Morphophysiological Diversity and Haplotype Analysis of Saltol QTL Region in Diverse Rice Landraces for Salinity Tolerance

Rice is sensitive to salinity stress at both the seedling and reproductive stages.The present study used 145 rice genotypes comprising of 100 landraces and 45 advanced breeding lines collected from different regions of India.These genotypes were evaluated in hydroponics under control[electrical conductivity(ECe)~1.2 dS/m]and saline(ECe~10.0 dS/m)environments along with susceptible(IR29)and tolerant(FL478)checks.The stress susceptibility index for eight morphophysiological traits was estimated.Analysis of variance showed significant differences among the genotypes for all the parameters studied in control,stress and relative stress conditions.We identified 3 landraces(Kuttimanja,Tulasimog and IET-13713I)as tolerant and 14 lines as moderately tolerant to salt stress.Strong correlations in the morphological(root and shoot lengths)and physiological traits(shoot Na^(+),Ca^(2+)and Mg^(2+)contents,and Na^(+)/K^(+)ratio)were observed under all the conditions.The hierarchical cluster analysis grouped the genotypes into five clusters,among which cluster Ⅱ comprised salt-tolerant lines.Haplotyping of Saltol region using 11 simple sequence repeat markers on 17 saline tolerant and moderately tolerant lines was conducted.Markers AP3206F,RM10793 and RM3412b,located close to SKC1 gene(11.23‒12.55 Mb),displayed diverse allelic variations and they were not related to the FL478 type.In this region,tolerant lines like Kuttimanja,IET-13713I and Tulasimog have new alleles.As a result,these lines may be suitable candidates for novel genomic regions governing rice salinity tolerance.Salt-tolerance ability of Kuttimanja,Tulasimog and IET-13713I was validated in two years in three salinity stress environments.These promising lines can be used in breeding programs to broaden the genetic base of salinity tolerance in rice,and it may help to dissect key genomic regions responsible for salinity tolerance.

Development of a set of SSR markers for genetic polymorphism detection and interspecific hybrid jute breeding

Corchorus capsularis(white jute) and C. olitorius(dark jute) are the two principal cultivated spedes of jute that produce natural bast fiber of commercial importance. We have identified 4509 simple sequence repeat(SSR) loci from 34,163 unigene sequences of C. capsularis to develop a non-redundant set of 2079 flanking primer pairs. Among the SSRs, trinudeotide repeats were most frequent(60%) followed by dinucleotide repeats(37.6%). Annotation of the SSR-containing unigenes revealed their putative functions in various biological and molecular processes, including responses to biotic and abiotic signals. Eighteen expressed gene-derived SSR(eSSR) markers were successfully mapped to the existing single-nucleotide polymorphism(SNP) linkage map of jute,providing additional anchor points. Amplification of 72% of the 74 randomly selected primer pairs was successful in a panel of 24 jute accessions, comprising five and twelve accessions of C.capsularis and C. olitorius, respectively, and seven wild jute spedes. Forty-three primer pairs produced an average of 2.7 alleles and 58.1% polymorphism in a panel of 24 jute accessions. The mean PIC value was 0.34 but some markers showed PIC values higher than 0.5, suggesting that these markers can efficiently measure genetic diversity and serve for mapping of quantitative trait loci(QTLs) in jute. A primer polymorphism survey with parents of a wide-hybridized population between a cultivated jute and its wild relative revealed their efficacy for interspecific hybrid identificatioii. For ready accessibility of jute eSSR primers, we compiled all information in a user-friendly web database, JuteMarkerdb(http://jutemarkerdb.icar.gov.in/) for the first time in jute.This eSSR resource in jute is expected to be of use in characterization of germplasm, interspecific hybrid and variety identification, and marker-assisted breeding of superior-quality jute.

Haplotyping of Rice Genotypes Using Simple Sequence Repeat Markers Associated with Salt Tolerance

Salt stress is a major problem in most of the rice growing areas in the world. A major QTLSaltol associated with salt tolerance at the seedling stage has been mapped on chromosome 1 in rice.This study aimed to characterize the haplotype diversity at Saltol and additional QTLs associated withsalt tolerance. Salt tolerance at the seedling stage was assessed in 54 rice genotypes in the scale of 1to 9 score at EC = 10 dSm^-1 under controlled environmental conditions. Seven new breeding linesincluding three KMR3/O. rufipogon introgression lines showed similar salt tolerant ability as FL478 andcan be good sources of new genes/alleles for salt tolerance. Simple sequence repeat （SSR） markerRM289 showed only two alleles and RM8094 showed seven alleles. Polymorphic information contentvalue varied from 0.55 for RM289 to 0.99 for RM8094 and RM493. Based on 14 SSR markers, the 54lines were clearly separated into two major clusters. Fourteen haplotypes were identified based onSaltol linked markers with FL478 as the reference. Alleles of RM8094 and RM3412 can discriminatebetween the salt tolerant and susceptible genotypes clearly and hence can be useful in marker-assistedselection at the seedling stage. Other markers RM10720 on chromosome 1 and RM149 and RM264 onchromosome 8 can also distinguish tolerant and susceptible lines but with lesser stringency.

Draft genome sequence of a less-known wild Vigna: Beach pea(V. marina cv. ANBp-14-03)

Beach pea or beach cowpea(Vigna marina(Burm.)Merr.)belongs to the family Fabaceae.It is a close relative of cultivated Vigna species such as adzuki bean(V.angularis),cowpea(V.unguiculata),mung bean(V.radiata),and blackgram(V.mungo),and is distributed throughout the tropics.With its ability to tolerate salt stress,beach pea has great potential to contribute salt-tolerance genes for developing salt-tolerant cultivars in cultivated Vigna species.However,it is still underutilized in Vigna breeding programs.A draft genome sequence of beach pea was generated using a high-throughput next-generation sequencing platform,yielding 23.7 Gb of sequence from 79,929,868 filtered reads.A de novo genome assembly containing 68,731 scaffolds gave an N50 length of 10,272 bp and the assembled sequences totaled 365.6 Mb.A total of 35,448 SSRs,including 3574 compound SSRs,were identified and primer pairs for most of these SSRs were designed.Genome analysis identified 50,670 genes with mean coding sequence length 1042 bp.Phylogenetic analysis revealed highest sequence similarity with V.angularis,followed by V.radiata.Comparison with the V.angularis genome revealed 16,699 SNPs and 2253 InDels and comparison with the V.radiata genome revealed 17,538 SNPs and 2300 InDels.To our knowledge this is the first draft genome sequence of beach pea derived from an accession(ANBp-14-03)adapted locally in the Andaman and Nicobar Islands of India.The draft genome sequence may facilitate the genetic enhancement in cultivated Vigna species.

Influence of Bavistin and Silver Thiosulphate on <i>in Vitro</i>Regeneration of <i>Asclepias curassavica</i>(L). Using Nodal Explants

The effect of bavistin and the ethylene inhibitor (silver thiosulphate) on shoot regeneration using nodal explants of Asclepias curassavica (L). has been investigated. Among the different concentrations studied, highest number of shoots was obtained on MS media with 200 mg/L bavistin. Among the varying concentrations (10-100 μM/L) of silver thio-sulphate tested, highest number of shoots was obtained on MS medium amended with 60 μM/L silver thiosulphate without growth regulators. This study also establishes the stronger cytokinin like activity of bavistin. Effect of different growth additives like coconut milk, ascorbic acid and casein hydrolysate were tested on direct shoot regeneration. Among the different growth additives tested casein hydrolysate showed better and reproducible result at 0.025% in combination with 3 mg/l KN + 0.5 mg/l NAA. Antioxidants, activated charcoals and polyvinyl pyrrolidone were used to remove phenolics. Activated charcoal removed the phenolic exudates completely at 0.025% and prevented the browning of media and thus enhanced the frequency of regeneration (85%). The microshoots developed through in vitro regeneration were transferred to rooting media containing IBA alone and in combination with KN and the highest number of roots was observed on MS medium with IBA 1 mg/L + 0.2 mg/L KN.

Identification,development,and application of cross-species intron-spanning markers in lentil(Lens culinaris Medik.)

Lentil(Lens culinaris Medik) is one of the most important food legumes in the world. The use in lentil of molecular marker-assisted breeding is limited, owing to the low availability of polymorphic markers. In the present study, we developed a set of polymorphic intron-spanning markers(ISMs) using a cross-species mapping approach. In this approach, putative unique transcripts(PUTs) of L. culinaris were mapped onto the Medicago truncatula genome, exploiting its closeness with the lentil genome. Spliced alignment of the PUTs resulted in a total of 25,717 alignments, allowing the development of 1703 ISMs. From these, a subset of 105 ISMs were synthesized and validated with a 51% amplification success rate in 32 lentil genotypes. Of these ISMs, 40(74%) were polymorphic and generated 2–11 alleles per locus in a genetically diverse panel of 32 lentil genotypes including wild species.This set of polymorphic ISMs along with their functional annotation data will be useful in lentil breeding.

Genetic Dissection of Grain Size Traits Through Genome-Wide Association Study Based on Genic Markers in Rice

Grain size plays a significant role in rice,starting from affecting yield to consumer preference,which is the driving force for deep investigation and improvement of grain size characters.Quantitative inheritance makes these traits complex to breed on account of several alleles contributing to the complete trait expression.We employed genome-wide association study in an association panel of 88 rice genotypes using 142 new candidate gene based SSR(cgSSR)markers,derived from yield-related candidate genes,with the efficient mixed-model association coupled mixed linear model for dissecting complete genetic control of grain size traits.A total of 10 significant associations were identified for four grain size-related characters(grain weight,grain length,grain width,and length-width ratio).Among the identified associations,seven marker trait associations explain more than 10%of the phenotypic variation,indicating major putative QTLs for respective traits.The allelic variations at genes OsBC1L4,SHO1 and OsD2 showed association between 1000-grain weight and grain width,1000-grain weight and grain length,and grain width and length-width ratio,respectively.The cgSSR markers,associated with corresponding traits,can be utilized for direct allelic selection,while other significantly associated cgSSRs may be utilized for allelic accumulation in the breeding programs or grain size improvement.The new cgSSR markers associated with grain size related characters have a significant impact on practical plant breeding to increase the number of causative alleles for these traits through marker aided rice breeding programs.

Development of Permanent Mapping Populations RILs in Diploid A Genome

Recombinant inbred lines(RILs) serve as powerful tools for genetic mapping.RILs are obtained by crossing two inbred lines followed by repeated selfing or sib-mating to create a set of new

Comparative Analysis of Structure and Sequences of <i>Oryza sativa</i>Superoxide Dismutase

One of the major classes of antioxidant enzymes, which protect the cellular and subcellular components against harmful reactive oxygen species (ROS), is superoxide dismutase (SOD). SODs play pivotal role in scavenging highly reactive free oxygen radicals and protecting cells from toxic effects. In Oryza sativa three types of SODs are available based on their metal content viz. Cu-Zn SOD, Mn SOD and Fe SOD. In the present study attempts were made to critically assess the structure and phylogenetic relationship among Oryza sativa SODs. The sequence similarity search using local BLAST shows that Mn SODs and Fe SODs have greater degree of similarity compared with that of Cu-Zn SODs. The multiple alignment reveals that seven amino acids were found to be totally conserved. The secondary structure shows that Mn SODs and Fe SODs have similar helixes, sheets, turns and coils compared with that of Cu-Zn SODs. The comparative analysis also displayed greater resemblance in primary, secondary and tertiary structures of Fe SODs and Mn SODs. Comparison between the structure and sequence analysis reveals that Mn SOD and Fe SOD are found to be closely related whereas Cu-Zn SOD evolves independently.

Comparative Transmission Genetics of Introgressed Chromatin in Gossypium Species

Wild relatives and un-adapted germplasm of crop species harbors several adaptive traits.Introgression of genes for economic and adaptive traits brings in novel genetic variation into the gene

Carboxylesterase and Glutathione-S-Transferase （GST＇s） Induced Resistance to Bacillus thuringiensis Toxin CrylAb in Rice Leaf Folder, Cnaphalocrocis medinalis （Guenee） Populations

The rice leaf folder （RLF）, Cnaphalocrocis medinalis （Guenee） （lnsecta： Lepidoptera： Pyralidae）, is an important pest, widely distributed in many rice growing areas of Asia. The over-use of broad-spectrum chemical insecticides has been cited as a major cause of outbreaks of C. medinalis as excessive spraying of insecticide disrupts natural biological control insecticides still remain the major control tactics against leaf folder. Carbofuran and fenthion, bendiocarb, acephate, carbosulfan, quinolphos, monocrotophos, phosphamidon and fenvalerate are the common ones used against rice leaf folder. Genetically, modified rice lines expressing B. thuringiensis insecticidal crystal proteins produced are highly tolerant to leidopteran pests. Though economic and environmental benefits of GM crops is well established, the matter of concern is the possibility of target insect pest developing resistance to this B. thuringiensis insecticidal toxins, evident from many laboratory and field experiments against many insect pests. The involvement of GSH S-transferase, carboxylesterase, and microsomal monooxygenase in insecticide resistance has been reported in insecticide-resistant strains of many insect species. Hence, the present study was taken up to monitor for cross resistance between B. thuringiensis cry toxins and synthetic insecticides in larvae of leaf folder as it is mediated by carboxylesterase titre and other enzymes by bioassay for two selected rice leaf folder field populations at the Entomology division of Directorate of Rice Research which showed 2-fold resistance ratio. Qualitative and quantitative changes of carboxylesterase （CarE） and glutathione-s-transferase （GST＇s） were worked out with midguts extracts of the two C. medinalis populations in the presence of a-napthyl acetate and chlorodi-nitro benzene substrates.