defects by restoring the defect for sepal formation and significantly increasing second whorl petal production in Arabidopsis ap1 mutant plants. These results revealed that EgAP1 is the APETALA1 homolog in E. grandiflorum and that the function of EgAP1 is involved in floral induction and flower formation.     

Identification of candidate genes of QTLs for seed weight in <Emphasis Type="Italic">Brassica napus</Emphasis> through comparative mapping among <Emphasis Type="Italic">Arabidopsis</Emphasis> and <Emphasis Type="Italic">Brassica</Emphasis>species

Background

Map-based cloning of quantitative trait loci (QTLs) in polyploidy crop species remains a challenge due to the complexity of their genome structures. QTLs for seed weight in B. napus have been identified, but information on candidate genes for identified QTLs of this important trait is still rare.

Results

In this study, a whole genome genetic linkage map for B. napus was constructed using simple sequence repeat (SSR) markers that covered a genetic distance of 2,126.4 cM with an average distance of 5.36 cM between markers. A procedure was developed to establish colinearity of SSR loci on B. napus with its two progenitor diploid species B. rapa and B. oleracea through extensive bioinformatics analysis. With the aid of B. rapa and B. oleracea genome sequences, the 421 homologous colinear loci deduced from the SSR loci of B. napus were shown to correspond to 398 homologous loci in Arabidopsis thaliana. Through comparative mapping of Arabidopsis and the three Brassica species, 227 homologous genes for seed size/weight were mapped on the B. napus genetic map, establishing the genetic bases for the important agronomic trait in this amphidiploid species. Furthermore, 12 candidate genes underlying 8 QTLs for seed weight were identified, and a gene-specific marker for BnAP2 was developed through molecular cloning using the seed weight/size gene distribution map in B. napus.

Conclusions

Our study showed that it is feasible to identify candidate genes of QTLs using a SSR-based B. napus genetic map through comparative mapping among Arabidopsis and B. napus and its two progenitor species B. rapa and B. oleracea. Identification of candidate genes for seed weight in amphidiploid B. napus will accelerate the process of isolating the mapped QTLs for this important trait, and this approach may be useful for QTL identification of other traits of agronomic significance.

     

Genetic mapping of clubroot resistance genes in oilseed rape

Clubroot caused by the obligate biotrophic protist Plasmodiophora brassicae is a major disease of Brassica species. Clubroot resistances introduced from B. oleracea var. ‘Böhmerwaldkohl’ and resistance from B. rapa ECD-04 were genetically mapped in oilseed rape (B. napus L.). A doubled haploid (DH) population of rape seed was developed by crossing a resistant DH-line derived from a resynthesized B. napus with the susceptible cultivar ‘Express’. The DH population was tested in the greenhouse against seven P. brassicae isolates showing low and high virulence toward B. oleracea or/and B. rapa. DH-lines with highest or lowest disease scores were used in a bulked segregant analysis (BSA), and 43 polymorphic AFLPs were identified. A genetic map of the whole genome was constructed using 338 AFLP and 156 anchored SSR markers. Nineteen QTL were detected on chromosomes N02, N03, N08, N13, N15, N16 and N19 giving resistance to seven different isolates. Race-specific effects were observed for all QTL, none of the QTL conferred resistance to all isolates. The phenotypic variance explained by the respective QTL ranged between 10.3 and 67.5%. All QTL could be assigned to both ancestral genomes of B. napus. In contrast to previous reports, a clear differentiation into major QTL from B. rapa and minor QTL from B. oleracea could not be found. Composite interval mapping confirmed the linkage relationships determined by BSA, thus demonstrating that markers for oligogenic traits can be selected by merely testing the distributional extremes of a segregating population.     

Preferential retention,expression profile and potential functional diversity analysis of HD-Zip gene family in <Emphasis Type="Italic">Brassica rapa</Emphasis>

Homeodomain-Leu zipper (HD-Zip) gene family performs important biological functions related to organ development, photomorphogenesis and abiotic stress response in higher plants. However, systematic analysis of HD-Zip genes in Brassica rapa has not been performed. In the present study, a bioinformatics approach was used to identify and characterize the BraHD-Zip gene family in B. rapa. A total of 88 members were identified. All putative BraHD-Zip proteins contained a clear HD and LZ combined domain. Eighty-seven BraHD-Zips were non-randomly located on ten chromosomes. This gene family was mainly expanded following the whole genome triplication event and was preferentially over-retained relative to its neighboring genes in B. rapa. On phylogenetic analysis, the BraHD-Zips could be categorized into four distinct major groups (I–IV). Each group exhibited variant gene structures and motif distributions. Some syntenic orthologous gene pairs presented diverse expression profiles, which indicate that these gene pairs may be involved in the development of new functions during evolution. In summary, our analysis provided genome-wide insights into the expansion, preferential retention, expression profiles and functional diversity of BraHD-Zip genes following whole genome triplication in B. rapa.     

Genetic dissection of seed weight by QTL analysis and detection of allelic variation in Indian and east European gene pool lines of <Emphasis Type="Italic">Brassica juncea</Emphasis>

Key message

Seed weight QTL identified in different populations were synthesized into consensus QTL which were shown to harbor candidate genes by in silico mapping. Allelic variation inferred would be useful in breeding B. juncea lines with high seed weight.

Abstract

Seed weight is an important yield influencing trait in oilseed Brassicas and is a multigenic trait. Among the oilseed Brassicas, Brassica juncea harbors the maximum phenotypic variation wherein thousand seed weight varies from around 2.0 g to more than 7.0 g. In this study, we have undertaken quantitative trait locus/quantitative trait loci (QTL) analysis of seed weight in B. juncea using four bi-parental doubled-haploid populations. These four populations were derived from six lines (three Indian and three east European lines) with parental phenotypic values for thousand seed weight ranging from 2.0 to 7.6 g in different environments. Multi-environment QTL analysis of the four populations identified a total of 65 QTL ranging from 10 to 25 in each population. Meta-analysis of these component QTL of the four populations identified six ‘consensus’ QTL (C-QTL) in A3, A7, A10 and B3 by merging 33 of the 65 component Tsw QTL from different bi-parental populations. Allelic diversity analysis of these six C-QTL showed that Indian lines, Pusajaikisan and Varuna, hold the most positive allele in all the six C-QTL. In silico mapping of candidate genes with the consensus QTL localized 11 genes known to influence seed weight in Arabidopsis thaliana and also showed conserved crucifer blocks harboring seed weight QTL between the A subgenomes of B. juncea and B. rapa. These findings pave the way for a better understanding of the genetics of seed weight in the oilseed crop B. juncea and reveal the scope available for improvement of seed weight through marker-assisted breeding.

     

Development and characterization of <Emphasis Type="Italic">Brassica juncea – fruticulosa</Emphasis> introgression lines exhibiting resistance to mustard aphid (<Emphasis Type="Italic">Lipaphis erysimi</Emphasis> Kalt)

Background

Mustard aphid is a major pest of Brassica oilseeds. No source for aphid resistance is presently available in Brassica juncea . A wild crucifer, Brassica fruticulosa is known to be resistant to mustard aphid. An artificially synthesized amphiploid, AD-4 (B. fruticulosa × B. rapa var. brown sarson) was developed for use as a bridge species to transfer fruticulosa resistance to B. juncea. Using the selfed backcross we could select a large number of lines with resistance to mustard aphid. This paper reports cytogenetic stability of introgression lines, molecular evidence for alien introgression and their reaction to mustard aphid infestation.

Results

Majority of introgression lines had expected euploid chromosome number(2n= 36), showed normal meiosis and high pollen grain fertility. Well-distributed and transferable simple-sequence repeats (SSR) markers for all the 18 B. juncea chromosomes helped to characterize introgression events. Average proportions of recipient and donor genome in the substitution lines were 49.72 and 35.06%, respectively. Minimum alien parent genome presence (27.29%) was observed in the introgression line, Ad3K-280 . Introgressed genotypes also varied for their resistance responses to mustard aphid infestations under artificial release conditions for two continuous seasons. Some of the test genotypes showed consistent resistant reaction.

Conclusions

B.juncea-fruticulosa introgression set may prove to be a very powerful breeding tool for aphid resistance related QTL/gene discovery and fine mapping of the desired genes/QTLs to facilitate marker assisted transfer of identified gene(s) for mustard aphid resistance in the background of commercial mustard genotypes.

     

Morphological and genetic characterization of a new cytoplasmic male sterility system (<Emphasis Type="Italic">oxa</Emphasis> CMS) in stem mustard (<Emphasis Type="Italic">Brassica juncea</Emphasis>)

Key message

oxa CMS is a new cytoplasmic male sterility type in Brassica juncea.

Abstract

oxa CMS is a cytoplasmic male sterility (CMS) line that has been widely used in the production and cultivation of stem mustard in the southwestern China. In this study, different CMS-type specific mitochondrial markers were used to confirm that oxa CMS is distinct from the pol CMS, ogu CMS, nap CMS, hau CMS, tour CMS, Moricandia arvensis CMS, orf220-type CMS, etc., that have been previously reported in Brassica crops. Pollen grains of the oxa CMS line are sterile with a self-fertility rate of almost 0% and the sterility strain rate and sterility degree of oxa CMS is 100% due to a specific flower structure and flowering habit. Scanning electron microscopy revealed that most pollen grains in mature anthers of the oxa CMS line are empty, flat and deflated. Semi-thin section further showed that the abortive stage of anther development in oxa CMS is initiated at the late uninucleate stage. Abnormally vacuolated microspores caused male sterility in the oxa CMS line. This cytological study combined with marker-assisted selection showed that oxa CMS is a novel CMS type in stem mustard (Brassica juncea). Interestingly, the abortive stage of oxa CMS is later than those in other CMS types reported in Brassica crops, and there is no negative effect on the oxa CMS line growth period. This study demonstrated that this novel oxa CMS has a unique flower structure with sterile pollen grains at the late uninucleate stage. Our results may help to uncover the mechanism of oxa CMS in Brassica juncea.

     

Genome-wide analysis and stress-responsive expression of CCCH zinc finger family genes in <Emphasis Type="Italic">Brassica rapa</Emphasis>

Background

Ubiquitous CCCH nucleic acid-binding motif is found in a wide-variety of organisms. CCCH genes are involved in plant developmental processes and biotic and abiotic stress responses. Brassica rapa is a vital economic crop and classical model plant of polyploidy evolution, but the functions of CCCH genes in B. rapa are unclear.

Results

In this study, 103 CCCH genes in B. rapa were identified. A comparative analysis of the chromosomal position, gene structure, domain organization and duplication event between B. rapa and Arabidopsis thaliana were performed. Results showed that CCCH genes could be divided into 18 subfamilies, and segmental duplication might mainly contribute to this family expansion. C-X_7/8-C-X₅-C₃-H was the most commonly found motif, but some novel CCCH motifs were also found, along with some loses of typical CCCH motifs widespread in other plant species. The multifarious gene structures and domain organizations implicated functional diversity of CCCH genes in B. rapa. Evidence also suggested functional redundancy in at least one subfamily due to high conservation between members. Finally, the expression profiles of subfamily-IX genes indicated that they are likely involved in various stress responses.

Conclusion

This study provides the first genome-wide characterization of the CCCH genes in B. rapa. The results suggest that B. rapa CCCH genes are likely functionally divergent, but mostly involved in plant development and stress response. These results are expected to facilitate future functional characterization of this potential RNA-binding protein family in Brassica crops.

     

Identification of a nuclear-recessive gene locus for male sterility on A2 chromosome using the Brassica 60 K SNP array in non-heading Chinese cabbage

WS24-3 is a newly bred recessive genic male sterility line of the non-heading Chinese cabbage (Brassica rapa ssp. chinensis). Here, an F₂ population was produced from the cross between WS24-3 and a male-fertile breeding line (WS135). The Illumina Brassica 60 K single nucleotide polymorphism (SNP) array was used for SNPs detecting between sterile and fertile bulks from the F₂ population, and 62 SNPs were identified. BLAST analysis of the 62 SNPs revealed that the A2 chromosome of Brassica rapa genome contained 22 SNPs, whereas the other chromosomes did not contain more than 6 SNPs each. These data indicated that the potential target gene locus, named Bra2Ms, might be located on A2. Based on 10 of the 22 SNPs, allele-specific-polymerase chain reaction (AS-PCR) primers and single sequence repeat (SSR) primers were designed, 5 AS-PCR primers and 9 SSR primers showed difference between the bulks in electrophoretic determination. Analysis of these markers in F₂ population revealed that Bra2Ms was genetically delimited to a region of 1.2 cM. We also detected two co-segregated markers SSRa2-951 and SSRa2-960 in this region. The markers identified in our study might facilitate the transfer of recessive genic male sterility alleles to other favorable genetic backgrounds. Furthermore, these markers will support a map-based clone of Bra2Ms.     

Temporal and spatial regulation of anthocyanin biosynthesis provide diverse flower colour intensities and patterning in <Emphasis Type="Italic">Cymbidium</Emphasis> orchid

Main conclusion

This study confirmed pigment profiles in different colour groups, isolated key anthocyanin biosynthetic genes and established a basis to examine the regulation of colour patterning in flowers of Cymbidium orchid. Cymbidium orchid (Cymbidium hybrida) has a range of flower colours, often classified into four colour groups; pink, white, yellow and green. In this study, the biochemical and molecular basis for the different colour types was investigated, and genes involved in flavonoid/anthocyanin synthesis were identified and characterised. Pigment analysis across selected cultivars confirmed cyanidin 3-O-rutinoside and peonidin 3-O-rutinoside as the major anthocyanins detected; the flavonols quercetin and kaempferol rutinoside and robinoside were also present in petal tissue. β-carotene was the major carotenoid in the yellow cultivars, whilst pheophytins were the major chlorophyll pigments in the green cultivars. Anthocyanin pigments were important across all eight cultivars because anthocyanin accumulated in the flower labellum, even if not in the other petals/sepals. Genes encoding the flavonoid biosynthetic pathway enzymes chalcone synthase, flavonol synthase, flavonoid 3′ hydroxylase (F3′H), dihydroflavonol 4-reductase (DFR) and anthocyanidin synthase (ANS) were isolated from petal tissue of a Cymbidium cultivar. Expression of these flavonoid genes was monitored across flower bud development in each cultivar, confirming that DFR and ANS were only expressed in tissues where anthocyanin accumulated. Phylogenetic analysis suggested a cytochrome P450 sequence as that of the Cymbidium F3′H, consistent with the accumulation of di-hydroxylated anthocyanins and flavonols in flower tissue. A separate polyketide synthase, identified as a bibenzyl synthase, was isolated from petal tissue but was not associated with pigment accumulation. Our analyses show the diversity in flower colour of Cymbidium orchid derives not from different individual pigments but from subtle variations in concentration and pattern of pigment accumulation.

     

<Emphasis Type="Italic">SOC1</Emphasis> and <Emphasis Type="Italic">AGL24</Emphasis> interact with AGL18-1, not the other family members AGL18-2 and AGL18-3 in <Emphasis Type="Italic">Brassica juncea</Emphasis>

Many MCM1-AGAMOUS-DEFICIENS-SRF (MADS) genes have been proved to play an important role in the flowering time regulation of plants. The flowering-inhibiting factor AGAMOUS-LIKE 18 (AGL18) integrates into the two flowering-activating factors SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) and AGAMOUS-LIKE 24 (AGL24), which play an important role during the plant developmental stages of the flowering pathway. However, it remains unknown whether and how the AGL18 protein directly interacts with SOC1 and/or AGL24 genes to regulate flowering time in Brassica juncea. In this study, three members (AGL18-1 in florescence, AGL18-2 and AGL18-3 in young seedlings) of the AGL18 family, and SOC1 and AGL24 in florescence were cloned in Brassica juncea. Yeast One-Hybrid assays and Dual-Glo^® Luciferase assays showed that the SOC1 and AGL24 promoters interacted only with AGL18-1 protein, not AGL18-2 and AGL18-3. The typical conserved structure of the M-domain of AGL18-1 was the key region that mediated the interaction between the AGL18-1 protein and SOC1 promoter, and the I-domain, K-domain and C-domain did not regulate the interaction of AGL18-1/SOC1. In contrast, the K-domain and M-domain in AGL18-1 could mediate the interaction between the AGL18-1 protein and AGL24 promoter. This indicated that the AGL18-1 protein must have its unique functions that differed from AGL18-2 and AGL18-3. This work provides valuable information for in-depth studies into the molecular mechanisms of the AGL18 protein with SOC1 and AGL24 for flowering time control of Brassica juncea.     

Patterns of subspecies genetic diversity among oilseed <Emphasis Type="Italic">Brassica rapa</Emphasis> as revealed by agro-morphological traits and SSR markers

Brassica rapa (2n = 20, AA genome) is an important oil yielding species of the family Brassicaceae and characterized by wide range of genetic and morphological subtypes suitable for cultivation under diverse agro-climatic regions of India. In this study, genetic diversity among three subspecies of B. rapa including yellow sarson, toria and outlier brown sarson was estimated using various agro-morphological traits and simple sequence repeat (SSR) markers. Maximum variability was recorded for siliqua angle (Coefficient of variation = 30.9%), followed by seeds/siliqua (CV = 18.8%), leaf length (CV = 10%) and plant height (CV = 16.8%). Principal component analysis explained more than 50% of the total observed morphological variability for first two components. Of the 107 SSR markers tested, 80 generated reproducible, clear and distinct amplicons of which, 65 (81.25%) were found polymorphic. The number of alleles at each locus ranged from 2 to 7, with an average of 3.03 alleles per marker. A total of 197 alleles were detected at 65 SSR loci with average PIC value of 0.457 and a mean resolving power of 3.04. Neighbor-Joining cluster analysis based on morphological traits and SSR markers separately classified all the 28 genotypes into five major groups. The population structure analysis resulted into three sub-populations with certain extent of admixture among the earlier established taxonomic sub-groups. Twenty-three unique alleles were detected in thirteen B. rapa varieties. The clustering analysis and principal coordinate analysis outlined the genetic relationships among different varieties belonging to the three subspecies of B. rapa. Genetically diverse genotypes as illustrated by score plots and from the clustering patterns brought out the wide range of diversity present among B. rapa genotypes and the underlying options available for selecting parental genotypes for hybridization and developing high yielding cultivars suitable for Indian conditions.     

Identification and fine mapping of a stay-green gene (<Emphasis Type="Italic">Brnye1</Emphasis>) in pakchoi (<Emphasis Type="Italic">Brassica campestris</Emphasis> L. ssp. <Emphasis Type="Italic">chinensis</Emphasis>)

Key message

Using bulked segregant analysis combined with next-generation sequencing, we delimited the Brnye1 gene responsible for the stay-green trait of nye in pakchoi. Sequence analysis identified Bra019346 as the candidate gene.

Abstract

“Stay-green” refers to a plant trait whereby leaves remain green during senescence. This trait is useful in the cultivation of pakchoi (Brassica campestris L. ssp. chinensis), which is marketed as a green leaf product. This study aimed to identify the gene responsible for the stay-green trait in pakchoi. We identified a stay-green mutant in pakchoi, which we termed “nye”. Genetic analysis revealed that the stay-green trait is controlled by a single recessive gene, Brnye1. Using the BSA-seq method, a 3.0-Mb candidate region was mapped on chromosome A03, which helped us localize Brnye1 to an 81.01-kb interval between SSR markers SSRWN27 and SSRWN30 via linkage analysis in an F₂ population. We identified 12 genes in this region, 11 of which were annotated based on the Brassica rapa annotation database, and one was a functionally unknown gene. An orthologous gene of the Arabidopsis gene AtNYE1, Bra019346, was identified as the potential candidate for Brnye1. Sequence analysis revealed a 40-bp insertion in the second exon of Bra019346 in nye, which generated the TAA stop codon. A candidate gene-specific Indel marker in 1561 F₂ individuals showed perfect cosegregation with Brnye1 in the nye mutant. These results provide a foundation for uncovering the molecular mechanism of the stay-green trait in pakchoi.