Complete chloroplast genome sequences of <Emphasis Type="Italic">Solanum commersonii</Emphasis> and its application to chloroplast genotype in somatic hybrids with <Emphasis Type="Italic">Solanum tuberosum</Emphasis>

Key message

Chloroplast genome of Solanum commersonii and S olanum tuberosum were completely sequenced, and Indel markers were successfully applied to distinguish chlorotypes demonstrating the chloroplast genome was randomly distributed during protoplast fusion.

Abstract

Somatic hybridization has been widely employed for the introgression of resistance to several diseases from wild Solanum species to overcome sexual barriers in potato breeding. Solanum commersonii is a major resource used as a parent line in somatic hybridization to improve bacterial wilt resistance in interspecies transfer to cultivated potato (S. tuberosum). Here, we sequenced the complete chloroplast genomes of Lz3.2 (S. commersonii) and S. tuberosum (PT56), which were used to develop fusion products, then compared them with those of five members of the Solanaceae family, S. tuberosum, Capsicum annum, S. lycopersicum, S. bulbocastanum and S. nigrum and Coffea arabica as an out-group. We then developed Indel markers for application in chloroplast genotyping. The complete chloroplast genome of Lz3.2 is composed of 155,525 bp, which is larger than the PT56 genome with 155,296 bp. Gene content, order and orientation of the S. commersonii chloroplast genome were highly conserved with those of other Solanaceae species, and the phylogenetic tree revealed that S. commersonii is located within the same node of S. tuberosum. However, sequence alignment revealed nine Indels between S. commersonii and S. tuberosum in their chloroplast genomes, allowing two Indel markers to be developed. The markers could distinguish the two species and were successfully applied to chloroplast genotyping (chlorotype) in somatic hybrids and their progenies. The results obtained in this study confirmed the random distribution of the chloroplast genome during protoplast fusion and its maternal inheritance and can be applied to select proper plastid genotypes in potato breeding program.

     

Cloning and characterization of <Emphasis Type="Italic">Tabas1</Emphasis>-<Emphasis Type="Italic">B1</Emphasis> gene associated with flag leaf chlorophyll content and thousand-grain weight and development of a gene-specific marker in wheat

The chloroplast photosystem of flag leaves contributes the largest proportion of photosynthates to grain in crops and consequently affects grain weight. The plant 2-Cys peroxiredoxin BAS1 is involved in chlorophyll protection against chloroplast damage. In the present study, we cloned a Tabas1 gene in common wheat (Triticum aestivum L.), comprising seven exons and six introns with a complete sequence of 2847 bp and an open reading frame of 789 bp. The gene was located on chromosome 2B, and designated Tabas1-B1. A codominant gene-specific marker TaS1 was developed based on a 1-bp InDel (-/A) in the second intron of Tabas1-B1. Two alleles, Tabas1-B1a and Tabas1-B1b, at the Tabas1-B1 locus were identified by TaS1. Linkage and quantitative trait locus (QTL) mapping indicated that Tabas1-B1 was linked to Xcfa2278 (5.23 cM) and Xbarc167 (10.38 cM) on chromosome 2BL. A stable QTL co-segregating with Tabas1-B1 explained 9.0–19.2 % of phenotypic variations for chlorophyll content (ChlC) and 9.5–15.5 % for thousand-grain weight (TGW), respectively, across three environments. Association analysis further indicated a significant and positive effect of Tabas1-B1a on the ChlC of flag leaf post-anthesis and TGW in two populations across four environments. Geographic distribution analysis suggested a slightly higher frequency of Tabas1-B1a than Tabas1-B1b in the main wheat-growing regions of China. Selection of Tabas1-B1a may increase grain weight in wheat breeding.     

Characterization and mapping of <Emphasis Type="Italic">Dt1</Emphasis> locus which co-segregates with <Emphasis Type="Italic">CcTFL1</Emphasis> for growth habit in pigeonpea

Key message

We report growth habit profiling following SEM, genetic mapping and QTL analysis. Highlighted CcTFL1 , a candidate for determinacy in pigeonpea, since an Indel marker derived from this gene co-segregated with Dt1 locus.

Abstract

Pigeonpea (Cajanus cajan) is one of the most important legume crops grown in arid and semi-arid regions of the world. It is characterized with few unique features compared with other legume species, such as Lotus, Medicago, and Glycine. One of them is growth habit, an important agronomic trait. In the present study, identification of mutations affecting growth habit accompanied by a precise analysis of phenotype has been done which will shed more light upon developmental regulation in pigeonpea. A genetic study was conducted to examine the inheritance of growth habit and a genotyping by sequencing (GBS)-based genetic map constructed using F₂ mapping population derived from crossing parents ICP 5529 and ICP 11605. Inheritance studies clearly demonstrated the dominance of indeterminate (IDT) growth habit over determinate (DT) growth habit in F₂ and F_2:3 progenies. A total of 787 SNP markers were mapped in the genetic map of 1454 cM map length. Growth habit locus (Dt1) was mapped on the CcLG03 contributing more than 61% of total phenotypic variations. Subsequently, QTL analysis highlighted one gene, CcTFL1, as a candidate for determinacy in pigeonpea, since an Indel marker derived from this gene co-segregated with the Dt1 locus. Ability of this Indel-derived marker to differentiate DT/IDT lines was also validated on 262 pigeonpea lines. This study clearly demonstrated that CcTFL1 is a candidate gene for growth habit in pigeonpea and a user-friendly marker was developed in the present study which will allow low-cost genotyping without need of automation.

     

Characterization of an IAA-glucose hydrolase gene <Emphasis Type="Italic">TaTGW6</Emphasis> associated with grain weight in common wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

In rice, the TGW6 gene determines grain weight and encodes a protein with indole-3-acetic acid (IAA)-glucose hydrolase activity. Its homolog in wheat, TaTGW6, is considered as a candidate gene related to grain development. To amplify this gene, we designed primers based on a homologous conserved domain of the rice TGW6 gene. Sequence analysis indicated that TaTGW6 comprises only one exon, with 1656 bp in total and an open reading frame of 1035 bp. Three alleles at TaTGW6 locus detected by the primer pair TG23 were designated as TaTGW6-a, TaTGW6-b and TaTGW6-c, respectively. Compared with TaTGW6-a, TaTGW6-b had a 6-bp InDel at the position 170 downstream of initiation codon, and TaTGW6-c was a null mutant. Both TaTGW6-b and TaTGW6-c could significantly increase grain size and weight other than TaTGW6-a; however, the former two alleles showed a low frequency distribution in modern varieties. TaTGW6 was located on chromosome 4AL using a recombinant inbred line population and a set of Chinese Spring nullisomic-tetrasomic lines. It was linked to the SSR locus Xbarc1047 with a genetic distance of 6.62 cM and explained 15.8–21.0 % of phenotypic variation of grain weight in four environments. Association analysis using a natural population and Chinese wheat mini-core collections additionally validated the relationship of TaTGW6 with grain weight; the gene could explain 7.7–12.4 % of phenotypic variation in three environments. Quantitative real-time PCR revealed that TaTGW6-b showed relatively lower expression than TaTGW6-a in immature grain at 20 and 30 days post-anthesis and in mature grain. The low expression of TaTGW6 generally associated with low IAA content, but with high grain weight. The novel functional marker, designated as TG23, can be used for marker-assisted selection to improve grain weight in wheat and also provides insights into the regulatory mechanism underlying grain weight.     

Molecular characterization of a novel vernalization allele <Emphasis Type="Italic">Vrn-B1d</Emphasis> and its effect on heading time in Chinese wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) landrace Hongchunmai

Flowering time of wheat cultivars contributes greatly to the adaptability to environmental conditions and it is largely controlled by vernalization genes. In this study, 262 Chinese mini-core wheat cultivars were used to identify the allelic variation at VRN-B1 locus. A novel dominant allele Vrn-B1d was found in Chinese spring wheat landrace cultivar Hongchunmai. This allele contained several genetic divergence within the first intron comparing to the recessive allele vrn-B1, including one large 6850-bp deletion (670–7519 bp), one small 187-bp deletion (7851–8037 bp), one unique SNP (T to C, 7845 bp), and one 4-bp mutation (TTTT to ACAA, 7847–7850 bp). Meanwhile, it was also different from the three known dominant alleles at VRN-B1 locus. Two pairs of primers were designed to identify the novel allele Vrn-B1d and other four known alleles of VRN-B1. A multiplex PCR was established to discriminate all five alleles simultaneously. The greenhouse experiment with high temperature (non-vernalizing condition) and long light showed that F₂ plants containing Vrn-B1d allele headed significantly earlier than those with recessive vrn-B1 allele, suggesting that Vrn-B1d is a dominant allele conferring the spring growth habit. This study provides a useful germplasm and molecular markers for wheat breeding.     

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 a cryptic promoter from <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> reveals bidirectionality

Cryptic promoter elements play a significant role in evolution of plant gene expression patterns and are prospective tools for creating gene expression systems in plants. In a previous report, a 452 bp promoter fragment designated as cryptic root-specific promoter (AY601849) was identified immediately upstream to T-DNA insertion, in the intergenic region between divergent genes SAHH1 and SHMT4, in T-DNA tagged mutant M57 of Arabidopsis thaliana. In silico analysis of 452 bp promoter revealed typical eukaryotic promoter architecture, presence of root-specific motifs and other cis-regulatory motifs responsible for the spatial and temporal expression. GUS expression driven by 452 bp in M57 was developmentally as well as light-regulated. The AT-rich 452 bp promoter does not show homology to any known sequences. The 452 bp promoter was further proved cryptic and detailed molecular characterization of the promoter carried out through serial 5′ and 3′ deletion analysis, by cloning the promoter fragments upstream to promoter-less GUS vector. A 279 bp fragment obtained by deleting 173 bp from 5′ end of 452 bp was capable of driving root-specific expression, similar to that of full-length promoter. Further, root tip-specific, root-specific and core-regulatory motifs for root-specific expression were identified at positions 173–227, 251–323 and 408–452 bp, respectively, from the 5′ end of 452 bp. The 452 bp promoter was equally functional in inverse orientation, hence bidirectional and symmetric. In heterologous systems, such as Brassica juncea and Oryza sativa, the promoter activity was not significant since GUS was not visually detected in transient assays.     

Complete mitochondrial genome sequence of <Emphasis Type="Italic">Cucullaea labiata</Emphasis> (Arcoida: Cucullaeidae) and phylogenetic implications

The complete mitochondrial genome of Cucullaea labiata (Arcoida: Cucullaeidae) was firstly determined in this study in order to better understand the phylogenetic relationship between Cucullaeidae and Arcidae. The C. labiata mitochondrial genome was 25,845 bp in size and contained 12 protein-coding genes, 2 rRNA and 22 tRNA genes. The number and the location of the tRNA genes were different from three Arcidae species (Scapharca broughtonii, Scapharca kagoshimensis and Tegillarca granosa). Gene arrangement also differed dramatically. The length of the non-coding regions was 10,559 bp, in which the largest one (6057 bp) included eight point nine copies of a 659 bp repeat motif. The number of repeated sequences was different in different individuals, similar to the findings from the mitochondrial genome of S. broughtonii and Placopecten magellanicus. One intron was found in cox1 gene both in CL_98 and in CL_99 individuals of C. labiata. The reason why mitochondrial introns are retained so scarcely in bivalve taxa needs further research. Phylogenetic analyses based on 12 concatenated amino acid sequences of protein-coding genes supported Cucullaeidae was the sister group of Arcidae.     

Complete mitochondrial DNA sequence and phylogenetic analysis of Zhikong scallop <Emphasis Type="Italic">Chlamys farreri</Emphasis> (Bivalvia: Pectinidae)

The complete mitochondrial genome of Zhikong scallop Chlamys farreri is 21,695 bp in length and contains 12 protein-coding genes (the atp8 gene is absent, as in most bivalves), 2 ribosomal RNA genes, and 22 transfer RNA genes. The heavy strand has an overall A+T content of 58.7%. GC and AT skews for the mt genome of C. farreri are 0.337 and ?0.184, respectively, indicating the nucleotide bias against C and A. The mitochondrial gene order of C. farreri differs drastically from the scallops Argopecten irradians, Mimachlamys nobilis and Placopecten magellanicus, which belong to the same family Pectinidae. 6623 bp non-coding nucleotides exist intergenically in the mitogenome of C. farreri, with a large continuous sequence (4763 bp) between tRNA ^Val and tRNA ^Asn . Two repeat families are found in the large continuous sequence, which seems to be a common feature of scallops. Phylogenetic analysis based on 12 concatenated amino acid sequences of protein-coding genes supports the monophyly of Pectinidae and paraphyletic Pteriomorphia with respect to Heteroconchia.     

Molecular characterization and expression analysis of <Emphasis Type="Italic">Hsp90</Emphasis> in <Emphasis Type="Italic">Schizothorax prenanti</Emphasis>

Aquatic animals suffer from various environmental stresses because the aquatic environment is a very complex system. To monitor the health status of fish, Hsp90 a potential early warning marker was determined in Schizothorax prenanti after infection with a bacterium. In this study, we cloned Hsp90 from S. prenanti for the first time. The full-length cDNA sequence of SpHsp90 was 2663 bp, contains an open reading frame of 2181 bp, and has a gene encoding 726 amino acids, an estimated molecular mass of 83.38 kDa, and a theoretical isoelectric point of 4.91. The SpHsp90 amino acid sequence has five conserved HSP90 family signatures and shares 87.0–95.5 % identity with other vertebrates. Phylogenetic analysis and structure comparison indicated that SpHsp90 should be a β isoform of the HSP90 family. SpHsp90 was ubiquitously expressed in all examined tissues, and the highest level of expression was in the kidney. After Streptococcus agalactiae infection, the level of SpHsp90 expression had significant changes (P < 0.05) in the hepatopancreas, spleen, kidney, and blood. The expression increased to the highest level at 6 h in the blood and at 24 h in the hepatopancreas, spleen, and kidney. The results suggested that the SpHsp90 gene could be induced by S. agalactiae in S. prenanti and that SpHsp90 may be involved in resistance to bacterial infection and provide an early warning information. The kidney is the most suitable for detecting SpHsp90 after bacterial infection.     

Association between <Emphasis Type="Italic">cagA</Emphasis>, <Emphasis Type="Italic">vacAi</Emphasis>, and <Emphasis Type="Italic">dupA</Emphasis> genes of <Emphasis Type="Italic">Helicobacter pylori</Emphasis> and gastroduodenal pathologies in Chilean patients

In addition to the already known cagA gene, novel genetic markers have been associated with Helicobacter pylori (H. pylori) virulence: the dupA and vacAi genes. These genes might play an important role as specific markers to determine the clinical outcome of the disease, especially the vacAi gene, which has been expected to be a good marker of severe pathologies like gastric adenocarcinoma. In the present study, the association of cagA, dupA, and vacAi genes with gastroduodenal pathologies in Chilean patients was studied. One hundred and thirty-two patients positive for H. pylori were divided into two groups—non-severe and severe gastric pathologies—and investigated for the presence of cagA, dupA, and vacAi H. pylori virulence genes by PCR. The cagA gene was detected in 20/132 patients (15.2%), the vacAi1 gene was detected in 54/132 patients (40.9%), the vacAi2 gene was detected in 26/132 patients (19.7%), and the dupA gene was detected in 50/132 (37.9%) patients. Logistic regression model analysis showed that the vacAi1 isoform gene in the infected strains and the severity of the diseases outcome were highly associated, causing severe gastric damage that may lead to gastric cancer (p < 0.0001; OR = 8.75; 95% CI 3.54–21.64). Conversely, cagA (p = 0.3507; OR = 1.62; 95% CI 0.59–4.45) and vacAi2 (p = 0.0114; OR = 3.09; 95% CI 1.26–7.60) genes were not associated with damage, while the dupA gene was associated significantly with non-severe clinical outcome (p = 0.0032; OR = 0.25; 95% CI 0.09–0.65). In addition, dupA gene exerts protection against severe gastric pathologies induced by vacAi1 by delaying the outcome of the disease by approximately 20 years.     

Functional marker related to germination vigor of maize seed

Germination and synchronous seedling emergence are necessary for crop yield. One differentially expressed Cupin protein during maize seed germination had been identified in our previous study. To elucidate the functional sites in its coding gene, ZmGLP, a diverse maize association population was assayed. In alignment with B73, the ZmGLP gene contains five exons and encodes 522 amino acids, and a potential signal peptide domain from the 7 to 22 amino acids and a typical Cupin domain from the 332 to 477 amino acids were identified. The ZmGLP has a quick linkage disequilibrium decay with about 300 bp physical distance in the analyzed sequences, which implied that human selection might be undone in this gene. High genetic variations were evidenced in ZmGLP gene with 118 polymorphic sites in the association population. An Indel9 (18 bp) in the fifth exon of the intermotif region of the second Cupin domain, which influences the formation of C coil in the barrel folding, was detected significantly associated with germination vigor of maize seeds. The 18-bp insertion, which might help keeping the barrel folding intact, was a favorable allele for relative high germination vigor. Evolution analysis showed that this 18-bp deletion was a loss-of-function mutation. However, the action mode of ZmGLP is still yet to be further studied for maize improvement.     

Locating QTLs controlling overwintering seedling rate in perennial glutinous rice 89-1 (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

A new cold tolerant germplasm resource named glutinous rice 89-1 (Gr89-1, Oryza sativa L.) can overwinter using axillary buds, with these buds being ratooned the following year. The overwintering seedling rate (OSR) is an important factor for evaluating cold tolerance. Many quantitative trait loci (QTLs) controlling cold tolerance at different growth stages in rice have been identified, with some of these QTLs being successfully cloned. However, no QTLs conferring to the OSR trait have been located in the perennial O. sativa L. To identify QTLs associated with OSR and to evaluate cold tolerance. 286 F₁₂ recombinant inbred lines (RILs) derived from a cross between the cold tolerant variety Gr89-1 and cold sensitive variety Shuhui527 (SH527) were used. A total of 198 polymorphic simple sequence repeat (SSR) markers that were distributed uniformly on 12 chromosomes were used to construct the linkage map. The gene ontology (GO) annotation of the major QTL was performed through the rice genome annotation project system. Three main-effect QTLs (qOSR2, qOSR3, and qOSR8) were detected and mapped on chromosomes 2, 3, and 8, respectively. These QTLs were located in the interval of RM14208 (35,160,202 base pairs (bp))–RM208 (35,520,147 bp), RM218 (8,375,236 bp)–RM232 (9,755,778 bp), and RM5891 (24,626,930 bp)–RM23608 (25,355,519 bp), and explained 19.6%, 9.3%, and 11.8% of the phenotypic variations, respectively. The qOSR2 QTL displayed the largest effect, with a logarithm of odds score (LOD) of 5.5. A total of 47 candidate genes on the qOSR2 locus were associated with 219 GO terms. Among these candidate genes, 11 were related to cell membrane, 7 were associated with cold stress, and 3 were involved in response to stress and biotic stimulus. OsPIP1;3 was the only one candidate gene related to stress, biotic stimulus, cold stress, and encoding a cell membrane protein. After QTL mapping, a total of three main-effect QTLs—qOSR2, qOSR3, and qOSR8—were detected on chromosomes 2, 3, and 8, respectively. Among these, qOSR2 explained the highest phenotypic variance. All the QTLs elite traits come from the cold resistance parent Gr89-1. OsPIP1;3 might be a candidate gene of qOSR2.     

Genome sequence of <Emphasis Type="Italic">Candida versatilis</Emphasis> and comparative analysis with other yeast

The genome of Candida versatilis was sequenced to understand its characteristics in soy sauce fermentation. The genome size of C. versatilis was 9.7 Mb, the content of G + C was 39.74 %, scaffolds of N50 were 1,229,640 bp in length, containing 4711 gene. There were predicted 269 tRNA genes and 2201 proteins with clear function. Moreover, the genome information of C. versatilis was compared with another salt-tolerant yeast Zygosaccharomyces rouxii and the model organism Saccharomyces cerevisiae. C. versatilis and Z. rouxii genome size was close and both smaller than 12.1 for the Mb of S. cerevisiae. Using the OrthoMCL protein, three genomes were divided into 4663 groups. There were about 3326 homologous proteins in C. versatilis, Z. rouxii and S. cerevisiae.