Penetrance of creeping-rootedness in clonal progenies of lucerne and observations on underground morphology of plants differing for this character

Elaeis oleifera or ‘caiaué’, a close relative of oil palm (E. guineensis), has some agronomic traits of great interest for the oil palm genetic breeding such as slow growth, oil quality (mostly unsaturated) and disease resistance. An analysis of a Brazilian oil palm germplasm collection was carried out using RAPD (Random Amplified Polymorphic DNA) markers with the objective of understanding the genetic variation of ‘caiaué’ accessions collected in the Amazon Forest in the last two decades. A sample of 175 accessions obtained along the Amazon River Basin was analyzed and compared to 17 accessions of oil palm from Africa. Ninety-six RAPD markers were used in the analysis, of which fourteen were shown to be specific to oil palm, while twelve were specific to ‘caiaué’. Results showed that the Brazilian ‘caiaué’ accessions studied have moderate levels of genetic diversity as compared to oil palm accessions. The data allowed the establishment of similarity groups for ‘caiaué’ accessions, which is useful for selecting parental plants for population breeding. Cluster analysis showed that, in general, genetic similarities are not correlated to geographical distances, but consistent with geographical dispersal along the Amazon River network. AMOVA showed that most of the genetic variation is found within populations, as expected for anallogamous and long-lived perennial species. The study provides important information to define strategies for future collection expeditions, for germplasm conservation and for the use of E. oleifera in breeding programs. This revised version was published online in August 2006 with corrections to the Cover Date.     

Suitability of RAPD analysis for the detection of somaclonal variants in oil palm (Elaeis guineensis Jacq)

Random amplified polymorphic DNA (RAPD) analysis using arbitrary 10-mer oligonucleotide primers was employed in order to investigate the genetic fidelity of somatic embryogenesis-derived regenerants of oil palm (Elaeis guineensis Jacq). Clonal palms bearing the ‘mantled’ phenotype were identified in the field and the ability of RAPD markers to distinguish these variants from palms of the normal type was assessed. Of the 387 arbitrary primers used, 259 (67%) were successfully used to amplify oil palm DNA genomic fragments with consistently reproducible banding. Of these 387 primers, 73 (19%) primers enabled the identification of polymorphism between clones. No intraclonal variability and no differences between mother palms and regenerants could be identified using the total number of markers scored (8900). Twenty-four of these 73 primers were chosen for use in a larger experiment aimed at comparing, first, the mother palm genome with that of its clonal offspring and, second, true-to-type and variant regenerants. Thus, the regeneration protocol based on somatic embryogenesis set up for oil palm clonal propagation does not induce any gross genetic changes. The results obtained revealed however, that the RAPD approach is not suitable for the detection of the ‘mantled’ variant phenotype. The use of RAPD markers for the detection of somaclonal variation in oil palm is discussed.     

Analysis of growth in the oil palm II. Estimation of genetic variances of growth parameters and yield of fruit bunches

Summary Vegetative and yield measurements were made in oil palm (Elaeis guineensis Jacq.) breeding experiments leading to estimates of genetic variance for yield of fruit bunches, Crop Growth Rate, Leaf Area, Leaf Area Index, Leaf Area Ratio, Vegetative Dry Matter Production and Bunch Index (dry matter in fruit bunches/total dry matter). Results suggest the presence of genetic variability for all the traits examined, and indications are that BI, LAI and VDM have a reasonable level of genetic control. The data are discussed in relation to breeding and it is suggested that instead of straight-forward selection for high yield of individual palms, a more promising approach may be to select for palms that at high planting densities maintain reasonable BI values.     

Prediction of precocity in the oil palm (Elaeis guineensis Jacq.)

Summary Precocity in the oil palm (Elaeis guineensis Jacq.) is defined, for the purpose of this study, as the percentage of palms bearing female or hermaphrodite inflorescences 36 months after germination. A method of calculating breeding values for precocity is described and it is shown that the character is predictable. An upper limit for heritability (h²) of 0.68 and a correlation between observed and predicted values of r=+0.77 (significant at P=0.01) have been calculated from the available data. The available information suggests that precocity could easily be adjusted to the desired level by breeding and selection.     

A novel aphid resistance locus in cowpea identified by combining SSR and SNP markers

The utility of combining simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) marker genotyping was determined for genetically mapping a novel aphid (Aphis craccivora) resistance locus in cowpea breeding line SARC 1‐57‐2 and for introgressing the resistance into elite cultivars by marker‐assisted backcrossing (MABC). The locus was tagged with codominant SSR marker CP 171F/172R with a recombination fraction of 5.91% in an F₂ population from ‘Apagbaala’ x SARC 1‐57‐2. A SNP‐genotyped biparental recombinant inbred line population was genotyped for CP 171F/172R, which was mapped to position 11.5 cM on linkage group (LG) 10 (physical position 30.514 Mb on chromosome Vu10). Using CP 171F/172R for foreground selection and a KASP‐SNP‐based marker panel for background selection in MABC, the resistance from SARC 1‐57‐2 was introduced into elite susceptible cultivar ‘Zaayura’. Five BC₄F₃ lines of improved ‘Zaayura’ that were isogenic except for the resistance locus region had phenotypes similar to SARC 1‐57‐2. This study identified a novel aphid resistance locus and demonstrated the effectiveness of integrating SSR and SNP markers for trait mapping and marker‐assisted breeding.     

Genetic diversity of tropical maize inbred lines combining resistance to Striga hermonthica with drought tolerance using SNP markers

Striga hermonthica and drought are the major stresses limiting maize yields in sub‐Saharan Africa. The search for diverse maize lines’ tolerance to drought and resistance to S. hermonthica (DTSTHR) is very crucial for yield improvement in areas affected by the two stresses. Understanding the genetic diversity among the lines is important to develop cultivars resistant to S. hermonthica and tolerant to drought. The lines were developed from biparental crosses of drought‐tolerant and Striga‐resistant lines. A total of 128 DTSTHR maize lines were characterized using single‐nucleotide polymorphism (SNP) markers. Results of the cluster analysis based on 3297 SNP markers showed four distinct groups consistent with the pedigrees of the lines. Furthermore, model‐based analysis also formed the same groups of the DTSTHR lines. Integrating the pedigree information with combining ability and the SNP analyses may provide defined heterotic groups for maize improvement work in West and Central Africa. These results also help breeders to utilize DTSTHR lines present at IITA for developing biparental crosses without disrupting the heterotic groups they have established in their breeding programmes.     

Effects of inbreeding on growth and yield of oil palm

The effects of inbreeding were studied in three oil palm (Elaeis guineensis Jacq.) trials in the Democratic Republic of Congo (Congo). In selfings, marked inbreeding depression was observed for yield of fruit bunches, mean bunch weight and bunch number, but there was little effect on bunch composition. Most vegetative measurements were significantly affected by selfing, but leaf production rate and leaf area ratio were unaffected. Sib-crossing had less depressive effect than selfing, and in some families sib-crosses were superior to outcrosses. Where selection needs to be done within inbred families, our study suggests that leaf area ratio and bunch composition would be useful criteria.     

Identification and confirmation of quantitative trait loci for stachyose content in soybean seed

Stachyose is an unfavorable sugar in soybean meal that causes flatulence for non‐ruminant animals. Understanding the genetic control of stachyose in soybean will facilitate the modification of stachyose content at the molecular level. The objective of this study was to identify quantitative trait loci (QTL) associated with seed stachyose content using simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) markers. A normal stachyose cultivar, ‘Osage’, was crossed with a low stachyose line, V99‐5089, to develop a QTL mapping population. Two parents were screened with 33 SSR and 37 SNP markers randomly distributed on chromosome 10, and 20 SSR and 19 SNP markers surrounding a previously reported stachyose QTL region on chromosome 11. Of these, 5 SSR and 16 SNP markers were used to screen the F_3:4 lines derived from ‘Osage’ x V99‐5089. Seed samples from F_3:5 and F_3:6 lines were analyzed for stachyose content using high‐performance liquid chromatography (HPLC). Composite interval mapping analysis indicated that two stachyose QTL were mapped to chromosome 10 and 11, explaining 11% and 79% of phenotypic variation for stachyose content, respectively. The SSR/SNP markers linked to stachyose QTL could be used in breeding soybean lines with desired stachyose contents. Chi‐square tests further indicated that these two QTL probably represent two independent genes for stachyose content. Therefore, a major QTL was confirmed on chromosome 11 and a novel QTL was found on chromosome 10 for stachyose content.     

Development of gene‐based markers for the Turnip mosaic virus resistance gene retr02 in Brassica rapa

Turnip mosaic virus (TuMV) is responsible for a serious disease that affects the production of Chinese cabbage. Previous studies have cloned a series of TuMV resistance genes and developed molecular markers. In this study, a derived cleaved amplified polymorphism sequence (dCAPS) marker and a Kompetitive Allele Specific PCR (KASP) marker were developed based on a single recessive gene, retr02, which confers broad‐spectrum TuMV resistance in Chinese cabbage by means of an additional G at the junction of exon 1 and intron 1. The two markers were able to detect the retr02 allele in Chinese cabbage accessions used in breeding programmes. Compared with the dCAPS marker, the KASP marker was flexible, cost‐effective and quick to process, which is likely to be beneficial in establishing high‐throughput assays for marker‐assisted selection.     

Genetic analysis and fine mapping of tms9, a novel thermosensitive genic male‐sterile gene in rice (Oryza sativa L.)

Two‐line hybrid rice as a novel hybrid breeding method has huge potential for yield increasing and utilization of intersubspecific heterosis, and it is of major significance for the food security of rice‐consuming populations. Zhu1S is a thermosensitive genic male‐sterile line of rice with low critical temperature and excellent combining ability, which has been widely exploited as a female parent in Chinese two‐line hybrid rice breeding. Here, genetic mapping in F₂ populations was used to show that its male sterility is inherited as a single recessive gene and that responsible gene (termed tms9) lies on the short arm of chromosome 2. A high‐resolution linkage analysis which was based on the Zhu1S/R173 F₂ population found that the thermosensitive genic male‐sterile gene tms9 of Zhu1S was fine mapped between insertion–deletion (Indel) markers Indel 37 and Indel 57, and the genetic distance from the tms9 to the two markers was 0.12 and 0.31 cM, respectively. The physical distance between the two markers was about 107.2 kb. Sequence annotation databases showed that the two Indel markers (Indel 37 and Indel 57) were located on two BAC clones (B1307A11 and P0027A02). There are sixteen open reading frames (ORF) present in this region. The results of this study are of great significance for further cloning tms9 and molecular marker–assisted selection.     

QTL analysis for cooking traits of super rice with a high‐density SNP genetic map and fine mapping of a novel boiled grain length locus

Hybrid rice has contributed substantially to the improvement of grain production worldwide, yet its poor cooking and tasting characteristics have long been recognized. In this study, 132 recombinant inbred lines derived from LYPJ were used to identify quantitative trait loci (QTLs) for 12 cooking traits with the high‐density SNP linkage map recently developed by our team. We identified 17 QTLs on chromosomes 1, 2, 4, 5, 6, 7, 8, 9 and 11, which accounted for 7.50% to 23.50% of the phenotypic variations. A novel major QTL qBGL7 for boiled grain length was further fine‐mapped to an interval of 440 Kb between the two markers RM21906 and gl3 using a BC₃F₂ population. Two near‐isogenic lines with extreme boiled grain length, GX5‐176 and GX5‐101, could be directly used in improving cooking quality. We also identified a QTL for soaked grain width expansion rate, qSGWE6, in the Wx gene region on chromosome 6. The Wx differential regulation coincided with sequential variation between the two parents. Our work offered a theoretical basis for molecular breeding of high‐quality hybrid rice.     

A SNP genetic linkage map based on the ‘Hamilton’ by ‘Spencer’ recombinant inbred line population identified QTL for seed isoflavone contents in soybean

Soybean is one of the most important crops worldwide for its protein and oil as well as the health beneficial phytoestrogens or isoflavone. This study reports a relatively dense single nucleotide polymorphism (SNP)‐based genetic map based on ‘Hamilton’ by ‘Spencer’ recombinant inbred line population and quantitative trait loci (QTL) for seed isoflavone contents. The genetic map is composed of 1502 SNP markers and covers about 1423.72 cM of the soybean genome. Two QTL for seed isoflavone contents have been identified in this population. One major QTL that controlled both daidzein (qDZ1) and total isoflavone contents (qTI1) was found on LG C2 (Chr 6). And a second QTL for glycitein content (qGT1) was identified on the LG G (Chr 18). These two QTL in addition to others identified in soybean could be used in soybean breeding to optimize isoflavone content. This newly assembled soybean linkage map is a useful tool to identify and map QTL for important agronomic traits and enhance the identification of the genes involved in these traits.     

Identification of novel QTL for leaf traits in soybean

Several leaf traits of soybean (Glycine max L. Merr.), including leaf area (LA), leaf shape (LS) and specific leaf weight (SLW) may be related to soybean yield. The objective of this study was to identify novel quantitative trait loci (QTL) for LA, LS and SLW in a recombinant inbred line (RIL) population. The phenotype data were collected in 2011 and 2012 for 93 F_7:10 RILs using a randomized complete block design with 2 replicates each year. Five hundred and sixteen single‐nucleotide polymorphism (SNP) markers and the phenotype data were used to detect QTL using single marker analysis (SMA) and composite interval mapping (CIM). Single markers analysis identified 26 QTL for the three traits, of which 17 were novel and the rests were previously reported QTL. Most of these QTL were also identified by CIM. Most QTL reported in this study were in close proximity (<1 cM) of one or more SNP markers. These publicly available SNP markers with close linkage to LA, LS and SLW should be useful for marker‐assisted breeding for these traits.     

MicroRNA‐based molecular markers: a novel PCR‐based genotyping technique in Brassica species

DNA‐based molecular markers play a significant role in gene mapping, genetic diversity analysis, germplasm evaluation and molecular marker‐assisted selection. A combination of desirable marker characteristics such as abundant polymorphism, good stability, ease of production and high efficiency is difficult to achieve when utilizing traditional molecular marker systems. MicroRNAs are a type of endogenous non‐coding RNAs prevalent in the genomes of many organisms. The high conservation of miRNA and pre‐miRNA sequences provides an opportunity to develop a novel molecular marker type. We downloaded 82 miRNA sequences from the Brassica miRBase database and designed 46 single miRNA‐based primers which could be randomly combined to generate primer pairs. A proportion of these primer pairs were validated for transferability and polymorphism using DNA from 16 varieties of six Brassica species. The percentage of polymorphic primer pairs were 28.1%, and the average polymorphism information content (PIC) value for the 34 primer pairs was 0.43. Good transferability was verified across species. These results indicate that miRNA‐based markers provide a novel genotyping technique with low costs, high efficiency, stability and good transferability.     

Association mapping of leaf traits in spinach (Spinacia oleracea L.)

Spinach (Spinacia oleracea L.) is an important leafy vegetable crop grown worldwide. Leaf traits, surface texture (smooth, savoy or semi‐savoy), petiole colour (different shades of green vs. purple) and edge shape (serrate vs. entire), are important commercial traits of spinach. Association mapping for the three traits was conducted on 323 USDA spinach germplasm accessions, originally collected from 33 countries and representing the entire USDA spinach germplasm collection. The majority of accessions were from Europe (36.3%), Asia (25.3%) and North America (15.8%). The majority of the spinach accessions (82.0%) were smooth (unwrinkled types), whereas the savoy and semi‐savoy types (wrinkled types) accounted for 18.0%. The collection contained 74.9% green petiole types, while the purple petioles consisted of 25.1%. The collection consisted of 27.2% serrated leaf types and 72.8% entire leaf edge types. Genotyping‐by‐sequencing (GBS) was used for single nucleotide polymorphism (SNP) discovery, and SNPs were used as genotypic data to conduct genetic diversity and association mapping of the three leaf traits. Five genetic subpopulations and principal components (PCs) were postulated by structure 2 and JMP Genomics 7 for this association panel. Five, seven and 14 SNPs were identified to be associated with surface texture, edge shape and petiole colour, respectively. This study provides us an approach to identify SNP markers through association analysis in spinach and thus leads to select these three leaf traits through marker‐assisted selection in spinach breeding programme.     

Identification of candidate genes associated with resistance to bruchid (Callosobruchus maculatus) in cowpea

Cowpea is an important legume crop widely grown in sub‐Saharan Africa for food and feed. However, it is largely challenged by bruchid, a serious storage pest resulting in losses in quantity and quality of grains. Therefore, this research was designed to contribute to the breeding of cowpea resistance to bruchid through the identification of candidate genes associated with resistance to bruchid. A total of 217 mini‐core cowpea accessions were genotyped and phenotyped for their reactions to bruchid. To determine the genomic regions linked with bruchid resistance, 41,948 polymorphic SNP markers were used. Genome‐wide association study identified 11 SNPs linked to the average number of eggs, holes, insect emergence and development period and Dobie susceptibility index. Gene search via Phytozome identified six candidate genes (Vigun08g132300, Vigun08g158000, Vigun06g053700, Vigun02g131000, Vigun01g234900 and Vigun01g201900) associated with the resistance traits. These candidate genes could be incorporated into the farmers preferred but susceptible cowpea varieties to bruchid. The SNP markers associated with the resistance traits can be used in marker‐assisted breeding for accurate and rapid screening of cowpea resistant genotypes to bruchid.     

Independent target region amplification polymorphism and single‐nucleotide polymorphism marker utility in genetic evaluation of sugarcane genotypes

The independent target region amplification polymorphism (TRAP) and single‐nucleotide polymorphism (SNP) marker s were used for genetic evaluation of different selected 47 sugarcane genotypes. A total of 23 pairs of TRAP markers generated 925 alleles, of which 74% alleles were polymorphic. Polymorphism was generally high (>50%), ranging from 54 to 98%. The polymorphism information content (PIC) values 0.20 varied among the primer combination ranging from 0.17 in SAI + Arbi 2 to 0.31 in GL 2+ Arbi 1 with an average of 0.24. However, the Pearson correlation between PIC and power of discrimination (PD) was found to be less significant. Single‐nucleotide polymorphisms were used first time for the assessment of genetic diversity among different species of Saccharum and cultivated sugarcane varieties. The SNPs were detected from 454 sequencing. A total of 245 SNP markers were assayed across the 47 genotypes, and 167 SNPs were found to be polymorphic. The PIC values ranged from 0.04 to 0.38 with an average of 0.21, and their respective PD varied from 0.58 to 0.04 with an average value of 0.31. The obtained results relatively significant were compared with the other marker systems through genetic similarity and the clusters formed in different unweighted pair group method with arithmetic mean clustering dendrogram. The clustering analysis established genetic relationship in the order of Erianthus > Sclerostachya > Narenga > Saccharum spontaneum > S. robustum > S. barberi > S. officinarum/cultivars. These results ratify TRAP and SNP marker systems for assessing genetic diversity studies, and more diversified Erianthus spp. can contribute substantially towards sugarcane varietal improvement through breeding with Saccharum spp. or hybrid cultivars.     

Development and utilization of genomic and genic microsatellite markers in Assam tea (Camellia assamica ssp. assamica) and related Camellia species

Microsatellite or simple sequence repeat (SSR) markers are valuable tools for many purposes, such as phylogenetic, fingerprinting and molecular breeding studies. However, such marker resources are unavailable in Assam tea (Camellia assamica ssp. assamica; Masters). With an objective to enrich the repertoire of microsatellite markers in traditional tea, 185 novel microsatellite (150 genomic and 35 genic) markers were identified from (GA)n‐enriched genomic libraries and public expressed sequence data in Assam tea. High‐quality 0.412‐Mb non‐redundant (NR) genomic data set derived from nucleotide sequencing of 1297 (GA)n‐enriched genomic positive clones and 2723 unigenes (1.33 Mb) predicted from 10 803 random public expressed sequence tags (ESTs) in C. assamica ssp. assamica were utilized for identification of genomic and genic microsatellite markers, respectively. The average number of alleles and polymorphic information content (PIC) recorded for the newly developed SSR markers were 6.17 and 0.398, respectively. The average observed (H_o) and expected (H_e) heterozygosity varied from 0.626 to 0.697, respectively. These markers were found to be highly transferable (74.5–100%) to cultivated (C. sinensis, C. assamica ssp. lasiocalyx) and five wild Camellia species. Genetic diversity coefficient detected a high level of divergence in 24 cultivated tea accessions (69.3%). Phylogenetic analysis revealed that major groupings were broadly in accordance with taxonomic classification of tea, and all the wild Camellia species remained as an out‐group. The high polymorphic content coupled with high rate of cross‐transferability demonstrates wider applicability of novel microsatellite markers in genotyping, genetic diversity, genome mapping and evolutionary studies in various Camellia species.     

Analysis of growth of the oil palm (Elaeis guineensis Jacq.) I. Estimation of growth parameters and application in breeding

Summary Non-destructive methods for estimation of Crop Growth Rate and Harvest Index of the oil plam (Elaeis guineensisJacq.) are described. These methods were applied in a selection experiment, and the importance of considering Harvest Index, Leaf Area Index and planting density in selecting for increased yields is discussed.