Solanum pennellii (LA5240) backcross inbred lines (BILs) for high resolution mapping in tomato.

Wild species are an invaluable source of new traits for crop improvement. Over the years, the tomato community bred cultivated lines that carry introgressions from different species of the tomato tribe to facilitate trait discovery and mapping. The next phase in such projects is to find the genes that drive the identified phenotypes. This can be achieved by genotyping a few thousand individuals resulting in fine mapping that can potentially identify the causative gene. To couple trait discovery and fine mapping, we are presenting large, recombination-rich, Backcross Inbred Line (BIL) populations involving an unexplored accession of the wild, green-fruited species Solanum pennellii (LA5240; the 'Lost' Accession) with two modern tomato inbreds: LEA, determinate, and TOP, indeterminate. The LEA and TOP BILs are in BC2F6-8 generation and include 1400 and 500 lines, respectively. The BILs were genotyped with 5000 SPET markers, showing that in the euchromatic regions there was one recombinant every 17-18 Kb while in the heterochromatin a recombinant every 600-700 Kb (TOP and LEA respectively). To gain perspective on the topography of recombination we compared five independent members of the Self-pruning gene family with their respective neighboring genes; based on PCR markers, in all cases we found recombinants. Further mapping analysis of two known morphological mutations that segregated in the BILs (self-pruning and hairless) showed that the maximal delimited intervals were 73 Kb and 210 Kb, respectively, and included the known causative genes. The 'Lost'_BILs provide a solid framework to study traits derived from a drought-tolerant wild tomato.

Fine Mapping and Candidate Gene Analysis of Pm36, a Wild Emmer-Derived Powdery Mildew Resistance Locus in Durum Wheat.

Powdery mildew (PM) is an economically important foliar disease of cultivated cereals worldwide. The cultivation of disease-resistant varieties is considered the most efficient, sustainable and economical strategy for disease management. The objectives of the current study were to fine map the chromosomal region harboring the wild emmer PM resistance locus Pm36 and to identify candidate genes by exploiting the improved tetraploid wheat genomic resources. A set of backcross inbred lines (BILs) of durum wheat were genotyped with the SNP 25K chip array and comparison of the PM-resistant and susceptible lines defined a 1.5 cM region (physical interval of 1.08 Mb) harboring Pm36. The genetic map constructed with F2:3 progenies derived by crossing the PM resistant line 5BIL-42 and the durum parent Latino, restricted to 0.3 cM the genetic distance between Pm36 and the SNP marker IWB22904 (physical distance 0.515 Mb). The distribution of the marker interval including Pm36 in a tetraploid wheat collection indicated that the positive allele was largely present in the domesticated and wild emmer Triticum turgidum spp. dicoccum and ssp. dicoccoides. Ten high-confidence protein coding genes were identified in the Pm36 region of the emmer, durum and bread wheat reference genomes, while three added genes showed no homologous in the emmer genome. The tightly linked markers can be used for marker-assisted selection in wheat breeding programs, and as starting point for the Pm36 map-based cloning.

A Solanum neorickii introgression population providing a powerful complement to the extensively characterized Solanum pennellii population.

We present a complementary resource for trait fine-mapping in tomato to those based on the intra-specific cross between cultivated tomato and the wild tomato species Solanum pennellii, which have been extensively used for quantitative genetics in tomato over the last 20 years. The current population of backcross inbred lines (BILs) is composed of 107 lines derived after three backcrosses of progeny of the wild species Solanum neorickii (LA2133) and cultivated tomato (cultivar TA209) and is freely available to the scientific community. These S. neorickii BILs were genotyped using the 10K SolCAP single nucleotide polymorphism chip, and 3111 polymorphic markers were used to map recombination break points relative to the physical map of Solanum lycopersicum. The BILs harbor on average 4.3 introgressions per line, with a mean introgression length of 34.7 Mbp, allowing partitioning of the genome into 340 bins and thereby facilitating rapid trait mapping. We demonstrate the power of using this resource in comparison with archival data from the S. pennellii resources by carrying out metabolic quantitative trait locus analysis following gas chromatography-mass spectrometry on fruits harvested from the S. neorickii BILs. The metabolic candidate genes phenylalanine ammonia-lyase and cystathionine gamma-lyase were then tested and validated in F2 populations and via agroinfiltration-based overexpression in order to exemplify the fidelity of this method in identifying the genes that drive tomato metabolic phenotypes.

Analysis of the MIR160 gene family and the role of MIR160a_A05 in regulating fiber length in cotton.

MAIN CONCLUSION: The MIR160 family in Gossypium hirsutum and G. barbadense was characterized, and miR160a_A05 was found to increase cotton-fiber length by downregulating its target gene (ARF17) and several GH3 genes. Cotton fiber is the most important raw material for the textile industry. MicroRNAs are involved in regulating cotton-fiber development, but a role in fiber elongation has not been demonstrated. In this study, miR160a was found to be differentially expressed in elongating fibers between two interspecific (between Gossypium hirsutum and G. barbadense) backcross inbred lines (BILs) with different fiber lengths. The gene MIR160 colocalized with a previously mapped fiber-length quantitative trait locus. Its target gene ARF17 was differentially expressed between the two BILs during fiber elongation, but in the inverse fashion. Bioinformatics was used to analyze the MIR160 family in both G. hirsutum and G. barbadense. Moreover, qRT-PCR analysis identified MIR160a as the functional MIR160 gene encoding the miR160a precursor during fiber elongation. Using virus-induced gene silencing and overexpression, overexpressed MIR160a_A05 resulted in significantly longer fibers compared with wild type, whereas suppression of miR160 resulted in significantly shorter fibers. Expression levels of the target gene auxin-response factor 17 (ARF17) and related genes GH3 in the two BILs and/or the virus-infected plants demonstrated similar changes in response to modulation of miR160a level. Finally, overexpression or suppression of miR160 increased or decreased, respectively, the cellular level of indole-3-acetic acid, which is involved in fiber elongation. These results describe a specific regulatory mechanism for fiber elongation in cotton that can be utilized for future crop improvement.

Solanum pennellii backcross inbred lines (BILs) link small genomic bins with tomato traits.

We present a resource for fine mapping of traits derived from the wild tomato species Solanum pennellii (LA0716). The population of backcross inbred lines (BILs) is composed of 446 lines derived after a few generations of backcrosses of the wild species with cultivated tomato (cultivar M82; LA3475), followed by more than seven generations of self-pollination. The BILs were genotyped using the 10K SOL-CAP single nucleotide polymorphism (SNP) -Chip, and 3700 polymorphic markers were used to map recombination break points relative to the physical map of Solanum lycopersicum. The BILs carry, on average, 2.7 introgressions per line, with a mean introgression length of 11.7 Mbp. Whereas the classic 76 introgression lines (ILs) partitioned the genome into 106 mapping bins, the BILs generated 633 bins, thereby enhancing the mapping resolution of traits derived from the wild species. We demonstrate the power of the BILs for rapid fine mapping of simple and complex traits derived from the wild tomato species.

Identification of candidate genes for fiber length quantitative trait loci through RNA-Seq and linkage and physical mapping in cotton.

BACKGROUND: Cotton (Gossypium spp.) fibers are single-celled elongated trichomes, the molecular aspects of genetic variation in fiber length (FL) among genotypes are currently unknown. In this study, two backcross inbred lines (BILs), i.e., NMGA-062 ("Long") and NMGA-105 ("Short") with 32.1 vs. 27.2 mm in FL, respectively, were chosen to perform RNA-Seq on developing fibers at 10 days post anthesis (DPA). The two BILs differed in 4 quantitative trait loci (QTL) for FL and were developed from backcrosses between G. hirsutum as the recurrent parent and G. barbadense. RESULTS: In total, 51.7 and 54.3 million reads were obtained and assembled to 49,508 and 49,448 transcripts in the two genotypes, respectively. Of 1551 differentially expressed genes (DEGs) between the two BILs, 678 were up-regulated and 873 down-regulated in "Long"; and 703 SNPs were identified in 339 DEGs. Further physical mapping showed that 8 DEGs were co-localized with the 4 FL QTL identified in the BIL population containing the two BILs. Four SNP markers in 3 DEGs that showed significant correlations with FL were developed. Among the three candidate genes encoding for proline-rich protein, D-cysteine desulfhydrase, and thaumatin-like protein, a SNP of thaumatin-like protein gene showed consistent correlations with FL across all testing environments. CONCLUSIONS: This study represents one of the first investigations of positional candidate gene approach of QTL in cotton in integrating transcriptome and SNP identification based on RNA-Seq with linkage and physical mapping of QTL and genes, which will facilitate eventual cloning and identification of genes responsible for FL QTL. The candidate genes may serve as the foundation for further in-depth studies of the molecular mechanism of natural variation in fiber elongation.

A comparative transcriptome analysis of two sets of backcross inbred lines differing in lint-yield derived from a Gossypium hirsutum × Gossypium barbadense population.

Upland cotton (Gossypium hirsutum L.) is the most important fiber crop, and its lint-yield improvement is impeded due to its narrow genetic base and the lack of understanding of the genetic basis of yield. Backcross inbred lines (BILs) or near-isogenic lines (NILs) in the same genetic background differing in lint yield, developed through advanced backcrossing, provide an important genomic resource to study the molecular genetic basis of lint yield. In the present study, a high-yield (HY) group and a low-yield (LY) group each with three BILs were selected from a BIL population between G. hirsutum and G. barbadense. Using a microarray-based comparative transcriptome analysis on developing fibers at 10 days post-anthesis (DPA) between the two groups, 1486 differentially expressed genes (DEGs) were identified. A total of 212 DEGs were further mapped in the regions of 24 yield QTL and 11 yield trait QTL hotspots as reported previously, and 81 DEGs mapped with the 7 lint-yield QTL identified in the BIL population from which the two sets of BILs were selected. Gene Ontology annotations and Blast-Mapping-Annotation-KEGG analysis via Blast2GO revealed that more DEGs were associated with catalytic activity and binding, followed by transporters, nucleic acid binding transcription factors, structural molecules and molecular transducer activities. Six DEGs were chosen for a quantitative RT-PCR assay, and the results were consistent with the microarray analysis. The development of DEGs-based markers revealed that 7 single strand conformation polymorphism-based single nucleotide polymorphic (SSCP-SNP) markers were associated with yield traits, and 3 markers with lint yield. In the present study, we identified a number of yield and yield component QTL-co-localizing DEGs and developed several DEG-based SSCP-SNP markers for the traits, thereby providing a set of candidate genes for molecular breeding and genetic manipulation of lint yield in cotton.

Genetic dissection of seed storability using two different populations with a same parent rice cultivar N22.

Seed storability in rice (Oryza sativa L.) is an important agronomic trait. Two segregating populations with N22 (indica) as a common parent, viz. a set of 122 backcross-inbred lines (BILs) derived from the backcross Nanjing35 (japonica)/N22//Nanjing35 and another population comprising 189 recombinant inbred lines (RILs) from the cross of USSR5 (japonica) and N22, were studied to detect quantitative trait loci (QTL) controlling seed storability. Germination percentage (GP) was used to evaluate seed storability after aging treated under three different conditions, viz. natural, artificial and combined aging treatments. A total of seven QTLs were identified on chromosomes 1, 2, 5, 6 and 9. Among them, a major QTL, qSSn-9, was common in the two populations. In contrast, four QTLs (qSSnj-2-1, qSSn-2-2, qSSn-5 and qSSn-6) were detected in BILs and the QTL qSSn-1 was identified in RILs, which was a new QTL for seed storability. The N22-derived alleles increased the seed storability at all the loci except qSSnj-2-1. We also investigated the effect of QTLs using five selected lines with high storability from BILs and verified qSSn-5 with a near-isogenic line (NIL). These results provide an opportunity for pyramiding or map-based cloning major QTLs for seed storability in rice.

Detection of Chromosomal Segments Introgressed from Wild Species of Carrot into Cultivars: Quantitative Trait Loci Mapping for Morphological Features in Backcross Inbred Lines.

Cultivated carrot is thought to have been domesticated from a wild species, and various phenotypes developed through human domestication and selection over the past several centuries. Little is known about the genomic contribution of wild species to the phenotypes of present-day cultivars, although several studies have focused on identifying genetic loci that contribute to the morphology of storage roots. A backcross inbred line (BIL) population derived from a cross between the wild species Daucus carota ssp. carota "Songzi" and the orange cultivar "Amsterdam forcing" was developed. The morphological features in the BIL population became more diverse after several generations of selfing BC2F1 plants. Only few lines retained features of wild parent. Genomic resequencing of the two parental lines and the BILs resulted in 3,223,651 single nucleotide polymorphisms (SNPs), and 13,445 bin markers were generated using a sliding window approach. We constructed a genetic map with 2027 bins containing 154,776 SNPs; the total genetic distance was 1436.43 cM and the average interval between the bins was 0.71 cm. Five stable QTLs related to root length, root shoulder width, dry material content of root, and ratio of root shoulder width to root middle width were consistently detected on chromosome 2 in both years and explained 23.4-66.9% of the phenotypic variance. The effects of introgressed genomic segments from the wild species on the storage root are reported and will enable the identification of functional genes that control root morphological traits in carrot.

Quantitative Trait Locus Analysis and Identification of Candidate Genes Affecting Seed Size and Shape in an Interspecific Backcross Inbred Line Population of Gossypium hirsutum × Gossypium barbadense.

Seed size and shape are key agronomic traits affecting seedcotton yield and seed quality in cotton (Gossypium spp.). However, the genetic mechanisms that regulate the seed physical traits in cotton are largely unknown. In this study, an interspecific backcross inbred line (BIL) population of 250 BC1F7 lines, derived from the recurrent parent Upland CRI36 (Gossypium hirsutum) and Hai7124 (Gossypium barbadense), was used to investigate the genetic basis of cotton seed physical traits via quantitative trait locus (QTL) mapping and candidate gene identification. The BILs were tested in five environments, measuring eight seed size and shape-related traits, including 100-kernel weight, kernel length width and their ratio, kernel area, kernel girth, kernel diameter, and kernel roundness. Based on 7,709 single nucleotide polymorphic (SNP) markers, a total of 49 QTLs were detected and each explained 2.91-35.01% of the phenotypic variation, including nine stable QTLs mapped in at least three environments. Based on pathway enrichment, gene annotation, genome sequence, and expression analysis, five genes encoding starch synthase 4, transcription factor PIF7 and MYC4, ubiquitin-conjugating enzyme E27, and THO complex subunit 4A were identified as candidate genes that might be associated with seed size and shape. Our research provides valuable information to improve seed physical traits in cotton breeding.

Quantitative Trait Locus Analysis and Identification of Candidate Genes for Micronaire in an Interspecific Backcross Inbred Line Population of Gossypium hirsutum × Gossypium barbadense.

Cotton is the most important fiber crop and provides indispensable natural fibers for the textile industry. Micronaire (MIC) is determined by fiber fineness and maturity and is an important component of fiber quality. Gossypium barbadense L. possesses long, strong and fine fibers, while upland cotton (Gossypium hirsutum L.) is high yielding with high MIC and widely cultivated worldwide. To identify quantitative trait loci (QTLs) and candidate genes for MIC in G. barbadense, a population of 250 backcross inbred lines (BILs), developed from an interspecific cross of upland cotton CRI36 × Egyptian cotton (G. barbadense) Hai7124, was evaluated in 9 replicated field tests. Based on a high-density genetic map with 7709 genotyping-by-sequencing (GBS)-based single-nucleotide polymorphism (SNP) markers, 25 MIC QTLs were identified, including 12 previously described QTLs and 13 new QTLs. Importantly, two stable MIC QTLs (qMIC-D03-2 on D03 and qMIC-D08-1 on D08) were identified. Of a total of 338 genes identified within the two QTL regions, eight candidate genes with differential expression between TM-1 and Hai7124 were identified. Our research provides valuable information for improving MIC in cotton breeding.

Identification of QTLs for Salt Tolerance at the Germination and Seedling Stages in Rice.

Rice is highly sensitive to salinity stress during the seedling establishment phase. Salt stress is widely occurring in cultivated areas and severely affects seed germination ability and seedling establishment, which may result in a complete crop failure. The objective of the present study is to identify quantitative trait loci (QTLs) related to salt tolerance of the germination and seedling stages in a rice backcross inbred line (BIL) population that was derived from a backcross of an Africa rice ACC9 as donor and indica cultivar Zhenshan97 (ZS97) as the recurrent parent. Under salt stress, ACC9 exhibited a higher germination percentage, but more repressed seedling growth than ZS97. Using the BIL population, 23 loci for germination parameters were detected at the germination stage and 46 loci were identified for several morphological and physiological parameters at the seedling stage. Among them, nine and 33 loci with the ACC9 alleles increased salt tolerance at the germination and seedling stages, respectively. Moreover, several major QTLs were found to be co-localized in the same or overlapping regions of previously reported genes for salt stress. These major loci will facilitate improving salt-tolerance rice in genome-breeding programs.

Quantitative Trait Loci Mapping of Mineral Element Contents in Brown Rice Using Backcross Inbred Lines Derived From Oryza longistaminata.

Mineral elements play an extremely important role in human health, and are worthy of study in rice grain. Wild rice is an important gene pool for rice improvement including grain yield, disease, and pest resistance as well as mineral elements. In this study, we identified 33 quantitative trait loci (QTL) for Fe, Zn, Se, Cd, Hg, and As contents in wild rice Oryza longistaminata. Of which, 29 QTLs were the first report, and 12 QTLs were overlapped to form five clusters as qSe1/qCd1 on chromosome 1, qCd4.2/qHg4 on chromosome 4, qFe5.2/qZn5.2 on chromosome 5, qFe9/qHg9.2/qAs9.2 on chromosome 9, and qCd10/qHg10 on chromosome 10. Importantly, qSe1/qCd1, can significantly improve the Se content while reduce the Cd content, and qFe5.2/qZn5.2 can significantly improve both the Fe and Zn contents, they were delimited to an interval about 53.8 Kb and 26.2 Kb, respectively. These QTLs detected from Oryza longistaminata not only establish the basis for subsequent gene cloning to decipher the genetic mechanism of mineral element accumulation, but also provide new genetic resource for rice quality improvement.

Genetic Dissection of Seed Dormancy in Rice (Oryza sativa L.) by Using Two Mapping Populations Derived from Common Parents.

BACKGROUND: Seed dormancy, a quality characteristic that plays a role in seed germination, seedling establishment and grain yield, is affected by multiple genes and environmental factors. The genetic and molecular mechanisms underlying seed dormancy in rice remain largely unknown. RESULTS: Quantitative trait loci (QTLs) for seed dormancy were identified in two different mapping populations, a chromosome segment substitution line (CSSL) and backcross inbred line (BIL) population, both derived from the same parents Nipponbare, a japonica cultivar with seed dormancy, and 9311, an indica cultivar lacking seed dormancy. A total of 12 and 27 QTL regions for seed dormancy were detected in the CSSLs and BILs, respectively. Among these regions, four major loci (qSD3.1, qSD3.2, qSD5.2 and qSD11.2) were commonly identified for multiple germination parameters associated with seed dormancy in both populations, with Nipponbare alleles delaying the seed germination percentage and decreasing germination uniformity. Two loci (qSD3.1 and qSD3.2) were individually validated in the near-isogenic lines containing the QTL of interest. The effect of qSD3.2 was further confirmed in a CSSL-derived F2 population. Furthermore, both qSD3.1 and qSD3.2 were sensitive to abscisic acid and exhibited a significant epistatic interaction to increase seed dormancy. CONCLUSIONS: Our results indicate that the integration of the developed CSSLs and BILs with high-density markers can provide a powerful tool for dissecting the genetic basis of seed dormancy in rice. Our findings regarding the major loci and their interactions with several promising candidate genes that are induced by abscisic acid and specifically expressed in the seeds will facilitate further gene discovery and a better understanding of the genetic and molecular mechanisms of seed dormancy for improving seed quality in rice breeding programs.

Genetic Dissection and Validation of Chromosomal Regions for Transmission Ratio Distortion in Intersubspecific Crosses of Rice.

Transmission ratio distortion (TRD) refers to a widespread phenomenon in which one allele is transmitted by heterozygotes more frequently to the progeny than the opposite allele. TRD is considered as a mark suggesting the presence of a reproductive barrier. However, the genetic and molecular mechanisms underlying TRD in rice remain largely unknown. In the present study, a population of backcross inbred lines (BILs) derived from the cross of a japonica cultivar Nipponbare (NIP) and an indica variety 9311 was utilized to study the genetic base of TRD. A total of 18 genomic regions were identified for TRD in the BILs. Among them, 12 and 6 regions showed indica (9311) and japonica (NIP) alleles with preferential transmission, respectively. A series of F2 populations were used to confirm the TRD effects, including six genomic regions that were confirmed by chromosome segment substitution line (CSSL)-derived F2 populations from intersubspecific allelic combinations. However, none of the regions was confirmed by the CSSL-derived populations from intrasubspecific allelic combination. Furthermore, significant epistatic interaction was found between TRD1.3 and TRD8.1 suggesting that TRD could positively contribute to breaking intersubspecific reproductive barriers. Our results have laid the foundation for identifying the TRD genes and provide an effective strategy to breakdown TRD for breeding wide-compatible lines, which will be further utilized in the intersubspecific hybrid breeding programs.

The dual role of phytoene synthase genes in carotenogenesis in carrot roots and leaves.

Carrot (Daucus carota L.) is an important food crop and is useful for studying carotenogenesis due to the quantity and diversity of carotenoids in its roots. Phytoene synthase catalyzes the first committed step in the carotenoid biosynthesis pathway, and its overexpression is the main driving force in the orange phenotype. At present, we lack fundamental knowledge of the role of these genes and their effects on carotenoid accumulation in leaves. In the present study, three backcross inbred lines (BC2S4) with different colored roots derived from a cross between the orange inbred line (Af) and related wild species were used to investigate the role of the duplicated DcPSY genes in root carotenogenesis. Promoter analysis showed that DcPSY genes have diverged substantially in their regulatory sequences after gene duplication. Expression levels of DcPSY1 and DcPSY2 were generally positively correlated with carotenoid content during root development. In mature leaves, total carotenoid content was higher than that in the roots, DcPSY1 expression increased extremely higher than DcPSY2 expression compared with roots, and DcPSY1 was more sensitive than DcPSY2 during leaf de-etiolation under sunlight. These results suggest that DcPSY1 seems to make an important contribution to carotenoid accumulation in the leaves and is important for photosynthesis and photoprotection, but they are not the determining factors of root color. This expands our understanding of the regulation of carotenoid biosynthesis in carrot.