Mendelian Segregation Ratio Research Articles

Selfing Promotes Spread and Introgression of Segregation Distorters in Hermaphroditic Plants.

Segregation distorters (SDs) are genetic elements that distort the Mendelian segregation ratio to favor their own transmission and are able to spread even when they incur fitness costs on organisms carrying them. Depending on the biology of the host organisms and the genetic architecture of the SDs, the population dynamics of SDs can be highly variable. Inbreeding is considered an effective mechanism for inhibiting the spread of SDs in populations, and can evolve as a defense mechanism against SDs in some systems. However, we show that inbreeding in the form of selfing in fact promotes the spread of SDs acting as pollen killers in a toxin-antidote system in hermaphroditic plants by two mechanisms: (i) By reducing the effective recombination rate between killer and antidote loci in the two-locus system and (ii) by increasing the proportion of SD alleles in individual flowers, rather than in the general gene-pool. We also show that in rice (Oryza sativa L.), a typical hermaphroditic plant, all molecularly characterized SDs associated with pollen killing were involved in population hybridization and have introgressed across different species. Paradoxically, these loci, which are associated with hybrid incompatibility and can be thought of as Bateson-Dobzhansky-Muller incompatibility loci are expected to reduce gene-flow between species, in fact cross species boundaries more frequently than random loci, and may act as important drivers of introgression.

Analysis of inheritance in a novel seedling stage zebra leaf mutant, Pusa Zebra 18 in rice (Oryza sativa L.)

A novel zebra leaf mutant, Pusa Zebra 18 derived spontaneously from F2 intercross population of Pusa 44 near-isogenic lines (NILs) exhibits distinguishable yellowish cross bands across the green leaf blade, specifically expressed in the seedling stage from 6 DAT, which diffuses progressively, leading to the recovery of the normal green leaf by 30 DAT. We elucidated the genetics and nature of inheritance for zebra trait in F2 and BC1F2 populations by crossing Pusa 6B with Pusa Zebra 18. The leaves of the F1 were a normal green color, indicating that the seedling stage zebra leaf trait in Pusa Zebra 18 is recessive. Chi-square goodness of fit analysis of these segregating populations showed that the segregation ratio was fitting to the Mendelian segregation ratio of 3:1. The seedling stage zebra leaf mutation in Pusa Zebra 18 is monogenic recessive in nature. Further, mapping, fine mapping, cloning, and functional characterization need to be carried out, which will help gain insight into the mechanism involved in chlorophyll biosynthesis and regulation studies in rice.

Development of activation-tagged gain-of-functional mutants in indica rice line (BPT 5204) for sheath blight resistance.

The development of sheath blight (ShB) resistance varieties has been a challenge for scientists for long time in rice. Activation tagging is an efficient gain-of-function mutation approach to create novel phenotypes and to identify their underlying genes. In this study, a mutant population was developed employing activation tagging in the recalcitrant indica rice (Oryza sativa L.) cv. BPT 5204 (Samba Mahsuri) through activation tagging. In this study, we have generated more than 1000 activation tagged lines in indica rice, from these mutant population 38 (GFP- RFP+) stable Ds plants were generated through germinal transposition at T2 generation based on molecular analysis and seeds selected on hygromycin (50mg/L) containing medium segregation analyses confirmed that the transgene inherited as mendelian segregation ratio of 3:1 (3 resistant: 1 susceptible). Of them, five stable activation tagged Ds lines (M-Ds-1, M-Ds-2, M-Ds-3, M-Ds-4 and M-Ds-5) were selected based on phenotypic observation through screening for sheath blight (ShB) resistance caused by fungal pathogen Rhizoctonia solani (R. solani),. Among them, M-Ds-3 and M-Ds-5 lines showed significant resistance for ShB over other tagged lines and wild type (WT) plants. Furthermore, analysed for launch pad insertion through TAIL-PCR results and mapped on corresponding rice chromosomes. Flanking sequence and gene expression analysis revealed that the upregulation of glycoside hydrolase-OsGH or similar to Class III chitinase homologue (LOC_Os08g40680) in M-Ds-3 and a hypothetical protein gene (LOC_Os01g55000) in M-Ds-5 are potential candidate genes for sheath blight resistance in rice. In the present study, we developed Ac-Ds based ShB resistance gain-of-functional mutants through activation tagging in rice. These activation tagged mutant lines can be excellent sources for the development of ShB resistant cultivars in rice.

Ectopic expression of pigeonpea Orf147 gene imparts partial sterility in Cicer arietinum

Orf147, a cytotoxic peptide, has been found to cause cytoplasmic male sterility (CMS) in Cajanus cajanifolius (pigeonpea). In our study, Orf147 was introduced into self-pollinating Cicer arietinum (chickpea) using Agrobacterium-mediated transformation for induction of CMS. The stable integration and expression of the transgene has been assessed through PCR and qRT-PCR analysis. In addition, phenotypic sterility analysis has been performed, considering developmental parameters like flower development, pod formation and flower drop. Transgene inheritance analysis demonstrates that out of the five PCR positive events in the T0 generation, two events have segregated according to the Mendelian segregation ratio (3:1) in the T2 generation. Further, pollen viability test using microscopic analysis confirms the induction of partial CMS in transgenic chickpea. The study holds significant value regarding the heterosis of self-pollinating legumes like chickpea. As a part of the prospect, exploring inducible promoters of species-specific or related legumes would be the next step to developing a two-line hybrid system.

Genetic analysis of a DROOPING LEAF mutant allele dl-6 associated with a twisted and folded leaf base caused by a deficiency in midrib development in rice

The failure of midrib formation in rice leaf blades results in the drooping leaf (dl) phenotype. A normal DROOPING LEAF (DL) gene is necessary for leaf homeotic transformation, which affects midrib and pistil development. Genetic analysis was performed on a new drooping leaf (dl) mutant named dl-6 in rice. The dl-6 allelic mutant exhibited drooping leaves that were severely folded and twisted at the base but had normal flower structure. The dl-6 allele is a nuclear recessive trait that fits a 3:1 Mendelian segregation ratio. The dl-6 mutant leaves displayed an abnormal main vein (midrib-less) with undeveloped aerenchyma and vascular bundles, resulting in severe leaf drooping. The lack of a midrib in dl-6 caused weak mechanical support, which resulted in folding at the collar junction of the leaf base and downward bending. Through genetic mapping, the dl-6 allele was identified at approximately 28.2 cM on rice chromosome 3. The allele was caused by mutations within the DL (LOC_Os03g11600.1) gene, with specific amino acid substitutions and additions in the encoded protein of the YABBY transcription factor. The dl-6 mutant is a recessive allele encoding a dysfunctional YABBY transcription factor that regulates leaf midrib development and aerenchymatous clear cell structures, leading to a drooping leaf phenotype in rice.

Conjunctive Analyses of Bulk Segregant Analysis Sequencing and Bulk Segregant RNA Sequencing to Identify Candidate Genes Controlling Spikelet Sterility of Foxtail Millet.

Foxtail millet has gradually become a model gramineous C4 crop owing to its short growth period and small genome. Research on the development of its spikelets is not only directly related to the yield and economic value of foxtail millet but also can provide a reference for studying the fertility of other C4 crops. In this study, a hybrid population containing 200 offspring was constructed from the Xinong8852 and An15 parental lines, and two extreme trait populations were constructed from the F2 generation for analysis of the spikelet sterility. The F2 population conformed to a 3:1 Mendelian segregation ratio, and it was thus concluded that this trait is likely controlled by a single recessive gene. Bulk segregant analysis sequencing (BSA-Seq) was used to determine the candidate regions and candidate genes related to the development of foxtail millet spikelets. Additionally, the functional analysis of differentially expressed genes in populations with different traits was conducted by bulk segregant RNA sequencing (BSR-Seq). Finally, conjunctive analysis of BSA-Seq and BSR-Seq results, combined with biological information analysis, revealed six genes on chromosome VII that were ultimately identified as candidate genes controlling foxtail millet spikelet development. This study provides a new reference for research on foxtail millet sterility and lays a solid foundation for the examination of fertility in other gramineous crops.

Fine Mapping of Two Interacting Loci for Transmission Ratio Distortion in Rice (Oryza sativa L.)

Transmission ratio distortion (TRD) denotes the observed allelic or genotypic frequency deviation from the expected Mendelian segregation ratios in the offspring of a heterozygote. TRD can severely hamper gene flow between and within rice species. Here, we report the fine mapping and characterization of two loci (TRD4.1 and TRD4.2) for TRD using large F2 segregating populations, which are derived from rice chromosome segment substitution lines, each containing a particular genomic segment introduced from the japonica cultivar Nipponbare (NIP) into the indica cultivar Zhenshan (ZS97). The two loci exhibited a preferential transmission of ZS97 alleles in the derived progeny. Reciprocal crossing experiments using near-isogenic lines harboring three different alleles at TRD4.1 suggest that the gene causes male gametic selection. Moreover, the transmission bias of TRD4.2 was diminished in heterozygotes when they carried homozygous TRD4.1ZS97. This indicates an epistatic interaction between these two loci. TRD4.2 was mapped into a 35-kb region encompassing one candidate gene that is specifically expressed in the reproductive organs in rice. These findings broaden the understanding of the genetic mechanisms of TRD and offer an approach to overcome the barrier of gene flow between the subspecies in rice, thus facilitating rice improvement by introgression breeding.

Genetic Transformation for Pod Borer Resistance in Dolichos Bean [Lablab purpureus (L.) Sweet

Background: Agrobacterium mediated genetic transformation experiments were carried out in Dolichos bean Cv. (Konkan Bhushan) showing better regenerability. Methods: Three cry genes viz. cry1Aabc, cry1Fa1 and cry2Aa were used for the transformation each of which were linked to CaMV35S promoter and nptII gene under control of nos promoter and terminator. A vector system consisting of the disarmed hyper virulent Agrobacterium tumefaciens strain EHA-105 harboring pBinAR or BinBt3 was used. Mature embryo axis with single cotyledon was used as explant. Kanamycin as well as PCR screening was carried out to assess the transformation frequency. Progeny analysis using PCR was also carried out to assess the transgene segregation and stable transformation.Result: Kanamycin concentration of 500 mg/l was found as optimum for selection of a transgenic turning leaf blades albino. Among five methods of colonization used, the method employing mild injury to explant with dipping in Agrobacterium culture for 20 minutes followed by co-cultivation for 48 hours, cefotaxime washing and sowing in soil resulted in maximum survival (74.80%) associated with maximum transformation frequency through PCR analysis (2.13%). Among three cry genes, the gene cry2Aa was found the most effective in transforming Dolichos bean. The progeny analysis of transformants has shown the 3:1 mendelian segregation ratio confirming stable transformation of transgene.

Inheritance Pattern of Mungbean Yellow Mosaic Virus (MYMV) Disease Resistance in Blackgram [Vigna mungo (L.) Hepper

Background: Mungbean yellow mosaic virus (MYMV) disease is the most destructive disease in blackgram. Development of MYMV resistant varieties is one of the best possible solutions to avoid the yield reduction in blackgram. There are conflicting reports on the genetics of resistance to MYMV disease claiming that it is controlled by both dominant and recessive genes. Hence the present study was aimed to understand the inheritance pattern of the MYMV disease resistance in eight crosses of blackgram.Methods: Parents, F1 and F2 generation of eight cross combinations were raised during July - Sep, 2018 at National Pulses Research Centre, Tamil Nadu Agricultural University, Vamban, Tamil Nadu. An infector row of CO 5 was raised to intensify the MYMV disease pressure after every eight rows. Based on disease incidence on 60th day after sowing, two phenotypic classes were formed among F2 plants with the scales of (1 to 3) as resistant phenotype and (4 to 9) as susceptible phenotype. The goodness of fit to Mendelian segregation ratio for MYMV disease resistance in the segregating population was tested by Chi square test (Stansfield, 1991).Result: The MYMV disease incidence was tri-genically controlled with inhibitory gene action in four crosses viz., MDU 1 x Mash 114, CO5 x Mash 114, MDU 1 x VBN 6 and CO 5 x VBN 6. Complementary gene action with two genes was observed in four crosses viz., MDU 1 x Mash 1008, CO 5 x Mash 1008, MDU 1 x VBN 8 and CO 5 x VBN 8. Differences in number of genes were observed due to the presence of recessive inhibitory gene in both male and female parents of the crosses which had complementary gene action for MYMV disease. The putative gene symbols assigned for the six genotypes viz., S1S1S2S2ii (MDU 1 and CO 5), s1s1s2s2II (Mash 114 and VBN 6) and s1s1s2s2ii (Mash 1008 and VBN 8), respectively.

Maximum Likelihood Estimation for Ascertainment Bias in Sampling Siblings

When there is a rare disease in a population, it is inefficient to take a random sample to estimate a parameter. Instead one takes a random sample of all nuclear families with the disease by ascertaining at least one affected sibling (proband) of each family. In these studies, an estimate of the proportion of siblings with the disease will be inflated. For example, studies of the issue of whether a rare disease shows an autosomal recessive pattern of inheritance, where the Mendelian segregation ratios are of interest, have been investigated for several decades. How do we correct for this ascertainment bias? Methods, primarily based on maximum likelihood estimation, are available to correct for the ascertainment bias. We show that for ascertainment bias, although maximum likelihood estimation is optimal under asymptotic theory, it can perform badly. The problem is exasperated in the situation where the proband probabilities are allowed to vary with the number of affected siblings. We use two data sets to illustrate the difficulties of maximum likelihood estimation procedure, and we use a simulation study to assess the quality of the maximum likelihood estimators.

Introgression and confirmation of everbearing trait in strawberry (Fragaria x ananassa Duch.)

The online version of this article (10.1007/s12298-020-00916-w) contains supplementary material, which is available to authorized users.

Genetic Mapping and Nucleotide Diversity of Two Powdery Mildew Resistance Loci in Melon (Cucumis melo).

Powdery mildew (PM) significantly and negatively affects the yield and quality of melon (Cucumis melo) worldwide. Race 2F is the predominant physiological race of the pathogen Podosphaera xanthii in many regions. We used accessions PMR 6 (P1; resistant to PM) and M1-7 (P2; susceptible to PM) to analyze the inheritance of resistance to PM (race 2F). The ratio between resistant and susceptible individuals fits a Mendelian segregation ratio of 13:3 in a total of 256 F2 individuals and 1:1 in BC1P2. The resistance to PM in PMR 6 was governed by two genes: a dominant (AA) gene with an epistatic effect and a recessive gene (bb). Only individuals with aaBB or aaBb genotypes were susceptible to PM. Two PM resistance loci, Pm2.1 and pm12.1, were mapped on chromosomes 2 and 12 by bulked segregant analysis and secondary mapping by quantitative trait loci analysis with 18 markers. A new marker-assisted selection system to identify melon genotypes resistant or susceptible to PM was developed and tested in 93 melon accessions. Nucleotide diversity (π) and fixation index (Fst) for the two PM resistance loci were estimated using resequencing data of 336 melons from three groups: C. melo subsp. agrestis, Cucumis melo subsp. melo, and the intermediate type. The lowest π was observed in C. melo ssp. agrestis, and the highest Fst value was between C. melo ssp. agrestis and C. melo ssp. melo. The findings provide a promising tool that can be used to accelerate breeding for durable resistance to PM.

Mapping QTL for important seed traits in an interspecific F2 population of pigeonpea

Seed traits present important breeding targets for enhancing grain yield and quality in various grain legume crops including pigeonpea. The present study reports significant genetic variation for six seed traits including seed length (SL), seed width (SW), seed thickness (ST), seed weight (SWT), electrical conductivity (EC) and water uptake (WU) among Cajanus cajan (L.) Millspaugh acc. ICPL 20340 and Cajanus scarabaeoides (L.) Thouars acc. ICP 15739 and an F2 population derived from this interspecific cross. Maximum phenotypic values recorded for the F2 population were higher than observed in the parent ICPL 20340 [F2 max vs ICPL 20340: SW (7.05 vs 5.38), ST (4.63 vs 4.51), EC (65.17 vs 9.72), WU (213.17 vs 109.5)], which suggested contribution of positive alleles from the wild parent, ICP 15739. Concurrently, to identify the QTL controlling these seed traits, we assayed two parents and 94 F2 individuals with 113 polymorphic simple sequence repeat (SSR) markers. In the F2 population, 98 of the 113 SSRs showed Mendelian segregation ratio 1:2:1, whereas significant deviations were observed for 15 SSRs with their χ 2 values ranging between 6.26 and 20.62. A partial genetic linkage map comprising 83 SSR loci was constructed. QTL analysis identified 15 marker-trait associations (MTAs) for seed traits on four linkage groups i.e. LG01, LG02, LG04 and LG05. Phenotypic variations (PVs) explained by these QTL ranged from 4.4 (WU) to 19.91% (EC). These genomic regions contributing significantly towards observed variability of seed traits would serve as potential candidates for future research that aims to improve seed traits in pigeonpea.

Construction of genetic linkage map and identification of QTLs related to agronomic traits in DH population of maize (Zea mays L.) using SSR markers.

In this study, we used phenotypic and genetic analysis to investigate Double haploid (DH) lines derived from normal corn parents (HF1 and 11S6169). DH technology offers an array of advantages in maize genetics and breeding as follows: first, it significantly shortens the breeding cycle by development of completely homozygous lines in two or three generations; and second, it simplifies logistics, including requiring less time, labor, and financial resources for developing new DH lines compared with the conventional RIL population development process. In our study, we constructed a maize genetic linkage map using SSR markers and a DH population derived from a cross of normal corn (HF1) and normal corn (11S6169). The DH population used in this study was developed by the following methods: we crossed normal corn (HF1) and normal corn (11S6169), which are parent lines of a normal corn cultivar, in 2014; and the next year, the F1 hybrids were crossed with a tropicalized haploid inducer line (TAIL), which is homozygous for the dominant marker gene R1-nj (Nanda and Chase in Crop Sci 6:213-215, 1966), and we harvested seeds of the haploid lines. A total of 200 SSR markers were assigned to 10 linkage groups that spanned 1145.4cM with an average genetic distance between markers of 5.7cM. 68 SSR markers showed Mendelian segregation ratios in the DH population at a 5% significance threshold. A total of 15 quantitative trait loci (QTLs) for plant height (PH), ear height (EH), ear height ratio (ER), leaf length (LL), ear length (EL), set ear length (SEL), set ear ratio (SER), ear width (EW), 100 kernel weight (100KW), and cob color (CC) were found in the 121 lines in the DH population. The results of this study may help to improve the detection and characterization of agronomic traits and provide great opportunities for maize breeders and researchers using a DH population in maize breeding programs.

Construction of a genetic linkage map based on RAPD, AFLP, and SSR markers for tea plant (Camellia sinensis)

Based on double pseudo-testcross theory, a population of 79 F1 clones, which was derived from a crossing of two tea cultivars using a Japanese tea cultivar “Fushun” (Camellia sinensis) as female parent and a Korean tea cultivar “Kemsull” (C. sinensis) as male parent, was applied to construct a genetic linkage map with different molecular markers. Previously, 143 random amplified polymorphism DNA (RAPD) markers and 11 simple sequence repeat (SSR) loci developed from 41 decamer random primers and 60 published SSR primer pairs, respectively, were used into our mapping analysis. In the study of amplified fragment length polymorphism (AFLP), 2439 bands were generated from 27 primer combinations with an average number of 90.3 bands for each, of which 495 (20.3%) were polymorphic. The majority of those polymorphic markers segregated in accordance with Mendelian segregation ratios at p < 0.01 for 400 (80.8%) or at p < 0.05 for 382 (77.2%), of which 136 (34.0%) or 131 (34.3%) were at 3:1 segregation ratios and 264 (66.0%) or 251 (65.7%) were at 1:1 segregation ratios at p < 0.01 and p < 0.05, respectively. For developing more SSR markers, the transcriptome sequences of flowers and leaves of two parents were obtained using high throughput RNA sequencing and compared. Afterwards, 1800 potential polymorphic SSR markers were successfully developed and 296 of them were selected and experimentally validated with a subset of tea plants (including two parents and six F1 offspring), from which 75 (25.3%) were repeatably amplified and polymorphic between two parents. From that, 29 (38.7%) newly mined SSR markers were heterozygous in “Fushun” and/or “Kemsull” and showed segregant genotypes in F1 seedlings and were adoptable by JoinMap 4.0. Totally, 678 markers including 143 RAPDs, 11 public SSRs, 495 AFLPs, and 29 newly mined SSRs were conjointly used to construct a combined linkage map for tea plant. The new genetic map located 79 RAPDs, 5 public SSRs, 214 AFLPs, and 11 new SSRs developed from RNA seq technique and covered 1441.6 cM with an average distance of 4.7 cM between two adjacent markers. This map will lay a foundation for qualitative or quantitative trait loci (QTLs) analysis of important agronomic traits for tea plant in the future study.

Genetic background of resistance to gall mite in Ribes species

Resistance to gall mite is an important genetic trait of Ribes. P and Ce genes, responsible for gall mite resistance, were established in Ribes species and interspecific hybrids using molecular markers. Resistance in R. americanum is determined by P gene and in R. sanguineum by Ce gene. Both molecular markers were absent in R. dikuscha genome. Molecular markers related to P and Ce genes were identified in the genome of R. aureum. Resistance to gall mite in the field conditions in R. nigrum x R. americanum, R. nigrum x R. aureum and R. nigrum x R. sanguineum F3 hybrids fitted an expected Mendelian segregation ratio of 1:1, 3:1 and 1:1, respectively. 75.0% of hybrids with a pyramidal resistance to gall mite carrying markers related to Ce and P genes were obtained in the cross combination R. nigrum x R. aureum and will be included in the future breeding programs.

A high-density SNP genetic map consisting of a complete set of homologous groups in autohexaploid sweetpotato (Ipomoea batatas)

Sweetpotato (Ipomoea batatas) is an autohexaploid species with 90 chromosomes (2n = 6x = 90) and a basic chromosome number of 15, and is therefore regarded as one of the most challenging species for high-density genetic map construction. Here, we used single nucleotide polymorphisms (SNPs) identified by double-digest restriction site-associated DNA sequencing based on next-generation sequencing technology to construct a map for sweetpotato. We then aligned the sequence reads onto the reference genome sequence of I. trifida, a likely diploid ancestor of sweetpotato, to detect SNPs. In addition, to simplify analysis of the complex genetic mode of autohexaploidy, we used an S1 mapping population derived from self-pollination of a single parent. As a result, 28,087 double-simplex SNPs showing a Mendelian segregation ratio in the S1 progeny could be mapped onto 96 linkage groups (LGs), covering a total distance of 33,020.4 cM. Based on the positions of the SNPs on the I. trifida genome, the LGs were classified into 15 groups, each with roughly six LGs and six small extra groups. The molecular genetic techniques used in this study are applicable to high-density mapping of other polyploid plant species, including important crops.

Evaluation of SRAP markers for mapping of Pisum sativum L.

Linkage maps have become important tools for genetic studies. With the aim of evaluating the SRAP (sequence-related amplified polymorphism) technique for linkage mapping in Pisum sativum L., a F2 mapping population derived from an initial cross between cvs. DDR11 and Zav25 was generated. A total of 25 SRAP primer combinations were evaluated in 45 F2 plants and both parental lines, generating 208 polymorphic bands/markers. The markers were analyzed by the chi-square goodness-of-fit test to check the expected Mendelian segregation ratio. The resulting linkage map consists of 112 genetic markers distributed in 7 linkage groups (LGs), covering a total of 528.8 cM. The length of the LGs ranged from 47.6 to 144.3 cM (mean 75.54 cM), with 9 to 34 markers. The linkage map developed in this study indicates that the SRAP marker system could be applied to mapping studies of pea.

Identification of loci associated with embryo yield in microspore culture of Brassica rapa by segregation distortion analysis.

We identified three physical positions associated with embryo yield in microspore culture of Brassica rapa by segregation distortion analysis. We also confirmed their genetic effects on the embryo yield. Isolated microspore culture is well utilized for the production of haploid or doubled-haploid plants in Brassica crops. Brassica rapa cv. 'Ho Mei' is one of the most excellent cultivars in embryo yield of microspore culture. To identify the loci associated with microspore embryogenesis, segregation analysis of 154 DNA markers anchored to B. rapa chromosomes (A01-A10) was performed using a population of microspore-derived embryos obtained from an F1 hybrid between 'CR-Seiga', a low yield cultivar in microspore-derived embryos, and 'Ho Mei'. Three regions showing significant segregation distortion with increasing 'Ho Mei' alleles were detected on A05, A08 and A09, although these regions showed the expected Mendelian segregation ratio in an F2 population. The additive effect of alleles in these regions on embryo yield was confirmed in a BC3F1 population. One region on A08 containing Br071-5c had a higher effect than the other regions. Polymorphism of nucleotide sequences around the Br071-5c locus was investigated to find the gene possibly responsible for efficient embryogenesis from microspores.

Rapid and efficient Agrobacterium-mediated transformation of sorghum (Sorghum bicolor) employing standard binary vectors and bar gene as a selectable marker.

A rapid and efficient Agrobacterium -mediated transformation system in sorghum has been developed employing standard binary vectors and bar gene as a selectable marker. Sorghum (Sorghum bicolor) is an important food and biofuel crop worldwide, for which improvements in genetic transformation are needed to study its biology and facilitate agronomic and commercial improvement. Here, we report optimization of regeneration and transformation of public sorghum genotype P898012 using standard binary vectors and bar gene as a selectable marker. The tissue culture regeneration time frame has been reduced to 7-12weeks with a yield of over 18 plants per callus, and the optimized transformation system employing Agrobacterium tumefaciens strain AGL1 and the bar with a MAS promoter achieved an average frequency over 14%. Of randomly analyzed independent transgenic events, 40-50% carry single copy of integrated T-DNA. Some independent transgenic events were derived from the same embryogenic callus lines, but a 3:1 Mendelian segregation ratio was found in all transgenic events with single copy as estimated by Southern blots. The system described here should facilitate studies of sorghum biology and agronomic improvement.