Patterns of subspecies genetic diversity among oilseed <Emphasis Type="Italic">Brassica rapa</Emphasis> as revealed by agro-morphological traits and SSR markers

Brassica rapa (2n = 20, AA genome) is an important oil yielding species of the family Brassicaceae and characterized by wide range of genetic and morphological subtypes suitable for cultivation under diverse agro-climatic regions of India. In this study, genetic diversity among three subspecies of B. rapa including yellow sarson, toria and outlier brown sarson was estimated using various agro-morphological traits and simple sequence repeat (SSR) markers. Maximum variability was recorded for siliqua angle (Coefficient of variation = 30.9%), followed by seeds/siliqua (CV = 18.8%), leaf length (CV = 10%) and plant height (CV = 16.8%). Principal component analysis explained more than 50% of the total observed morphological variability for first two components. Of the 107 SSR markers tested, 80 generated reproducible, clear and distinct amplicons of which, 65 (81.25%) were found polymorphic. The number of alleles at each locus ranged from 2 to 7, with an average of 3.03 alleles per marker. A total of 197 alleles were detected at 65 SSR loci with average PIC value of 0.457 and a mean resolving power of 3.04. Neighbor-Joining cluster analysis based on morphological traits and SSR markers separately classified all the 28 genotypes into five major groups. The population structure analysis resulted into three sub-populations with certain extent of admixture among the earlier established taxonomic sub-groups. Twenty-three unique alleles were detected in thirteen B. rapa varieties. The clustering analysis and principal coordinate analysis outlined the genetic relationships among different varieties belonging to the three subspecies of B. rapa. Genetically diverse genotypes as illustrated by score plots and from the clustering patterns brought out the wide range of diversity present among B. rapa genotypes and the underlying options available for selecting parental genotypes for hybridization and developing high yielding cultivars suitable for Indian conditions.     

Inheritance and gene mapping of the white flower trait in <Emphasis Type="Italic">Brassica juncea</Emphasis>

Despite being a unique marker trait, white flower inheritance in Brassica juncea remains poorly understood at the gene level. In this study, we investigated a B. juncea landrace with white petal in China. The white petal phenotype possessed defective chromoplasts with less plastoglobuli than the yellow petal phenotype. Genetic analysis confirmed that two independent recessive genes (Bjpc1 and Bjpc2) controlled the white flower trait. We then mapped the BjPC1 gene in a BC₄ population comprising 2295 individuals. We identified seven AFLP (amplified fragment length polymorphism) markers closely linked to the white flower gene. BLAST search revealed the sequence of AFLP fragments were highly homologous with the Scaffold000085 and Scaffold000031 sequences on the A02 chromosome in the Brassica rapa genome. Based on this sequence homology, we developed simple sequence repeat (SSR) primer pairs and identified 13 SSRs linked to the BjPC1 gene, including two that were co-segregated (SSR9 and SSR10). The two closest markers (SSR4 and SSR11) were respectively 0.9 and 0.4 cM on either side of BjPC1. BLAST analysis revealed that these marker sequences corresponded highly to A02 in B. juncea. They were mapped within a 33 kb genomic region on B. rapa A02 (corresponds to a 40 kb genomic region on B. juncea A02) that included three genes. Sequence BjuA008406, homologous to AtPES2 in Arabidopsis thaliana and Bra032956 in B. rapa, was the most likely candidate for BjPC1. These results should accelerate BjPC1 cloning and facilitate our understanding of the molecular mechanisms controlling B. juncea petal color.     

Development of an STS map of an interspecific progeny of <Emphasis Type="Italic">Malus</Emphasis>

Simple sequence repeat (SSR) markers developed from Malus, as well as Prunus, Pyrus and Sorbus, and some other sequence-tagged site (STS) loci were analysed in an interspecific F₁ apple progeny from the cross ‘Fiesta’ × ‘Totem’ that segregated for several agronomic characters. A linkage map was constructed using 259 STS loci (247 SSRs, four SCARs and eight known-function genes) and five genes for agronomic traits—scab resistance (Vf), mildew resistance (Pl-2), columnar growth habit (Co), red tissues (Rt) and green flesh background colour (Gfc). Ninety SSR loci and three genes (ETR1, Rt and Gfc) were mapped for the first time in apple. The transferability of markers from other Maloideae to Malus was found to be around 44%. The loci are spread across 17 linkage groups, corresponding to the basic chromosome number of Malus and cover 1,208 cM, approximately 85% of the estimated length of the apple genome. Interestingly, we have extended the top of LG15 with eight markers covering 25 cM. The average map density is 4.7 cM per marker; however, marker density varies greatly between linkage groups, from 2.5 in LG14 to 8.9 in LG7, with some areas of the genome still in need of further STS markers for saturation.     

Genetic dissection of seed weight by QTL analysis and detection of allelic variation in Indian and east European gene pool lines of <Emphasis Type="Italic">Brassica juncea</Emphasis>

Key message

Seed weight QTL identified in different populations were synthesized into consensus QTL which were shown to harbor candidate genes by in silico mapping. Allelic variation inferred would be useful in breeding B. juncea lines with high seed weight.

Abstract

Seed weight is an important yield influencing trait in oilseed Brassicas and is a multigenic trait. Among the oilseed Brassicas, Brassica juncea harbors the maximum phenotypic variation wherein thousand seed weight varies from around 2.0 g to more than 7.0 g. In this study, we have undertaken quantitative trait locus/quantitative trait loci (QTL) analysis of seed weight in B. juncea using four bi-parental doubled-haploid populations. These four populations were derived from six lines (three Indian and three east European lines) with parental phenotypic values for thousand seed weight ranging from 2.0 to 7.6 g in different environments. Multi-environment QTL analysis of the four populations identified a total of 65 QTL ranging from 10 to 25 in each population. Meta-analysis of these component QTL of the four populations identified six ‘consensus’ QTL (C-QTL) in A3, A7, A10 and B3 by merging 33 of the 65 component Tsw QTL from different bi-parental populations. Allelic diversity analysis of these six C-QTL showed that Indian lines, Pusajaikisan and Varuna, hold the most positive allele in all the six C-QTL. In silico mapping of candidate genes with the consensus QTL localized 11 genes known to influence seed weight in Arabidopsis thaliana and also showed conserved crucifer blocks harboring seed weight QTL between the A subgenomes of B. juncea and B. rapa. These findings pave the way for a better understanding of the genetics of seed weight in the oilseed crop B. juncea and reveal the scope available for improvement of seed weight through marker-assisted breeding.

     

Simple Sequence Repeat and <Emphasis Type="Italic">S</Emphasis>-locus Genotyping to Explore Genetic Variability in Polyploid <Emphasis Type="Italic">Prunus spinosa</Emphasis> and <Emphasis Type="Italic">P. insititia</Emphasis>

Polyploid Prunus spinosa (2n = 4×) and P. insititia (2n = 6×) represent enormous genetic potential in Central Europe, which can be exploited in breeding programmes. In Hungary, 17 cultivar candidates were selected from wild-growing populations including 10 P. spinosa, 4 P. insititia and three P. spinosa × P. domestica hybrids (2n = 5×). Their taxonomic classification was based on their phenotypic characteristics. Six simple sequence repeats (SSRs) and the multiallelic S-locus genotyping were used to characterize genetic variability and reliable identification of the tested accessions. A total of 98 SSR alleles were identified, which presents 19.5 average allele number per locus, and each of the 17 genotypes could be discriminated based on unique SSR fingerprints. A total of 23 S-RNase alleles were identified. The complete and partial S-genotype was determined for 8 and 9 accessions, respectively. The identification of a cross-incompatible pair of cultivar candidates and several semi-compatible combinations help maximize fruit set in commercial orchards. Our results indicate that the S-allele pools of wild-growing P. spinosa and P. insititia are overlapping in Hungary. A phylogenetic and principal component analysis confirmed the high level of diversity and genetic differentiation present within the analysed genotypes and helped clarify doubtful taxonomic identities. Our data confirm that S-locus genotyping is suitable for diversity studies in polyploid Prunus species. The analysed accessions represent huge genetic potential that can be exploited in commercial cultivation.     

Development of a large number of SSR and InDel markers and construction of a high-density genetic map based on a RIL population of pepper (<Emphasis Type="Italic">Capsicum annuum</Emphasis> L.)

Capsicum annuum, the most widely cultivated species of pepper, is used worldwide for its important nutritional and medicinal values. The construction of an intraspecific high-density genetic linkage map would be of practical value for pepper breeding. However, the numbers of PCR-based simple sequence repeat (SSR) and insertion/deletion (InDel) markers that are available are limited, and there is a need to develop a saturated, intraspecific linkage map. The non-redundant Capsicum species’ expressed sequence tag (EST) database from the National Center for Biotechnology Information was used in this study to develop a total of 902 usable EST-SSR markers. Additionally, 177,587 SSR loci were identified based on the pepper genomic information, including 9182 SSR loci 500 bp both upstream and downstream of coding regions. Another 4497 stable and reliable InDel loci were also developed. From 9182 SSR and 4497 InDel loci, 3356 pairs of genomic SSR primers and 1400 pairs of InDel primers that were evenly distributed in 12 chromosomes were selected. A high-density intraspecific genetic map of C. annuum was constructed using the F₁₀-generation recombinant inbred line of parents PM702 and FS871 as the mapping population, screening the selected 3356 pairs of genomic SSR primers and 1400 pairs of InDel primers and the 902 EST-SSR markers developed earlier, and 524 published SSR markers and 299 orthologous markers (including 263 COSII markers and 36 tomato-derived markers) used previously to develop an interspecific genetic map (C. annuum × C. frutescens). Eventually, a high-density complete genetic intraspecific linkage map of C. annuum containing 12 linkage groups and 708 molecular markers with a length of 1260.00 cM and an average map distance of 1.78 cM was produced. This intraspecific, high-density, complete genetic linkage map of C. annuum contains the largest number of SSR and InDel markers and the highest amount of saturation so far, and it will be of considerable significance for the breeding of improved cultivars of this important field crop in the future.     

<Emphasis Type="Italic">Miscanthus</Emphasis> sp.: Genetic Diversity and Phylogeny in China

Miscanthus genetic resources are widely distributed throughout China. However, genetic studies on Miscanthus lagged far behind other crops (e.g., sorghum, maize). To establish the comprehensive genetics knowledge of Miscnathus in China, here we report the genetic and phylogenetic diversity of 174 domestic Miscanthus accessions, along with an external Miscanthus × giganteus control. Cytological observations and flow cytometry analyses indicated that there were two major Miscanthus cytotypes in China: diploid (86.86%) and tetraploid (12.57%) without triploid. A total of 108 polymorphic loci generated from 25 SSR primers were used to evaluate the genetic variation. Large variations in genetic similarity coefficients (GSCs), ranging from 0.08 to 0.97 with a mean value of 0.39, were observed between these Miscanthus accessions. Our phylogenetic data revealed that these accessions were clustered into four main clades: M. section Miscanthus, M. section Diandranthus, M. section Triarrhena, and hybrids. The average percentage of polymorphic loci (P), gene diversity (H), and Shannon’s diversity index (I) among Miscanthus species are 70.93%, 0.22, and 0.34, respectively. These were consistent with the analysis of molecular variance (AMOVA) results, showing that 85% of genetic variation was found within clades. This study investigated the clear phylogenetic relationship of Miscanthus species in China, which will be valuable for further utilization of the germplasm in genetic improvement and hybrid breeding of Miscanthus.     

Microsatellite-based genetic diversity analyses of <Emphasis Type="Italic">sugary1</Emphasis>-, <Emphasis Type="Italic">shrunken2</Emphasis>- and double mutant- sweet corn inbreds for their utilization in breeding programme

Sweet corn has recently experienced sharp rise in demand worldwide. Recessive sugary1 (su1) and shrunken2 (sh2) that enhances kernel sweetness have been abundantly used in sweet corn breeding. Analyses of genetic diversity among sweet corn inbreds assume great significance for their effective utilization in hybrid breeding. A set of 48 diverse sweet corn genotypes encompassing su1su1, sh2sh2 and su1su1/sh2sh2 types were analyzed using 56 microsatellite markers. A total of 213 alleles with mean of 3.8 alleles per locus were generated. Two unique- and 12 rare- alleles were identified. The average PIC and genetic dissimilarity was 0.50 and 0.73, respectively. Cluster analysis grouped the inbreds into three major clusters, with each of the su1su1-, sh2sh2- and su1su1/sh2sh2-types were broadly clustered together. Principal coordinate analyses also depicted the diverse origin of the genotypes. The study identified inbreds for synthesis of pools and pedigree populations to develop novel inbreds. The study led to the identification of prospective heterotic combinations in various genetic backgrounds (sh2sh2 × sh2sh2, su1su1 × su1su1, su1su1/sh2sh2 × su1su1/sh2sh2, sh2sh2 × su1su1/sh2sh2 and su1su1 × su1su1/sh2sh2).     

Physiological characterization and allelic diversity of selected drought tolerant traditional rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) landraces of Koraput,India

Water-deficit stress tolerance in rice is important for maintaining stable yield, especially under rain-fed ecosystem. After a thorough drought-tolerance screening of more than 130 rice genotypes from various regions of Koraput in our previous study, six rice landraces were selected for drought tolerance capacity. These six rice landraces were further used for detailed physiological and molecular assessment under control and simulated drought stress conditions. After imposing various levels of drought stress, leaf photosynthetic rate (P_N), photochemical efficiency of photosystem II (Fv/Fm), SPAD chlorophyll index, membrane stability index and relative water content were found comparable with the drought-tolerant check variety (N22). Compared to the drought-susceptible variety IR64, significant positive attributes and varietal differences were observed for all the above physiological parameters in drought-tolerant landraces. Genetic diversity among the studied rice landraces was assessed using 19 previously reported drought tolerance trait linked SSR markers. A total of 50 alleles with an average of 2.6 per locus were detected at the loci of the 19 markers across studied rice genotypes. The Nei’s genetic diversity (He) and the polymorphism information content (PIC) ranged from 0.0 to 0.767 and 0.0 to 0.718, respectively. Seven SSR loci, such as RM324, RM19367, RM72, RM246, RM3549, RM566 and RM515, showed the highest PIC values and are thus, useful in assessing the genetic diversity of studied rice lines for drought tolerance. Based on the result, two rice landraces (Pandkagura and Mugudi) showed the highest similarity index with tolerant check variety. However, three rice landraces (Kalajeera, Machhakanta and Haldichudi) are more diverse and showed highest genetic distance with N22. These landraces can be considered as the potential genetic resources for drought breeding program.     

Development of microsatellite markers and their use in genetic diversity and population structure analysis in <Emphasis Type="Italic">Casuarina</Emphasis>

Casuarina is a widely cultivated plantation tree species in coastal India, primarily due to its fast growth, high productivity and suitable for pulp and paper production. However, genetic studies of Casuarina have been hindered by lack of genomic resources and genetic markers. Knowledge of the genetic diversity and population structure of Casuarina germplasms will provide the basis for utilizing and improving resource in the breeding program. Keeping this in view, in the present study, we have identified a total of 11,503 simple sequence repeat (SSR) makers from 86,415 expressed sequence tags (ESTs) of Casuarina equisetifolia and C. junghuhniana after redundancy elimination. Dinucleotide repeats were the most abundant accounting for 72.5 % of all microsatellites, followed by trimer (23.4 %), hexamer (1.7 %), tetramer (1.5 %), and very few pentamer (0.6 %) repeats. Of these, 50 markers were used to estimate genetic diversity and population structure among 96 accessions of C. cunninghamiana and C. junghuhniana. EST-SSR markers revealed high level of polymorphism, detecting a total of 829 alleles with an average of 17 alleles per locus. Polymorphic information content (PIC) values ranged from 0.32 to 0.93, with an average of 0.78 per locus. The average observed (H _o ) and expected heterozygosity (H _e ) obtained was high and fairly similar in C. cunninghamiana and C. junghuhniana, thereby suggesting highly heterogeneous nature of Casuarina. Population structure using a Bayesian model-based clustering approach identified clear delineation between C. cunninghamiana and C junghuhniana. Further, these markers were also evaluated in four species of Casuarina confirming high rate of cross-species transferability. The results of this study can provide valuable insights for genetic and genomic research in Casuarina.     

QTL analysis and the development of closely linked markers for days to flowering in spring oilseed rape (<Emphasis Type="Italic">Brassica napus</Emphasis> L.)

Days to flowering (DTF) is an important trait impacting cultivar performance in oilseed rape (Brassica napus L.), but the interaction of all loci controlling this trait in spring-type oilseed rape is not fully understood. We identified quantitative trait loci (QTL) for variation in DTF in a doubled haploid (DH) population from the Qinghai–Tibet Plateau that includes 217 lines derived from a cross between spring-type oilseed rape (B. napus L.) line No. 5246 and line No. 4512, the latter of which is responsive to the effective accumulated temperature (EAT). A linkage map was constructed for the DH population, using 202 SSR and 293 AFLP markers. At least 22 DTF QTL were found in multiple environments. Four major QTL were located on linkage groups A7, C2, C8 and C8. Among these QTL, cqDTFA7a and cqDTFC2a were identified in five environments and individually explained 10.4 and 23.0 % of the trait variation, respectively. cqDTFC8, a major QTL observed in spring environments, and a unique winter environment QTL, qDTFC8-3, were identified; these QTL explained 10.0 and 46.5 % of the phenotypic variation, respectively. Minor QTL (for example, cqDTFC2c) and epistatic interactions seemed evident in this population. Two closely linked SSR markers for cqDTFA7a and cqDTFC8 were developed (G1803 and S034). BnAP1, a B. napus gene with homology to Arabidopsis thaliana that was identified as a cqDTFA7a candidate gene, played a major role in this study. The allelic effects of the major and minor QTL on DTF were further validated in the DH population and in 93 breeding genotypes.