A QTL that enhances and broadens Bt insect resistance in soybean

Effective strategies are needed to manage insect resistance to Bacillus thuringiensis (Bt) proteins expressed in transgenic crops. To evaluate a multiple resistance gene pyramiding strategy, eight soybean (Glycine max) lines possessing factorial combinations of two quantitative trait loci (QTLs) from plant introduction (PI) 229358 and a synthetic Bt cry1Ac gene were developed using marker-assisted selection with simple sequence repeat markers. Field studies were conducted in 2000 and 2001 to evaluate resistance to corn earworm (Helicoverpa zea) and soybean looper (Pseudoplusia includens), and detached leaf bioassays were used to test antibiosis resistance to Bt-resistant and Bt-susceptible strains of tobacco budworm (TBW; Heliothis virescens). Based on defoliation in the field and larval weight gain on detached leaves, lines carrying a combination of cry1Ac and the PI 229358 allele at a QTL on linkage group M were significantly more resistant to the lepidopteran pests, including the Bt-resistant TBW strain, than were the other lines. This is the first report of a complementary additive effect between a Bt transgene and a plant insect resistance QTL with an uncharacterized mode of action that was introgressed using marker-assisted selection.     

Effects of defoliating insect resistance QTLs and a <Emphasis Type="Italic">cry1Ac</Emphasis> transgene in soybean near-isogenic lines

The crystal proteins coded by transgenes from Bacillus thuringiensis (Bt) have shown considerable value in providing effective insect resistance in a number of crop species, including soybean, Glycine max (L.) Merr. Additional sources of soybean insect resistance would be desirable to manage the development of tolerance/resistance to crystal proteins by defoliating insects and to sustain the deployment of Bt crops. The objective of this study was to evaluate the effects and interactions of three insect resistance quantitative trait loci (QTLs; QTL-M, QTL-H, and QTL-G) originating from Japanese soybean PI 229358 and a cry1Ac gene in a “Benning” genetic background. A set of 16 BC₆F₂-derived near isogenic lines (NILs) was developed using marker-assisted backcrosses and evaluated for resistance to soybean looper [SBL, Pseudoplusia includens (Walker)] and corn earworm [CEW, Helicoverpa zea (Boddie)] in field cage, greenhouse, and detached leaf assays. Both Bt and QTL-M had significantly reduced defoliation by both SBL and CEW and reduced larval weight of CEW. The antibiosis QTL-G had a significant effect on reducing CEW larval weight and also a significant effect on reducing defoliation by SBL and CEW in some assays. The antixenosis QTL-H had no main effect, but it appeared to function through interaction with QTL-M and QTL-G. Adding QTL-H and QTL-G further enhanced the resistance of the Bt and QTL-M combination to CEW in the field cage assay. These results should help guide the development of strategies for effective management of insect pests and for sustainable deployment of Bt genes.     

Identification of molecular markers linked to quantitative trait loci for soybean resistance to corn earworm

 One hundred and thirty nine restriction fragment length polymorphisms (RFLPs) were used to construct a soybean (Glycine max L. Merr.) genetic linkage map and to identify quantitative trait loci (QTLs) associated with resistance to corn earworm (Helicoverpa zea Boddie) in a population of 103 F₂-derived lines from a cross of ‘Cobb’ (susceptible) and PI229358 (resistant). The genetic linkage map consisted of 128 markers which converged onto 30 linkage groups covering approximately 1325 cM. There were 11 unlinked markers. The F₂-derived lines and the two parents were grown in the field under a plastic mesh cage near Athens, Ga., in 1995. The plants were artificially infested with corn earworm and evaluated for the amount of defoliation. Using interval-mapping analysis for linked markers and single-factor analysis of variance (ANOVA), markers were tested for an association with resistance. One major and two minor QTLs for resistance were identified in this population. The PI229358 allele contributed insect resistance at all three QTLs. The major QTL is linked to the RFLP marker A584 on linkage group (LG) ‘M’ of the USDA/Iowa State University public soybean genetic map. It accounts for 37% of the total variation for resistance in this cross. The minor QTLs are linked to the RFLP markers R249 (LG ‘H’) and Bng047 (LG ‘D1’). These markers explain 16% and 10% of variation, respectively. The heritability (h²) for resistance was estimated as 64% in this population. Received: 15 October 1997 / Accepted: 4 November 1997     

Inheritance of resistance in maize silks to the corn earworm

The corn earworm,Helicoverpa zea (Boddie), is a perennial economic pest of field crops in the United States. Maize,Zea mays L., is the major host crop promoting the build-up of devastating corn earworm populations that limit full production of cotton, soybean, peanut, and grain sorghum. Resistance to the corn earworm in maize and in particular sweet maize, would provide an environmentally safe, economical method of control for this pest insect. Antibiotic effects of corn silks on this insect are: small larvae, extended developmental period, and reduced fecundity. Silks from individual maize plants of resistant and susceptible lines and progeny in six generations consisting of parents (P₁, P₂), F₁, F₂, and backcrosses BC_1.1 (F₁ × P₁) and BC_1.2 (F₁ × P₂) from each of four crosses were used to determine the genetic basis of the antibiotic resistance of silks to the corn earworm. In the cross of Zapalote Chico × PI340856, genes controlling resistance in the silks to the corn earworm larvae are dominant in PI340856 to those in Zapalote Chico. The cross of Zapalote Chico × GT114 involves parents differing in degree of resistance, and possibly differing for the genetic mechanism by which the resistance is inherited. The inheritance of resistance may involve non-additive (dominance and epistasis) genetic variance. A digenic 6-parameter model indicated (1) the resistance in this cross is controlled by more than one pair of genes and (2) some or all of the genes interact to cause non-allelic interaction. Thus, the resistance in this cross may be controlled by both dominant and recessive genes. The resistance of Zapalote Chico × CI64, an intermediate inbred, is influenced by additive gene effects. The digenic model adequately predicts all generation means of the cross of GT3 × PI340856 except for the F1. Thus, it appears that the additive-dominance model is not satisfactory for this cross involving susceptible and resistant parents. Generation mean analysis indicates that resistance to silk-feeding by corn earworm larvae is under genetic control, but gene action differs from one type of cross to another.     

Development of transgenic sorghum for insect resistance against the spotted stem borer (Chilo partellus)

Transgenic sorghum plants expressing a synthetic cry1Ac gene from Bacillus thuringiensis (Bt) under the control of a wound-inducible promoter from the maize protease inhibitor gene (mpiC1) were produced via particle bombardment of shoot apices. Plants were regenerated from the transformed shoot apices via direct somatic embryogenesis with an intermittent three-step selection strategy using the herbicide Basta. Molecular characterisation based on polymerase chain reaction and Southern blot analysis revealed multiple insertions of the cry1Ac gene in five plants from three independent transformation events. Inheritance and expression of the Bt gene was confirmed in T₁ plants. Enzyme-linked immunosorbant assay indicated that Cry1Ac protein accumulated at levels of 1–8 ng per gram of fresh tissue in leaves that were mechanically wounded. Transgenic sorghum plants were evaluated for resistance against the spotted stem borer (Chilo partellus Swinhoe) in insect bioassays, which indicated partial resistance to damage by the neonate larvae of the spotted stem borer. Reduction in leaf damage 5 days after infestation was up to 60%; larval mortality was 40%, with the surviving larvae showing a 36% reduction in weight over those fed on control plants. Despite the low levels of expression of Bt -endotoxin under the control of the wound-inducible promoter, the transgenic plants showed partial tolerance against first instar larvae of the spotted stem borer.     

Field evaluation of soybean engineered with a synthetic cry1Ac transgene for resistance to corn earworm, soybean looper, velvetbean caterpillar (Lepidoptera: Noctuidae), and lesser cornstalk borer (Lepidoptera: Pyralidae)

A transgenic line of the soybean 'Jack', Glycine max (L.) Merrill, expressing a synthetic cry1Ac gene from Bacillus thuringiensis variety kurstaki (Jack-Bt), was evaluated for resistance to four lepidopteran pests in the field. Jack-Bt and genotypes serving as susceptible and resistant controls were planted in field cages and artificially infested with larvae of corn earworm, Helicoverpa zea (Boddie), and velvetbean caterpillar, Anticarsia gemmatalis (Hübner), in 1996, 1997, and 1998, and also with soybean looper, Pseudoplusia includens (Walker), in 1996. Susceptible controls included Jack (1996-1998), 'Cobb' (1996), and Jack-HPH (1996). GatIR 81-296 was used as the resistant control in all 3 yr. Compared with untransformed Jack, Jack-Bt showed three to five times less defoliation from corn earworm and eight to nine times less damage from velvetbean caterpillar. Defoliation of GatIR 81-296 was intermediate between that of Jack and Jack-Bt for corn earworm, and similar to that of Jack for velveltbean caterpillar. Jack-Bt exhibited significant, but lower resistance to soybean looper. Jack-Bt also showed four times greater resistance than Jack to natural infestations of lesser cornstalk borer, Elasmopalpus lignosellus (Zeller), in conventional field plots at two locations in 1998. Data from these experiments suggest that expression of this cry1Ac construct in soybean should provide adequate levels of resistance to several lepidopteran pests under field conditions.     

SSR mapping and confirmation of the QTL from PI96354 conditioning soybean resistance to southern root-knot nematode

Root-knot nematodes (Meloidogyne spp.) can cause severe yield loss of soybean [Glycine max (L.) Merr.] in the southern production region of the USA. Planting root-knot nematode-resistant cultivars is the most effective method of preventing yield loss. DNA marker-assisted breeding may accelerate the development of root-knot nematode-resistant cultivars. RFLP markers have previously been used to identify quantitative trait loci (QTLs) conferring resistance to southern root-knot nematode [Meloidogyne incognita (Kofoid and White) Chitwood] (Mi) in a F_2:3 soybean population created by crossing the resistant PI96354 and the susceptible ’Bossier.’ A major QTL on linkage group (LG) O conditioning 31% of the variation in Mi gall number and a minor QTL on LG-G conditioning 14% of the gall variation were reported. With the development of SSR markers for soybean improvement, a higher level of mapping resolution and semi-automated detection has become possible. The objectives of this research were: (1) to increase the marker density in the genomic regions of the QTLs for Mi resistance on LG-O and LG-G with SSR markers; and (2) to confirm the effect of the QTLs in a second population and a different genetic background. With SSR markers, the QTL on LG-O was flanked by Satt492 and Satt358, and on LG-G by Satt012 and Satt505. Utilizing SSR markers flanking the two QTLs, marker-assisted selection was performed in a second F_2:3 population of PI96354× Bossier. Results confirmed the effectiveness of marker-assisted selection to predict the Mi phenotypes. By screening the BC₂F₂ population of Prichard (3)×G93–9009 we confirmed that selection for the minor QTL on LG-G with flanking SSR markers would enhance the resistance of lines containing the major QTL (which is most-likely Rmi1). Received: 29 September 2000 / Accepted: 17 April 2001     

Seed yield of near-isogenic soybean lines with introgressed quantitative trait loci conditioning resistance to corn earworm (Lepidoptera: Noctuidae) and soybean looper (Lepidoptera: Noctuidae) from PI 229358

The development of superior soybean, Glycine max (L.) Merr., cultivars exhibiting resistance to insects has been hindered due to linkage drag, a common phenomenon when introgressing alleles from exotic germplasm. Simple-sequence repeat (SSR) markers were used previously to map soybean insect resistance (SIR) quantitative trait loci (QTLs) in a'Cobb' X PI 229358 population, and subsequently used to create near-isogenic lines (NILs) with SIR QTL i n a 'Benning' genetic background. SIR QTLs were mapped on linkage groups (LGs) M (SIRQTL-M), G (SIRQTL-G), and H (SIRQTL-H). The objectives of this study were to 1) evaluate linkage drag for seed yield by using Benning-derived NILs selected for SIRQTL-M, SIRQTL-H, and SIRQTL-G; 2) assess the amount of PI 229358 genome surrounding the SIR QTL in each Benning NIL; and 3) evaluate the individual effects these three QTLs on antibiosis and antixenosis to corn earworm, Helicoverpa zea (Boddie), and soybean looper, Pseudoplusia includens (Walker). Yield data collected in five environments indicated that a significant yield reduction is associated with SIRQTL-G compared with NILs without SIR QTL. Overall, there was no yield reduction associated with SIRQTL-M or SIRQTL-H. A significant antixenosis and antibiosis effect was detected for SIRQTL-M in insect feeding assays, with no effect detected in antixenosis or antibiosis assays for SIRQTL-G or SIRQTL-H without the presence of PI 229358 alleles at SIRQTL-M. These results support recent findings concerning these loci.     

Greenhouse and field evaluations of transgenic canola against diamondback moth, shape Plutella xylostella, and corn earworm, shape Helicoverpa zea

Canola (Brassica napus L.) cultivars Oscar and Westar, engineered with a Bacillus thuringiensis (Bt) cryIA(c) gene, were evaluated for resistance to lepidopterous pests, diamondback moth, Plutella xylostella L. (Plutellidae) and corn earworm, Helicoverpa zea (Boddie) (Noctuidae) in greenhouse and field conditions. In greenhouse preference assays conducted at vegetative and flowering plant stages, transgenic plants recorded very low levels of damage. A 100% diamondback moth mortality and 90% corn earworm mortality were obtained on transgenic plants in greenhouse antibiosis assays. The surviving corn earworm larvae on transgenic plants had reduced head capsule width and body weight. Mortality of diamondback moth and corn earworm were 100% and 95%, respectively, at different growth stages (seedling, vegetative, bolting, and flowering) on the transgenic plants in greenhouse tests. In field tests conducted during 1995–1997, plots were artificially infested with neonates of diamondback moth or corn earworm or left for natural infestation. Transgenic plants in all the treatments were highly resistant to diamondback moth and corn earworm larvae and had very low levels of defoliation. Plots infested with diamondback moth larvae had greater damage in both seasons as compared with corn earworm infested plots and plots under natural infestation. After exposure to defoliators, transgenic plants usually had higher final plant stand and produced more pods and seeds than non-transgenic plants. Diamondback moth injury caused the most pronounced difference in plant stand and pod and seed number between transgenic and non-transgenic plants. Our results suggest that transgenic canola could be used for effective management of diamondback moth and corn earworm on canola.     

Sequential transformation to pyramid two Bt genes in vegetable Indian mustard (<Emphasis Type="Italic">Brassica juncea</Emphasis> L.) and its potential for control of diamondback moth larvae

Vegetable Indian mustard (Brassica juncea cv. “Green Wave”) plants that control Plutella xylostella (diamondback moth) (DBM) were produced by introduction of one or two Bacillus thuringiensis (Bt) genes. A cry1Ac Bt gene associated with the nptII gene for kanamycin selection or a cry1C Bt gene with the hpt gene for hygromycin selection was introduced individually through Agrobacterium-mediated transformation of seedling explants. A cry1C line was then transformed with the cry1Ac gene to produce pyramided cry1Ac + cry1C plants. Sixteen cry1C, five cry1Ac, and six cry1Ac + cry1C plants were produced. PCR and Southern analyses confirmed the presence of the cry1C, cry1Ac or pyramided cry1Ac + cry1C genes in the Indian mustard genome. ELISA analysis showed that production of Bt proteins varied greatly among individual transgenic plants, ranging from undetectable to over 1,000 ng Bt/mg total soluble protein. The levels of the Bt proteins were correlated with the effectiveness of control of diamondback moth (DBM) larvae. Insect bioassays indicated that both the cry1C and cry1Ac plants were toxic to susceptible DBM. The cry1C plants also controlled Cry1A-resistant DBM while cry1Ac plants controlled Cry1C-resistant DBM, and the pyramided cry1Ac + cry1C plants effectively controlled all three types of DBM. These Bt-transgenic plants could be used either for direct control of DBM and other lepidopteran insect pests or for tests of “dead-end” trap crops as protection of high value non-transgenic crucifer vegetables such as cabbage.     

QTL mapping for bacterial wilt resistance in peanut (<Emphasis Type="Italic">Arachis hypogaea</Emphasis> L.)

Bacterial wilt (BW) caused by Ralstonia solanacearum is a serious, global, disease of peanut (Arachis hypogaea L.), but it is especially destructive in China. Identification of DNA markers linked to the resistance to this disease will help peanut breeders efficiently develop resistant cultivars through molecular breeding. A F₂ population, from a cross between disease-resistant and disease-susceptible cultivars, was used to detect quantitative trait loci (QTL) associated with the resistance to this disease in the cultivated peanut. Genome-wide SNPs were identified from restriction-site-associated DNA sequencing tags using next-generation DNA sequencing technology. SNPs linked to disease resistance were determined in two bulks of 30 resistant and 30 susceptible plants along with two parental plants using bulk segregant analysis. Polymorphic SSR and SNP markers were utilized for construction of a linkage map and for performing the QTL analysis, and a moderately dense linkage map was constructed in the F₂ population. Two QTL (qBW-1 and qBW-2) detected for resistance to BW disease were located in the linkage groups LG1 and LG10 and account for 21 and 12 % of the bacterial wilt phenotypic variance. To confirm these QTL, the F₈ RIL population with 223 plants was utilized for genotyping and phenotyping plants by year and location as compared to the F₂ population. The QTL qBW-1 was consistent in the location of LG1 in the F₈ population though the QTL qBW-2 could not be clarified due to fewer markers used and mapped in LG10. The QTL qBW-1, including four linked SNP markers and one SSR marker within 14.4-cM interval in the F₈, was closely related to a disease resistance gene homolog and was considered as a candidate gene for resistance to BW. QTL identified in this study would be useful to conduct marker-assisted selection and may permit cloning of resistance genes. Our study shows that bulk segregant analysis of genome-wide SNPs is a useful approach to expedite the identification of genetic markers linked to disease resistance traits in the allotetraploidy species peanut.     

Transfer of bacterial blight and blast resistance from the tetraploid wild rice Oryza minuta to cultivated rice,Oryza sativa

Summary Oryza minuta J. S. Presl ex C. B. Presl is a tetraploid wild rice with resistance to several insects and diseases, including blast (caused by Pyricularia grisea) and bacterial blight (caused by Xanthomonas oryzae pv. oryzae). To transfer resistance from the wild species into the genome of cultivated rice (Oryza sativa L.), backcross progeny (BC₁, BC₂, and BC₃) were produced from interspecific hybrids of O. sativa cv IR31917-45-3-2 (2n=24, AA genome) and O. minuta Acc. 101141 (2n=48, BBCC genomes) by backcrossing to the O. sativa parent followed by embryo rescue. The chromosome numbers ranged from 44 to 47 in the BC₁ progeny and from 24 to 37 in the BC₂ progeny. All F₁ hybrids were resistant to both blast and bacterial blight. One BC₁ plant was moderately susceptible to blast while the rest were resistant. Thirteen of the 16 BC₂ progeny tested were resistant to blast; 1 blast-resistant BC₂, plant 75-1, had 24 chromosomes. A 3 resistant: 1 susceptible segregation ratio, consistent with the action of a major, dominant gene, was observed in the BC₂F₂ and BC₂F₃ generations. Five of the BC₁ plants tested were resistant to bacterial blight. Ten of the 21 BC₂ progeny tested were resistant to Philippine races 2, 3, and 6 of the bacterial blight pathogen. One resistant BC₂, plant 78-1, had 24 chromosomes. The segregation of reactions of the BC₂F₂, BC₂F₃, and BC₂F₄ progenies of plant 78-1 suggested that the same or closely linked gene(s) conferred resistance to races 2, 3, 5, and 6 of the bacterial blight pathogen from the Philippines.     

Identification of agronomically favorable quantitative trait loci alleles from a dent corn inbred Dan232 using advanced backcross QTL analysis and comparison with the F<Subscript>2:3</Subscript> population in popcorn

Specific traits are an important consideration in plant breeding. In popcorn, inferior agronomic traits could be improved using dent or flint corn backcrossed with popcorn. In this study, we used advanced backcross quantitative trait locus (AB-QTL) analysis to identify trait-improving QTL alleles from a dent maize inbred Dan232, and compared the detection of QTL in the BC₂S₁ population with QTL results using F_2:3 families of the same population. Two hundred and twenty BC₂S₁ families developed from a cross between Dan232 and an elite popcorn inbred N04 were evaluated for nine plant traits in replicated field trials under two environments. Using composite interval mapping (CIM), a total of 28 significant QTL were detected, and of these, 23 (82.14%) had favorable alleles contributed by the dent corn parent Dan232. Nine QTL (32.14%) detected in the BC₂S₁ population were also located in or near the same chromosome intervals in the F_2:3 population. All of the favorable QTL alleles from Dan232 could be used in marker-assisted selection (MAS) to improve the respective plant traits in popcorn breeding. In addition, their near isogenic lines (QTL-NILs) could be obtained through selfing or another 1–2 backcross with N04. Also, N04 improved for the studied plant traits could be developed from the BC₂S₁ families used in this study. This study demonstrated that the AB-QTL method can be applied to identify favorable QTL from dent corn inbred in popcorn breeding and, once identified, the alleles could be used in marker-assisted selection to improve the respective plant traits.     

Expression of Cry1Ac in Transgenic Tobacco Plants Under the Control of a Wound-Inducible Promoter (AoPR1) Isolated from Asparagus officinalis to Control Heliothis virescens and Manduca sexta

Expression of cry1Ac gene from Bacillus thuringiensis (Bt) was evaluated under the control of a wound-inducible AoPR1 promoter from Asparagus officinalis in transgenic tobacco plants. The leaves of transgenic plants were mechanically wounded to evaluate the activity of the AoPR1 promoter in driving the expression of Cry1Ac protein at the wound site. Our results indicate that mechanical wounding of transgenic plants was effective in inducing the expression of Cry1Ac protein. As a result of this induction, the accumulated levels of Cry1Ac protein increased during 6–72 h post-wounding period. The leaves of transgenic tobacco plants were evaluated for resistance against Heliothis virescens and Manduca sexta in insect bioassays in two different ways. The detached tobacco leaves were either fed directly to the insect larvae or they were first mechanically wounded followed by a 72 h post-wounding feeding period. Complete protection of mechanically wounded leaves of transgenic plants was observed within 24 h of the bioassay. The leaves of transgenic plants fed directly (without pre-wounding) to the larvae achieved the same level of protection between 24 and 72 h of the bioassay.     

Expression of GFP and Bt transgenes in Brassica napus and hybridization with Brassica rapa

It is possible to monitor the movement of transgenes by tagging them with green fluorescent protein (GFP). In order to develop a model to study transgene flow, canola (Brassica napus cv Westar) was transformed with two GFP constructs, mGFP5er (GFP only) and pSAM 12 [GFP linked to a synthetic Bacillus thuringiensis (Bt) cry1Ac endotoxin gene]. Transformed callus sectors that fluoresced green were preferentially selected in the tissue culture process. Four independent GFP canola events and 12 events of GFP/Bt canola were regenerated through tissue culture. GFP fluorescence was macroscopically detectable throughout the entire life cycle of canola. The GFP/Bt events were insecticidal to neonate corn earworm (Helicoverpa zea) larvae and prevented herbivory damage. Fluorescence intensity at 508 nm varied between the independent transformation events, and ranged from 7.6×10⁵ to 13.8×10⁵ (counts per second) in contrast with the wild-type at 5.3×10⁵ cps. Nine GFP/Bt and three GFP events were hybridized with three wild accessions of B. rapa. The resultant hybrids fluoresced green and were insecticidal to neonate corn earworm larvae to the same degree as the transgenic canola parents. However, fluorescence intensities of the hemizygous F₁ hybrid lines were lower than the respective original homozygous canola parents. Each F₁ hybrid line was backcrossed by hand onto the B. rapa parent, and transgenic backcrosses were produced at rates ranging from 15% to 34%. These data suggest that GFP can be used as a tool to monitor transgene flow from crop species to wild relatives. Received: 11 September 2000 / Accepted: 1 February 2001     

Interspecific hybridization of cultivated rice, Oryza sativa L. with the wild rice, O. minuta Presl.

Crosses were made between four varieties (Mahsuri, Setanjung, MR84 and MR103) of Oryza sativa L. (2n=24, AA) and one accession of O. minuta (2n= 8, BBCC). The seed set obtained ranged between 9.5% and 25.1% depending on the rice variety used. By rescuing 14-day-old embryos and culturing them on 25%-strength MS medium we obtained a total of 414 F₁ hybrids. The F₁s were vigorous, tillered profusely, were perennial and male-sterile. The hybrids were triploid (ABC) with 36 chromosomes and showed irregular meiosis. The average frequency and range of chromosome associations at metaphase I or early anaphase I pollen mother cells of F₁ plants were 29.31(16–36) Is +3.32(0–10) IIs+0.016(0–1) IIIs+0.002(0–1) IVs. Upon backcrossing the original triploid hybrids and colchicine-treated hybrids to their respective recurrent parents, and further embryo rescue, 17 backcross-1 (BC₁) plants were obtained. Of all the crosses using MR84, no BC₁ plant was obtained even after pollinating 13 894 spikelets of the triploid hybrid. The BC₁s were similar in appearence to the F₁s and were male-sterile, their chromosome number ranged from 44 to 48. By backcrossing these BC₁s and nurturing them through embryo rescue, we obtained 32 BC₂ plants. Of these, however, only 18 plants grew vigorously. One of these plants has 24 chromosomes and the other 17 have chromosome numbers ranging between 30 and 37. The 24-chromosome plant was morphologically similar to the O. sativa parent and was partially fertile with a pollen and spikelet fertility of 58.8% and 12.5% respectively. All of the F₁ and BC₁ plants were found to be resistant to five Malaysian isolates (XO66, XO99, XO100, XO257 and XO319) of Xanthomonas campestris pv oryzae. Amongst the BC₂s, the reaction varied from resistant to moderately susceptible. The 24-chromosome BC₂ plant was resistant to the four isolates and moderately resistant to isolate XO100 to which the O. sativa parent was susceptible.Part of PhD thesis submitted by first author to Universiti Kebangsaan Malaysia, Bangi     

Breeding lines of the Indian mega-rice variety,MTU 1010, possessing protein kinase <Emphasis Type="Italic">OsPSTOL</Emphasis> (<Emphasis Type="Italic">Pup1</Emphasis>), show better root system architecture and higher yield in soils with low phosphorus

MTU 1010 is a high-yielding mega-variety of rice grown extensively in India. However, it does not perform well in soils with low phosphorus (P) levels. With an objective to improve MTU 1010 for tolerance to low soil P, we have transferred Pup1, a major quantitative trait locus (QTL) associated with tolerance from another mega-variety, Swarna, through marker-assisted backcross breeding (MABB). Foreground selection of the F₁ and backcross plants was performed with the co-dominant, closely linked CAPS marker, K20-2, while two flanking markers RM28011 and RM28157 were utilized for recombinant selection. At each backcross generation, positive plants were also analyzed with a set of 85 parental polymorphic SSR markers to identify the QTL-positive plants possessing maximum introgression of MTU 1010 genome. At BC₂F₁, the best backcross plant was selfed to generate BC₂F₂s. Among them, the plants homozygous for Pup1 (n?=?22) were reconfirmed using the functional marker for Pup1, viz., K46-1, and they were advanced through pedigree method of selection until BC₂F₆ generation. A total of five elite BC₂F₆ lines, possessing Pup1 and phenotypically similar to MTU 1010, were screened in the low soil P plot and normal plot (with optimum soil P levels) during wet season, 2016. All the selected lines showed better performance under low P soil with more number of productive tillers, better root system architecture, and significantly higher yield (>?390%) as compared to MTU 1010. Further, under normal soil, the lines were observed to be similar to or better than MTU 1010 for most of the agro-morphological traits and yield. This study represents the successful application of marker-assisted selection for improvement of tolerance to low soil P in a high-yielding Indian rice variety.     

Quantitative trait loci and candidate gene mapping of aluminum tolerance in diploid alfalfa

Aluminum (Al) toxicity in acid soils is a major limitation to the production of alfalfa (Medicago sativa subsp. sativa L.) in the USA. Developing Al-tolerant alfalfa cultivars is one approach to overcome this constraint. Accessions of wild diploid alfalfa (M. sativa subsp. coerulea) have been found to be a source of useful genes for Al tolerance. Previously, two genomic regions associated with Al tolerance were identified in this diploid species using restriction fragment length polymorphism (RFLP) markers and single marker analysis. This study was conducted to identify additional Al-tolerance quantitative trait loci (QTLs); to identify simple sequence repeat (SSR) markers that flank the previously identified QTLs; to map candidate genes associated with Al tolerance from other plant species; and to test for co-localization with mapped QTLs. A genetic linkage map was constructed using EST-SSR markers in a population of 130 BC₁F₁ plants derived from the cross between Al-sensitive and Al-tolerant genotypes. Three putative QTLs on linkage groups LG I, LG II and LG III, explaining 38, 16 and 27% of the phenotypic variation, respectively, were identified. Six candidate gene markers designed from Medicago truncatula ESTs that showed homology to known Al-tolerance genes identified in other plant species were placed on the QTL map. A marker designed from a candidate gene involved in malic acid release mapped near a marginally significant QTL (LOD 2.83) on LG I. The SSR markers flanking these QTLs will be useful for transferring them to cultivated alfalfa via marker-assisted selection and for pyramiding Al tolerance QTLs.