Showing papers in "Molecular Breeding in 2008"

Assessment of genetic diversity and relationship among a collection of US sweet sorghum germplasm by SSR markers

Abstract: Sweet sorghum (Sorghum bicolor L.) is a type of cultivated sorghums and has been recognized widely as potential alternative source of bio-fuel because of its high fermentable sugar content in the stalk. A substantial variation of sugar content and related traits is known to exist in US sweet sorghum. The objectives of the study were to assess the genetic diversity and relationship among the US sweet sorghum cultivars and lines using SSR markers and to examine the genetic variability within sweet sorghum accessions for sugar content. Sixty-eight sweet sorghum and four grain sorghum cultivars and lines were genotyped with 41 SSR markers that generated 132 alleles with an average of 3.22 alleles per locus. Polymorphism information content (PIC) value, a measure of gene diversity, was 0.40 with a range of 0.03-0.87. The genetic similarity co- efficient was estimated based on the segregation of the 132 SSR alleles. Clustering analysis based on the genetic similarity (GS) grouped the 72 sorghum accessions into 10 distinct clusters. Grouping based on clustering analysis was in good agreement with available pedigree and genetic background informa- tion. The study has revealed the genetic relationship of cultivars with unknown parentage to those with known parentage. A number of diverse pairs of sweet sorghum accessions were identified which were polymorphic at many SSR loci and significantly different for sugar content as well. Information generated from this study can be used to select parents for hybrid development to maximize the sugar content and total biomass, and development of segregating populations to map genes controlling sugar content in sweet sorghum.

Discovery of a new fragrance allele and the development of functional markers for the breeding of fragrant rice varieties

Abstract: The recessive fgr gene on chromosome 8 is associated with rice fragrance. It has been reported that this gene is a non-functional badh2 allele and that the functional Badh2 allele encoding putative betaine aldehyde dehydrogenase (BADH2) could render rice non-fragrant. Here we report the discovery of a new badh2 allele and the development of functional markers for the badh2 locus. A total of 24 fragrant and ten non-fragrant rice varieties were studied and sequenced for their Badh2/badh2 loci. Of the 24 fragrant rice varieties, 12 were found to have the known badh2 allele (badh2-E7), which has an 8-bp deletion and three single nucleotide polymorphisms (SNPs) in exon 7; the others had a novel null badh2 allele (badh2-E2), which has a sequence identical to that of the Badh2 allele in exon 7, but with a 7-bp deletion in exon 2. Both null badh2 alleles are responsible for rice fragrance. Based on sequence divergence amongst the functional Badh2 and two null badh2 alleles, we developed functional markers which can be easily used to distinguish non-fragrant from fragrant rice and to differentiate between two kinds of fragrant rice. These functional markers will find their usefulness in breeding for fragrant rice varieties via marker-assisted selection.

Identification of genes involved in the response of leaves of Phaseolus vulgaris to drought stress

Abstract: Common bean (Phaseolus vulgaris L.) is an important crop from the Fabaceae family that is cultivated worldwide for human consumption. It is also widely exposed to drought. The identification of genes whose expression is altered under conditions of drought is an important first step towards understanding the response of this species. Such a study has recently been published on roots of P. vulgaris (Torres et al. 2006, Plant Sci 171:300–307) and a complementary study is here reported on leaves. 19-day-old plants of 8 cultivars grown in a growth chamber and 21-day-old plants of four of these cultivars grown in a greenhouse under conditions nearer to those in the field, were subjected to progressive water withdrawal. Changes in gene expression in their leaves at different levels of dehydration were identified by differential display reverse transcriptase PCR and confirmed by quantitative real-time PCR. The levels of 15 transcripts were changed significantly (P < 0.05) in all cultivars under both growth conditions, 8 being increased and seven decreased. All these transcripts were related by blastp search to known plant proteins. At least six transcripts corresponded to those whose expression is altered under drought in Arabidopsis thaliana. Five of the genes identified have not previously been reported as being influenced by drought. The identified genes were different from those influenced by drought in roots of P. vulgaris. Only minor differences in expression of these 15 genes were found between the different cultivars.

Identification of QTL for sugar-related traits in a sweet × grain sorghum (Sorghum bicolor L. Moench) recombinant inbred population

Abstract: QTL for stem sugar-related and other agronomic traits were identified in a converted sweet (R9188) × grain (R9403463-2-1) sorghum population. QTL analyses were conducted using phenotypic data for 11 traits measured in two field experiments and a genetic map comprising 228 SSR and AFLP markers grouped into 16 linkage groups, of which 11 could be assigned to the 10 sorghum chromosomes (SBI-01 to SBI-10). QTL were identified for all traits and were generally co-located to five locations (SBI-01, SBI-03, SBI-05, SBI-06 and SBI-10). QTL alleles from R9188 were detected for increased sucrose content and sugar content on SBI-01, SBI-05 and SBI-06. R9188 also contributed QTL alleles for increased Brix on SBI-05 and SBI-06, and increased sugar content on SBI-03. QTL alleles from R9403463-2-1 were found for increased sucrose content and sucrose yield on SBI-10, and increased glucose content on SBI-07. QTL alleles for increased height, later flowering and greater total dry matter yield were located on SBI-01 of R9403463-2-1, and SBI-06 of R9188. QTL alleles for increased grain yield from both R9403463-2-1 and R9188 were found on SBI-03. As an increase in stem sugars is an important objective in sweet sorghum breeding, the QTL identified in this study could be further investigated for use in marker-assisted selection of sweet sorghum.

Application of multiplex-ready PCR for fluorescence-based SSR genotyping in barley and wheat

Abstract: Microsatellites (SSRs) are widely used in cereal research, and their use in marker assisted breeding has increased the speed and efficiency of germplasm improvement. Central to the application of SSRs for many purposes are methodologies enabling the low-cost acquisition of large quantities of genetic information for gene and genotype identification. In this study, multiplex-ready PCR was evaluated in barley and bread wheat as an approach for rapid and more automated SSR genotyping on a fluorescence-based DNA fragment analyzer. Multiplex-ready PCR is a method that allows SSR genotyping to be performed using a standardized protocol. The method enables flexible fluorescence labeling of SSRs, generates a relatively constant amount of PCR product for each marker, and has a high amenability to multiplex PCR (the simultaneous amplification of several SSRs in the same reaction). A high (92%) compatibility of published SSRs with multiplex-ready PCR is demonstrated, and the usefulness of the method for large scale genotyping is shown by its application for whole genome marker assisted breeding in barley. A database of more than 2,800 barley and wheat SSRs, and a suite of bio-informatic tools were developed to support the deployment of multiplex-ready PCR for various genetic applications, and are accessible at http://www.genica.net.au. Multiplex-ready PCR is broadly applicable to cereal genomics research and marker assisted breeding, and should be transferable to similar analyses of any animal or plant species.

An integrated DArT-SSR linkage map of durum wheat

Abstract: Genetic mapping in durum wheat (Triticum durum Desf.) is constrained by its large genome and allopolyploid nature. We developed a Diversity Arrays Technology (DArT) platform for durum wheat to enable efficient and cost-effective mapping and molecular breeding applications. Genomic representations from 56 durum accessions were used to assemble a DArT genotyping microarray. Microsatellite (SSR) and DArT markers were mapped on a durum wheat recombinant inbred population (176 lines). The integrated DArT-SSR map included 554 loci (162 SSRs and 392 DArT markers) and spanned 2022 cM (5 cM/marker on average). The DArT markers from durum wheat were positioned in respect to anchor SSRs and hexaploid wheat DArT markers. DArT markers compared favourably to SSRs to evaluate genetic relationships among the durum panel, with 1315 DArT polymorphisms found across the accessions. Combining DArT and SSR platforms provides an efficient and rapid method of generating linkage maps in durum wheat.

Whole genome mapping in a wheat doubled haploid population using SSRs and TRAPs and the identification of QTL for agronomic traits

Abstract: Genetic maps are useful for detecting quantitative trait loci (QTL) associated with quantitative traits and for marker-assisted selection (MAS) in breeding. In this research, we used the wheat × maize method to develop a doubled haploid (DH) population derived from the synthetic hexaploid wheat (SHW) line TA4152-60 and the North Dakota hard red spring wheat line ND495. The population consisted of 213 lines, of which a subset of 120 lines was randomly selected and used to construct linkage maps of all 21 chromosomes and for QTL detection. The whole genome maps consisted of 632 markers including 410 SSRs, 218 TRAPs, 1 RFLP, and 3 phenotypic markers, and spanned 3,811.5 cM with an average density of one marker per 6.03 cM. Telomere sequence-based TRAPs allowed us to define the ends of seven linkage groups. Analysis revealed major QTLs associated with the traits of days to heading on chromosomes 5A and 5B, plant height on chromosomes 4D and 5A, and spike characteristics on chromosomes 3D, 4A, 4D, 5A and 5B. The DH population and genetic map will be a useful tool for the identification of disease resistance QTL and agronomically important loci, and will aid in the identification and development of markers for MAS.

Inheritance of Fusarium wilt resistance introgressed from Solanum aethiopicum Gilo and Aculeatum groups into cultivated eggplant ( S. melongena ) and development of associated PCR-based markers

Abstract: The two eggplant relatives Solanum aethiopicum gr. Gilo and Solanum aethiopicum gr. Aculeatum (=Solanum integrifolium) carry resistance to the fungal wilt disease caused by Fusarium oxysporum f. sp. melongenae, a worldwide soil-borne disease of eggplant. To introgress the resistance trait into cultivated eggplant, the tetraploid somatic hybrids S. melongena + S. aethiopicum and S. melongena + S. integrifolium were used. An inheritance study of the resistance was performed on advanced anther culture-derived androgenetic backcross progenies from the two somatic hybrids. The segregation fitted a 3 resistant (R): 1 susceptible (S) ratio in the selfed populations and a 1R:1S ratio in the backcross progenies for the trait derived from S. aethiopicum and S. integrifolium. These ratios are consistent with a single gene, which we designated as Rfo-sa1, controlling the resistance to Fusarium oxysporum f. sp. melongenae. The allelic relationship between the resistance genes from S. aethiopicum and S. integrifolium indicate that these two genes are alleles of the same locus. Bulked Segregant Analysis (BSA) was performed with RAPD markers on the BC3/BC5 resistant advanced backcross progenies, and three RAPD markers associated with the resistance trait were identified. Cleaved Amplified Polymorphic Sequences (CAPSs) were subsequently obtained on the basis of the amplicon sequences. The evaluation of the efficiency of these markers in predicting the resistant phenotype in segregating progenies revealed that they represent useful tools for indirect selection of Fusarium resistance in eggplant.

A major QTL controlling seed dormancy and pre-harvest sprouting resistance on chromosome 4A in a Chinese wheat landrace

Abstract: Wheat pre-harvest sprouting (PHS) can cause significant reduction in yield and end-use quality of wheat grains in many wheat-growing areas worldwide To identify a quantitative trait locus (QTL) for PHS resistance in wheat, seed dormancy and sprouting of matured spikes were investigated in a population of 162 recombinant inbred lines (RILs) derived from a cross between the white PHS-resistant Chinese landrace Totoumai A and the white PHS-susceptible cultivar Siyang 936 Following screening of 1,125 SSR primers, 236 were found to be polymorphic between parents, and were used to screen the mapping population Both seed dormancy and PHS of matured spikes were evaluated by the percentage of germinated kernels under controlled moist conditions Twelve SSR markers associated with both PHS and seed dormancy were located on the long arm of chromosome 4A One QTL for both seed dormancy and PHS resistance was detected on chromosome 4AL Two SSR markers, Xbarc 170 and Xgwm 397, are 914 cM apart, and flanked the QTL that explained 283% of the phenotypic variation for seed dormancy and 306% for PHS resistance This QTL most likely contributed to both long seed dormancy period and enhanced PHS resistance Therefore, this QTL is most likely responsible for both seed dormancy and PHS resistance The SSR markers linked to the QTL can be used for marker-assisted selection of PHS-resistant white wheat cultivars

Use of doubled haploids in maize breeding: implications for intellectual property protection and genetic diversity in hybrid crops

Abstract: Through the concerted use of doubled haploidy (DH), molecular markers and off-season nurseries, maize (Zea mays L.) breeders have unprecedented capabilities to quickly and precisely create progeny with desired levels of similarity to either parents of a commercial hybrid. Genotypic data from both simulated and from actual populations created either by single seed descent or through doubled haploidy were examined for the initial and subsequent generations. Simulation data showed that DH progeny inherited larger blocks of parental chromosomes; approximately seven out of 10 chromosomes had intact segments of 50% or greater. By the third DH generation progeny can be selected that are more than 90% similar to either parent of the initial commercial hybrid. Actual marker data from the initial DH generation showed a maximum parental contribution of 88.4% compared to 78.7% for progeny developed by single seed descent (SSD). The number of intact chromosomes was higher among DH progeny than among progeny bred by SSD. Use of DH facilitates access to germplasm that is already present in commercial maize hybrids. Available technologies coupled with the intellectual property protection regime will influence decisions made by plant breeders in the balance of exotic compared to well-adapted germplasm they choose to access for further cycles.

Development of a genetic linkage map and identification of homologous linkage groups in sweetpotato using multiple-dose AFLP markers

Abstract: Sweetpotato genomic research is minimal compared to most other major crops despite its worldwide importance as a food crop. The development of a genetic linkage map in sweetpotato will provide valuable information about the genomic organization of this important species that can be used by breeders to accelerate the introgression of desired traits into breeding lines. We developed a mapping population consisting of 240 individuals of a cross between ‘Tanzania’, a cream-fleshed African landrace, and ‘Beauregard’, an orange-fleshed US sweetpotato cultivar. The genetic linkage map of this population was constructed using Amplified Fragment Length Polymorphism (AFLP) markers. A total of 1944 (‘Tanzania’) and 1751 (‘Beauregard’) AFLP markers, of which 1511 and 1303 were single-dose markers respectively, were scored. Framework maps consisting of 86 and 90 linkage groups for ‘Tanzania’ and ‘Beauregard’ respectively, were developed using a combination of JoinMap 3.0 and MAPMAKER/EXP 3.0. A total of 947 single-dose markers were placed in the final framework linkage map for ‘Tanzania’. The linkage map size was estimated as 5792 cM, with an average distance between markers of 4.5 cM. A total of 726 single-dose markers were placed in the final framework map for ‘Beauregard’. The linkage map length was estimated as 5276 cM, with an average distance between markers of 4.8 cM. Duplex and triple-dose markers were used to identify the corresponding homologous groups in the maps. Our research supports the hypothesis that sweetpotato is an autopolyploid. Distorted segregation in some markers of different dosages in this study suggests that some preferential pairing occurs in sweetpotato. However, strict allopolyploid inheritance in sweetpotato can be ruled out due to the observed segregation ratios of the markers, and the proportion of simplex to multiple-dose markers.

Marker-assisted selection for white mold resistance in common bean

Abstract: A marker-assisted selection (MAS) study was conducted on two recombinant inbred line (RIL) populations of common bean (Phaseolus vulgaris) to test the effectiveness of MAS for resistance to white mold (Sclerotinia sclerotiorum). Markers for quantitative trait loci (QTL) on linkage groups B2 and B7 that were previously associated with resistance and plant architectural avoidance traits in the resistant parent Bunsi were chosen. In the Bunsi/Midland population 10 RILs included in the MAS selected group developed significantly less disease than the control group based on two years of field evaluation under white mold pressure. Growth habit had no significant effect on disease severity or incidence. In the Bunsi/Raven RIL population, disease scores in the MAS selected group were significantly lower than scores in the control group over two years. Additional progress in enhancing resistance to white mold was detected when yield and plant architecture were included in the selection process. Lower disease scores among RILs were observed when comparisons were made to RILs selected using MAS alone. Yield is an important trait that should be considered when selecting for resistance to white mold. Finally the potential of Bunsi as a genetic donor of QTL for white mold resistance was confirmed in both populations studied. This study supported the effectiveness of MAS to enhance selection for a complexly inherited trait such as resistance to white mold in common bean.

QTL mapping of resistance to Fusarium ear rot using a RIL population in maize

Abstract: Fusarium ear rot is a prevalent disease in maize, reducing grain yields and quality. Resistance breeding is an efficient way to minimize losses caused by the disease. In this study, 187 lines from a RIL population along with the resistant (87-1) and susceptible (Zong 3) parents were planted in Zhengzhou and Beijing with three replications in years 2004 and 2006. Each line was artificially inoculated using the nail-punch method. Significant genotypic variation in response to Fusarium ear rot was detected in both years. Based on a genetic map containing 246 polymorphic SSR markers with average genetic distances of 9.1 cM, the ear-rot resistance QTL were firstly analyzed by composite interval mapping (CIM). Three QTL were detected in both Zhengzhou and Beijing in 2004; and three and four QTL, respectively, were identified in 2006. The resistant parent contributed all resistance QTL. By using composite interval mapping and a mixed model (MCIM), significant epistatic effects on Fusarium ear rot as well as interactions between mapped loci and environments were observed across environments. Two QTL on chromosome 3 (3.04 bin) were consistently identified across all environments by the two methods. The major resistant QTL with the largest effect was flanked by markers umc1025 and umc1742 on chromosome 3 (3.04 bin), explaining 13–22% of the phenotypic variation. The SSR markers closely flanking the major resistance QTL will facilitate marker-assisted selection (MAS) of resistance to Fusarium ear rot in maize breeding programs.

Dissecting of the FHB resistance QTL on the short arm of wheat chromosome 2D using a comparative genomic approach: from QTL to candidate gene

Abstract: Colinearity in gene content and order between rice and closely related cereal crops has been a powerful tool for gene identification. Using a comparative genomic approach, we have identified the rice genomic region syntenous to the region of the short arm of wheat chromosome 2D, on which quantitative trait loci (QTLs) for Fusarium head blight (FHB) resistance and for controlling accumulation of the mycotoxin deoxynivalenol (DON) are closely located. Utilizing markers known to reside near the FHB resistance QTL and data from several wheat genetic maps, we have limited the syntenous region to 6.8 Mb of the short arm of rice chromosome 4. From the 6.8-Mb sequence of rice chromosome 4, we found three putative rice genes that could have a role in detoxification of mycotoxins. DNA sequences of these putative rice genes were used in BLAST searches to identify wheat expressed sequence tags (ESTs) exhibiting significant similarity. Combined data from expression analysis and gene mapping of wheat homologues and results of analysis of DON accumulation using doubled haploid populations revealed that a putative gene for multidrug resistance-associated protein (MRP) is a possible candidate for the FHB resistance and/or DON accumulation controlling QTLs on wheat chromosome 2DS and can be used as a molecular marker to eliminate the susceptible allele when the Chinese wheat variety Sumai 3 is used as a resistance source.

Development of a seed DNA-based genotyping system for marker-assisted selection in maize

Abstract: Leaf collection from the field, labeling and tracking back to the source plants after genotyp- ing are rate limiting steps in leaf DNA-based genotyping. In this study, an optimized genotyping method using endosperm DNA sampled from single maize seeds was developed, which can be used to replace leaf DNA-based genotyping for both genetic studies and breeding applications. A similar approach is likely to be suitable for all plants with relatively large seeds. Part of the endosperm was excised from imbibed maize seeds and DNA extracted in 96-tube plates using individuals from eight F2 populations and seven inbreds. The quality of the resultant DNA was functionally comparable to DNA extracted from leaf tissue. Extraction from 30 mg of endosperm yields 3-10 lg DNA, which is sufficient for analysis of 200-400 agarose-gel PCR-based markers, with the potential for several million chip-based SNP marker analyses. By comparing endosperm DNA and leaf DNA for individuals from an F2 population, geno- typing errors caused by pericarp contamination and hetero-fertilization were found to average 3.8 and 0.6%, respectively. Endosperm sampling did not affect germination rates under controlled conditions, although under normal field conditions the germina- tion rate, seedling establishment, and growth vigor were significantly lower than that of non-sampled controls for some genotypes. However, careful field management can compensate for these effects. Seed DNA-based genotyping lowered costs by 24.6% compared to leaf DNA-based genotyping due to reduced field plantings and labor costs. A substantial advantage of this approach is that it can be used to select desirable genotypes before planting. As such it provides an opportunity for dramatic improvements in the efficiency and selective gain of breeding systems based on optimum combinations of marker- assisted selection and phenotypic selection within and between generations.

Colinearity between the barley grain protein content (GPC) QTL on chromosome arm 6HS and the wheat Gpc-B1 region

Abstract: Grain Protein Content (GPC) is an important determinant of grain quality in many crops, including barley and wheat. Recently, the map-based cloning of Gpc-B1, a wheat GPC quantitative trait locus (QTL), revealed a NAC transcription factor (TtNAM-B1) that was associated with increased grain protein, zinc, and iron content. In barley, a QTL for GPC was identified in a segregating population developed from a cross between ‘Karl’ (low GPC) and ‘Lewis’ (average GPC). This QTL was mapped near marker hvm74 on chromosome 6H and was suggested as a potential orthologue for Gpc-B1 on chromosome arm 6BS. In the current study, wheat genes that were previously mapped within a 0.8 cM segment spanning the TtNAM-B1 gene were converted into barley molecular markers. These new markers, together with the barley TtNAM-B1 orthologous gene (designated HvNAM-1 hereafter) were mapped on a 0.7 cM interval encompassing the peak of the barley QTL for GPC on chromosome arm 6HS. Sequence comparison of HvNAM-1 parental alleles showed two single nucleotide polymorphisms (SNPs) that result in two amino acid differences. Analysis of the allelic variation in a wild and cultivated barley collection revealed that the Karl haplotype was present only in nine out of 147 tested accessions. The colinearity between the wheat and barley GPC regions and the low frequency of the HvNAM-1 haplotype associated with low GPC suggest that the barley NAC transcription factor is responsible for the GPC QTL on barley chromosome 6H.

AB-QTL analysis in spring barley: III. Identification of exotic alleles for the improvement of malting quality in spring barley ( H. vulgare ssp. spontaneum )

Abstract: Malting quality is genetically determined by the complex interaction of numerous traits which are expressed prior to and, in particular, during the malting process. Here, we applied the advanced backcross quantitative trait locus (AB-QTL) strategy (Tanksley and Nelson, Theor Appl Genet 92:191–203, 1996), to detect QTLs for malting quality traits and, in addition, to identify favourable exotic alleles for the improvement of malting quality. For this, the BC2DH population S42 was generated from a cross between the spring barley cultivar Scarlett and the wild barley accession ISR42-8 (Hordeum vulgare ssp. spontaneum). A QTL analysis in S42 for seven malting parameters measured in two different environments yielded 48 QTLs. The exotic genotype improved the trait performance at 18 (37.5%) of 48 QTLs. These favourable exotic alleles were detected, in particular, on the chromosome arms 3HL, 4HS, 4HL and 6HL. The exotic allele on 4HL, for example, improved α-amylase activity by 16.3%, fermentability by 0.8% and reduced raw protein by 2.4%. On chromosome 6HL, the exotic allele increased α-amylase by 16.0%, fermentability by 1.3%, friability by 7.3% and reduced viscosity by 2.9%. Favourable transgressive segregation, i.e. S42 lines exhibiting significantly better performance than the recurrent parent Scarlett, was recorded for four traits. For α-amylase, fermentability, fine-grind extract and VZ45 20, 16, 2 and 26 S42 lines, respectively, surpassed the recurrent parent Scarlett. The present study hence demonstrates that wild barley does harbour valuable alleles, which can enrich the genetic basis of cultivated barley and improve malting quality traits.

Mapping and validation of a major QTL for yellow pigment content on 7AL in durum wheat (Triticum turgidum L. ssp. durum)

Abstract: Yellow pigment content in durum wheat (Triticum turgidum L. ssp. durum) is an important criterion for both pasta bright yellow color and human health because of antioxidant properties of carotenoids involved in this pigmentation. In the present study, QTLs for yellow pigment content in durum wheat were mapped in a population of 140 RILs developed from a intraspecific cross between a released variety (PDW 233) and a landrace (Bhalegaon 4). This trait was evaluated in one location for 3 years and in two more locations for one additional year (five different year × location combinations further called “environments”). Yellow pigment content was highly heritable across the five different environments. Analysis of variance showed the significant effect of genotype, environment and genotype × environment interaction on the trait. Five different QTLs linked to yellow pigment content were identified on chromosome 1A, 3B, 5B, 7A and 7B across five different environments. The strongest one located on the distal part of the long arm of chromosome 7A, QYp.macs-7A, explained 55.22% of the variation in the trait, while, remaining four QTLs explained 5–8.75% of phenotypic variation in yellow pigment content. Marker analysis revealed significant association of one ISSR and one AFLP fragment with the trait. These two markers were linked to the major QTL QYp.macs-7A and were converted into SCAR markers. These SCAR markers were further validated on another population as well as 38 diverse genotypes so as to prove their potential in marker assisted selection. These markers will be very useful for the marker assisted breeding of durum wheat for higher yellow pigment content.

Identification and validation of a core set of informative genic SSR and SNP markers for assaying functional diversity in barley

Abstract: A ‘core set’ of 28 simple sequence repeat (SSR) and 28 single nucleotide polymorphism (SNP) markers for barley was developed after screening six diverse genotypes (DGs) representing six countries (Afghanistan, Pakistan, Algeria, Egypt, Jordan and Syria) with 50 SSR and 50 SNP markers derived from expressed sequence tags (ESTs). The markers of the core set are single locus with very high quality amplifications, high polymorphism information content (PIC) and are distributed across the barley genome. PIC values for the selected SSR and SNP markers ranged between 0.32–0.72 (average 0.58) and 0.28–0.50 (average 0.42), respectively. To make the SNP genotyping cost effective, CAPS (cleaved amplified polymorphic sequence) and indel assays were developed for 23 markers and the remaining 5 SNP markers were optimized for pyrosequencing. A high coefficient of correlations (r = 0.96, P < 0.005) between the genetic similarity matrices of SSR and SNP genotyping data of the core set on diverse genotypes (DGs) and their similar groupings according to the geographical distribution in both SSR and SNP phenograms with high bootstrap values underline the utility and reliability of the core set. A comparative allelic and sequence diversity for SSR and SNP markers between the DGs and six elite parental genotypes (PGs) of mapping populations showed comparable diverse nature of two germplasm sets. However, unique SNPs and indels were observed in both germplasm sets providing more datapoints for analysing haplotypes in a better way for the corresponding SNP marker.

Mapping of quantitative trait loci for resistance to Mycosphaerella pinodes in Pisum sativum subsp. syriacum

Abstract: Aschochyta blight, caused by Mycosphaerella pinodes, is one of the most economically serious pea pathogens, particularly in winter sowings The wild Pisum sativum subsp syriacum accession P665 shows good levels of resistance to this pathogen Knowledge of the genetic factors controlling resistance to M pinodes in this wild accession would facilitate gene transfer to pea cultivars; however, previous studies mapping resistance to M pinodes in pea have never included this wild species The objective of this study was to identify quantitative trait loci (QTL) controlling resistance to M pinodes in P sativum subsp syriacum and to compare these with QTLs previously described for the same trait in P sativum A population formed by 111 F6:7 recombinant inbred lines derived from a cross between accession P665 and a susceptible pea cultivar (Messire) was analysed using morphological, isozyme, RAPD, STS and EST markers The map developed covered 1214 cM and contained 246 markers distributed in nine linkage groups, of which seven could be assigned to pea chromosomes Six QTLs associated with resistance to M pinodes were detected in linkage groups II, III, IV and V, which collectively explained between 31 and 75% of the phenotypic variation depending of the trait While QTLs MpIII1 and MpIII2 were detected both for seedlings and field resistance, MpV1 and MpII1 were specific for growth chamber conditions and MpIII3 and MpIV1 for field resistance Quantitative trait loci MpIII1, MpII1, MpIII2 and MpIII3 may coincide with other QTLs associated with resistance to M pinodes previously described in P sativum Four QTLs associated with earliness of flowering were also identified While dfIII2 and dfVI1, may correspond with other genes and QTLs controlling earliness in P sativum, dfIII1 and dfII1 may be specific to P sativum subsp syriacum Flowering date and growth habit were strongly associated with resistance to M pinodes in the field evaluations The relation observed between earliness, growth habit and resistance to M pinodes is discussed

Regulation of desaturase gene expression, changes in membrane lipid composition and freezing tolerance in potato plants

Abstract: Irreversible damage of the plant plasma membrane is the primary cause of freezing injury Changes in lipid unsaturation have being largely studied as one of the major components aimed at preserving the integrity and functionality of plasma membranes, and increasing freezing tolerance In the present study, the potato stearoyl-ACP desaturase (ω-9) gene, encoding the first enzyme involved in plant lipid unsaturation, was cloned and used to monitor its expression during cold acclimation in plants of two Solanum species, known to differ in their ability to cold acclimate Although up-regulated upon cold acclimation in both potato species, freezing tolerant S commersonii plants had a constitutive level of the ω-9 desaturase gene transcripts and of other known stress protective proteins (dhn2 and cpn60β) remarkably higher than plants of the not freezing tolerant potato species (S tuberosum) Transcript levels of oleoyl-desaturase (ω-6) and linoleyl CoA desaturase (ω-3), microsomal desaturases involved in further plasma membrane fatty acid (FA) unsaturation, did not vary appreciably during cold acclimation in both potato species S tuberosum potato plants overexpressing the ω-9 desaturase gene were generated, to change FA lipid composition and measure the effect on the basal level freezing tolerance of cultivated potato varieties Unsaturation level of total leaf polar lipids of one of best ω-9 desaturase overexpressing line, with a high transcript level of the exogenous gene and related protein, was slightly higher compared to potato plants transformed with the empty vector, with the notable appearance of cis-vaccenic acid (C18:1 Δ11), an unusual monoic FAs in plants Freezing tolerance, estimated as an increase in LT50 (2°C), derived from the electrolyte leakage test, enhanced in ω-9 desaturase overexpressing transgenic potato plants only upon cold acclimation These results indicate that modifications in lipid unsaturation account for only a small fraction of the acquired freezing tolerance, while interaction with other protective proteins (dhn2, cpn60β) is necessary to fulfil a higher level of freezing tolerance

High transformation efficiency in plum (Prunus domestica L.): a new tool for functional genomics studies in Prunus spp.

Abstract: An improved Agrobacterium-mediated transformation protocol for plum (Prunus domestica L.) hypocotyl slices was developed based on the addition of 2,4-d to the co-cultivation medium. This method increased transformation efficiency up to 10 × (42%) over previous reports with an average efficiency of 25% of hypocotyl slices producing transgenic plants. Timing of each step in the protocol was optimized producing self-rooted transgenic plants in the greenhouse in approximately 6 months. In order to test the system for its utility in functional genomic studies, we developed two hairpin constructs using a fragment of the peach (P. persica) Phytoene desaturase (PDS) gene. When A. tumefaciens with these constructs was used for targeted post-transcriptional gene silencing (PTGS), approximately 50% of the transformed plums were knockout PDS gene plants. The easy and efficient plum transformation protocol that we report here can be readily used for functional genomics studies in Prunus specifically, and Rosaceae and other woody species in general.

High-resolution mapping and gene prediction of Xanthomonas Oryzae pv. Oryzae resistance gene Xa7

Abstract: Bacterial blight (BB) caused by Xanthomonas oryzae pv. oryzae (Xoo) is a devastating disease in rice worldwide. The resistance gene Xa7, which provides dominant resistance against the pathogen with avirulence (Avr) gene AvrXa7, has proved to be durably resistant to BB. A set of SSR markers were selected from the “gramene” database based on the Xa7 gene initial mapping region on chromosome 6. These markers were used to construct a high-resolution genetic map of the chromosomal region surrounding the Xa7 gene. An F2 mapping population with 721 highly susceptible individuals derived from a cross between the near isogenic lines (NILs) IRBB7 and IR24 were constructed to localize the Xa7 gene. In a primary analysis with eleven polymorphic SSR markers, Xa7 was located in approximately the 0.28-cM region. To walk closer to the target gene, recombinant F2 individuals were tested using newly developed STMS (sequence tagged microsatellite) markers. Finally, the Xa7 gene was mapped to a 0.21-cM interval between the markers GDSSR02 and RM20593. The Xa7-linked markers were landed on the reference sequence of cv. Nipponbare through bioinformatics analysis. A contig map corresponding to the Xa7 gene was constructed. The target gene was assumed to span an interval of approximately 118.5-kb which contained a total of fourteen genes released by the TIGR Genome Annotation Version 5.0. Candidate-gene analysis of Xa7 revealed that the fourteen genes encode novel domains that have no amino acid sequence similar to other cloned Xa(xa) genes.

Genetic dissection of fruit quality components in melon (Cucumis melo L.) using a RIL population derived from exotic × elite US Western Shipping germplasm

Abstract: Growing environment dramatically influences melon (Cucumis melo L.; 2n = 2x = 24) fruit development and quality. Consequently, the characterization of quantitative trait loci (QTL) controlling melon fruit quality for application in marker-assisted selection (MAS) requires an assessment of genotype by environmental interactions, trait correlations, and QTL efficacy. Therefore, fruit quality traits [soluble solids content (SSC), mesocarp pressure (MP), fruit diameter (mesocarp + exocarp; FD), seed cavity diameter (endocarp; SCD), seed cavity to FD ratio (C:D), fruit shape (FS), and percentage of exocarp netting (PN) at time of harvest] were examined in 81 recombinant inbred lines (RIL) at two growing locations (California. and Wisconsin, USA) to identify the map position and consistency of QTL for MAS in a Group Cantalupensis U.S. Western Shipping market type background. RIL developed from a cross between U.S. Department of Agriculture line USDA-846-1 and ‘Top Mark’ were used to identify 57 QTL in both location tested (SSC = 10, MP = 8, FD = 6, SCD = 9, C:D = 8, PN = 6, and FS = 10). The QTL were distributed across 12 linkage groups and explained a significant portion of the associated phenotypic variation (R2 = 4–29%). Twelve of such QTL were consistently identified in the two locations tested [SSC (ssc7.4 and ssc10.8), MP (mp7.2, mp10.3, and mplg7.5), SCD (scd1.1, scd5.4, and scd8.5), C:D (cd2.1), and PN (pn2.1), FS (fs1.1 and fs2.3)]. The map positions of 18 QTL (FS = 7, SSC = 6, C:D = 3, SCD = 1, and PN = 1) were in equivalent (i.e., collinear) genomic regions with previous studies in Group Inodorus-based maps. Six of the collinear QTL were detected in both locations in our study (ssc7.4, ssc10.8, fs1.1, fs2.3, pn2.1, and scd5.4). The collinearity of these QTL with those identified in other maps, and their consistency across diverse growing environments portends their broad applicability in melon MAS.

Identification and validation of RAPD and SCAR markers linked to the gene Er3 conferring resistance to Erysiphe pisi DC in pea

Abstract: Three genes, er1, er2 and Er3, conferring resistance to powdery mildew (Erysiphe pisi) in pea have been described so far. Because single gene-controlled resistance tends to be overcome by evolution of pathogen virulence, accumulation of several resistance genes into a single cultivar should enhance the durability of the resistance. Molecular markers linked to genes controlling resistance to E. pisi may facilitate gene pyramiding in pea breeding programs. Molecular markers linked to er1 and er2 are available. In the present study, molecular markers linked to Er3 have been obtained. A segregating F2 population derived from the cross between a breeding line carrying the Er3 gene, and the susceptible cultivar ‘Messire’ was developed and genotyped. Bulk Segregant Analysis (BSA) was used to identify Random Amplified Polymorphic DNA (RAPD) markers linked to Er3. Four RAPD markers linked in coupling phase (OPW04_637, OPC04_640, OPF14_1103, and OPAH06_539) and two in repulsion phase (OPAB01_874 and OPAG05_1240), were identified. Two of these, flanking Er3, were converted to Sequence Characterized Amplified Region (SCAR) markers. The SCAR marker SCW4637 co-segregated with the resistant gene, allowing the detection of all the resistant individuals. The SCAR marker SCAB1874, in repulsion phase with Er3, was located at 2.8 cM from the gene and, in combination with SCW4637, was capable to distinguish homozygous resistant individuals from heterozygous with a high efficiency. In addition, the validation for polymorphism in different genetic backgrounds and advanced breeding material confirmed the utility of both markers in marker-assisted selection.

Salt stress alleviation in transgenic Vigna mungo L. Hepper (blackgram) by overexpression of the glyoxalase I gene using a novel Cestrum yellow leaf curling virus (CmYLCV) promoter

Abstract: A reproducible and efficient transformation system utilizing the nodal regions of embryonal axis of blackgram (Vigna mungo L. Hepper) has been established via Agrobacterium tumefaciens. This is a report of genetic transformation of Vigna mungo for value addition of an agronomic trait, wherein the gene of interest, the glyoxalase I driven by a novel constitutive Cestrum yellow leaf curling viral promoter has been transferred for alleviating salt stress. The overexpression of this gene under the constitutive CaMV 35S promoter had earlier been shown to impart salt, heavy metal and drought stress tolerance in the model plant, tobacco. Molecular analyses of four independent transgenic lines performed by PCR, Southern and western blot revealed the stable integration of the transgene in the progeny. The transformation frequency was ca. 2.25% and the time required for the generation of transgenic plants was 10–11 weeks. Exposure of T1 transgenic plants as well as untransformed control plants to salt stress (100 mM NaCl) revealed that the transgenic plants survived under salt stress and set seed whereas the untransformed control plants failed to survive. The higher level of Glyoxalase I activity in transgenic lines was directly correlated with their ability to withstand salt stress. To the best of our knowledge this is the only report of engineering abiotic stress tolerance in blackgram.

Heading date QTL in a spring × winter barley cross evaluated in Mediterranean environments

Abstract: Heading date is a key trait for the adaptation of barley to Mediterranean environments. We studied the genetic control of flowering time under Northern Spanish (Mediterranean) conditions using a new population derived from the spring/winter cross Beka/Mogador. A set of 120 doubled haploid lines was evaluated in the field, and under controlled temperature and photoperiod conditions. Genotyping was carried out with 215 markers (RFLP, STS, RAPD, AFLP, SSR), including markers for vernalization candidate genes, HvBM5 (Vrn-H1), HvZCCT (Vrn-H2), and HvT SNP22 (Ppd-H1). Four major QTL, and the interactions between them, accounted for most of the variation in both field (71–92%) and greenhouse trials (55–86%). These were coincident with the location of the major genes for response to vernalization and short photoperiod (Ppd-H2 on chromosome 1H). A major QTL, near the centromere of chromosome 2H was the most important under autumn sowing conditions. Although it is detected under all conditions, its action seems not independent from environmental cues. An epistatic interaction involving the two vernalization genes was detected when the plants were grown without vernalization and under long photoperiod. The simultaneous presence of the winter Mogador allele at the two loci produced a marked delay in heading date, beyond a mere additive effect. This interaction, combined with the effect of the gene responsive to short photoperiod, Ppd-H2, was found responsible of the phenomenon known as short-day vernalization, present in some of the lines of the population.

EST–SSR markers for asparagus genetic diversity evaluation and cultivar identification

Abstract: Simple sequence repeat (SSR) markers generated from expressed sequence tag (EST) sequences represent useful tools for genotyping and their development is relatively easy because of the public availability of EST databases. We report design and application of EST–SSRs to assess the level of genetic diversity among thirty-five asparagus cultivars and to fingerprint DePaoli, a new variety released by University of California, Riverside. DNA was isolated from bulks of pooled cladophylls coming from five plants of each variety to reduce the number of DNA extractions and PCR reactions. Allele frequencies were estimated from the intensity of the bands in two bulks and two individual plant samples for each variety. Although asparagus varieties derive from a limited germplasm pool, eight EST–SSR loci differentiated all of the analyzed cultivars. Moreover, UPGMA (unweighted pair group method with arithmetic mean) and neighbor-joining trees, as well as principal components analysis separated the cultivars into clusters corresponding to the geographical areas where they originated.

Plant gene responses to frequency-specific sound signals

Abstract: We identified a set of sound-responsive genes in plants using a sound-treated subtractive library and demonstrated sound regulation through mRNA expression analyses. Under both light and dark conditions, sound up-regulated expression of rbcS and ald. These are also light-responsive genes and these results suggest that sound could represent an alternative to light as a gene regulator. Ald mRNA expression increased significantly with treatment at 125 and 250 Hz, whereas levels decreased significantly with treatment at 50 Hz, indicating a frequency-specific response. To investigate whether the ald promoter responds to sound, we generated transgenic rice plants harboring a chimeric gene comprising a fusion of the ald promoter and GUS reporter. In three independent transgenic lines treated with 50 or 250 Hz for 4 h, GUS mRNA expression was up-regulated at 250 Hz, but down-regulated at 50 Hz. Thus, the sound-responsive mRNA expression pattern observed for the ald promoter correlated closely with that of ald, suggesting that the 1,506 bp ald promoter is sound-responsive. Therefore, we propose that in transgenic plants, specific frequencies of sound treatment could be used to regulate the expression of any gene fused to the ald promoter.

Reducing the content of nornicotine in tobacco via targeted mutation breeding

Abstract: Cultivated tobacco produces secondary alkaloids involved in the formation of nitrosamines with health concerns. The recent identification of target genes in nicotine and nornicotine biosynthetic pathways now allows biotechnological approaches for their control. We demonstrate here that mutation breeding can be used as an alternative to genetically modified (GM) plants for generating nornicotine-free tobacco. Ten alleles of the NtabCYP82E4 gene (nicotine N-demethylase) were identified by screening 1,311 M2 families of tobacco ethylmethane sulphonate (EMS) mutants. Alkaloid analysis indicated that the nornicotine contents of homozygous M2 plants carrying nonsense or missense alleles of NtabCYP82E4 were very low or near-null. Backcrossing with tobacco elite varieties yielded BC1 plants phenotypically undistinguishable from parental lines. This major objective of tobacco breeders in the last few decades could be reached in a period of less than 1.5 years, including the creation of highly mutagenised tobacco mutant collections and the detection of mutated alleles using a simple and versatile detection technology (capillary electrophoresis-single strand conformation polymorphism, CE-SSCP) accessible to most breeding companies and crop species.