Effect of VRN‐1 and PPD‐D1 genes on heading time in European bread wheat cultivars

The variation of the vernalization (VRN‐1) and photoperiod (PPD‐1) genes offers opportunities to adjust heading time and to maximize yield in crop species. The effect of these genes on heading time was studied based on a set of 245 predominantly spring cultivars of bread wheat from the main eco‐geographical regions of Europe. The genotypes were screened using previously published diagnostic molecular markers for detecting the dominant or recessive alleles of the major VRN‐1 loci such as: VRN‐A1, VRN‐B1, VRN‐D1 as well as PPD‐D1. We found that 91% of spring wheat cultivars contain the photoperiod sensitive PPD‐D1b allele. Photoperiod insensitive PPD‐D1a allele has been found mainly in southern region of Europe. For this region the monogenic control of vernalization by VRN‐B1 or VRN‐D1 dominant alleles is common, whereas in the remaining part of Europe, the combination of photoperiod sensitive PPD‐D1b allele with dominant VRN‐A1, VRN‐B1 and recessive vrn‐D1 alleles represents the most frequent genotype. Also, we revealed a significantly later (5–8 days) heading of the monogenically dominant genotypes at VRN‐B1 as compared to the digenic VRN‐A1 VRN‐B1 genotypes.     

A mutation in Waxy gene affects amylose content,starch granules and kernel characteristics of barley (Hordeum vulgare)

Waxy barley referred to as low‐amylose or amylose‐free has special advantages in nutrition composition and food processing. Waxy gene encoding granule–bound starch synthase I (GBSSI) is responsible for amylose synthesis in barley. The G³⁹³⁵‐to‐T in Waxy gene has been previously found in amylose–free barley. In this study, G³⁹³⁵‐to‐T was proved to co‐segregate with the waxy phenotype of barley, but has no obvious effect on GBSSI catalytic activity and starch chain length distribution. However, recombinant inbred lines with G³⁹³⁵‐to‐T in Waxy gene are of significant modification in starch granules morphology and pasting properties, increase of grain β‐glucan content, and decrease of thousand kernel weight along with lower kernel width. A polymerase chain reaction with confronting two–pair primers marker was developed for economic and efficient screening of G³⁹³⁵‐to‐T. This study provides the basis for cultivar improvement of waxy barley then fully developing its potential value and utility in food processing.     

Quantitative trait loci mapping of freezing tolerance and photosynthetic acclimation to cold in winter two‐ and six‐rowed barley

Photoacclimation (PA) and freezing tolerance (FT) have been identified as closely related traits, due to common mechanisms of environmental control. In this study, diversity array technology (DArT) was used for identification of the quantitative trait loci (QTL) of FT and PA in winter barley. Simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) markers were subsequently used to saturate QTL regions. Two F₂ mapping populations were created, for two‐rowed (P44) and six‐rowed barley (CaP). Different regions of the genome were responsible for differences in traits between parents in these two populations. Eleven QTLs were identified in the P44 population, including five typical for FT and PA, on chromosomes 2H, 3H and 7H. In the CaP population, only one QTL connected with PA and 10 connected with FT were found on all chromosomes except 2H. Our results demonstrate that different sets of markers should be applied in marker‐assisted selection for FT in two‐ and six‐rowed barley, as several loci determine FT at the level of biparental crosses.     

The effect of VRN1 genes on important agronomic traits in high‐yielding Canadian soft white spring wheat

For reproductive success, flowering time must synchronize with favourable environmental conditions. Vernalization genes play a major role in accelerating or delaying the time to flowering. We studied how different vernalization (VRN1) gene combinations alter days to flowering and maturity and consequently the effect on grain yield and other agronomic traits. The study focussed on the effect of the VRN1 gene series (Vrn‐A1, Vrn‐B1 and Vrn‐D1) and their combinations. The Vrn gene group Vrn‐A1a, Vrn‐B1, vrn‐D1 was the earliest to flower and mature, while Vrn‐A1b, Vrn‐B1, vrn‐D1 was the latest to flower. Spring wheat lines with vrn‐A1, Vrn‐B1, Vrn‐D1 were the highest yielding and matured at a similar time as those having vernalization genes Vrn‐A1a, Vrn‐B1 and Vrn‐D1. The findings of this study suggest that the presence of Vrn‐D1 has a direct or indirect role in producing higher grain yield. We therefore suggest the introduction of Vrn‐D1 allele into higher‐yielding classes within Canadian spring wheat germplasm.     

Evaluation of the waxy endosperm trait in proso millet (Panicum miliaceum)*

The entire USDA‐ARS maintained collection of 650 accessions of proso millet (Panicum miliaceum L.) was evaluated for the presence of accessions with waxy (amylose‐free) endosperm starch. Six accessions, five of which derived from mainland China, were identified. Segregation ratios for waxy endosperm were evaluated in F₂ and F₃ populations derived from crosses between two waxy accessions, PI 436625 (Lung Shu 16) and PI 436626 (Lung Shu 18), and several wild‐type accessions. The waxy trait was found to be under the control of duplicate recessive alleles at two loci, herein designated wx‐1 and wx‐2. Wild‐type alleles at these loci were designated Wx‐1 and Wx‐2. Iodine‐binding revealed a mean grain‐starch amylose concentration of 3.5% in waxy lines and 25.3% in wild‐type proso. Expression of the granule‐bound starch synthase (waxy protein) in waxy lines was reduced to approximately one‐tenth that of wild‐type accessions. The waxy accessions identified now are available for the introgression of this trait into breeding lines adapted to the Great Plains of North America.     

Marker‐assisted backcrossing of a null allele of the α‐subunit of Soybean (Glycine max) β‐conglycinin with a Chinese soybean cultivar (a). The development of improved lines

The development of soybean varieties that lack the β‐conglycinin α‐subunit is an attractive goal because the β‐conglycinin α‐subunit negatively influences the nutrition and gelation of tofu and is a major allergen. To remove this undesirable allergen and simultaneously improve the seed nutritional value and food‐processing quality, marker‐assisted background selection (MABS) was used in backcross breeding to incorporate cgy‐2, a null phenotype version of the gene encoding the β‐conglycinin α‐subunit, from the donor line ‘RiB’ into the genetic background of the Chinese cultivar ‘Dongnong47’ (DN47), a popular high‐oil superfine seed soybean cultivar from Heilongjiang Province, China. In each F₂ (F₂, BC_nF₂) generation of the breeding programme, the offspring that carried the introgressed cgy‐2 were identified by sodium dodecyl sulphate–polyacrylamide gel electrophoresis and rescreened by MABS using simple sequence repeat markers to accelerate recurrent parent genome recovery. Of the 49 advanced backcrossing breeding lines (ABLs), the three best lines, ABL1, ABL2 and ABL3, were selected from the BC₁, BC₂ and BC₃ populations, respectively. The ABLs were evaluated for desirable agronomic characteristics, yield‐related traits, amino acid composition, free amino acid composition and tofu‐processing quality in the mature seeds. All of the ABLs lacked the α‐subunit but grew and reproduced normally without deleterious effects on physiological processes such as seed development and germination. The free amino acid content of ABL1 was significantly higher than that of ‘DN47’, with arginine (Arg) being particularly enriched. Compared to the recurrent parent ‘DN47’, the total protein content of the three ABLs was higher, the amino acid composition of the seed proteins was markedly modified and the yield and hardness of the tofu that was made from the ABLs were significantly increased. MABS combined with stringent phenotypic selection in a backcross breeding programme is a feasible strategy for the genetic engineering of seed protein components to produce allergenic subunit‐deficient variant alleles.     

Genome‐wide association analysis reveals flowering‐related genes regulating rachis length in rice

Rachis length is correlated with panicle size in rice. Unveiling the genetic basis of rachis length is important for understanding the genetic regulation of panicle size. In this study, we performed a genomewide association study of rachis length using 529 rice accessions from two environments. In total, 20 loci were identified for rachis length and distributed across the 12 chromosomes except for chromosomes 2 and 5. Thirteen of the 20 loci were not linked to the cloned panicle size genes. Six flowering‐related genes, including the CCT domain‐containing genes Ghd7, Ghd7.1, Hd1, OsCCT1 and OsCO3 as well as Ehd1, were associated with rachis length in this study. These findings suggest that the network of these flowering‐related genes probably participates in the regulation of rachis length and thus affects panicle length and yield. Interestingly, haplotype analysis showed that OsCCT1 is a putative candidate gene which plays a key role in the variation of rachis length. Together, the genetic loci identified in this study could be used for rice breeding by marker‐assisted selection to improve grain yield.     

Marker-assisted backcross introgression of the Yd2 gene conferring resistance to barley yellow dwarf virus in barley

YLM, a codaominant polymerase chain reaction (PCR) marker linked to Yd2, could substantially improve the precision and efficiency of barley yellow dwarf virus (BYDV) resistance breeding. The aim of this study was to assess the effectiveness of YLM in a marker‐assisted introgression programme and to quantify associations between the presence of Yd2 and other agronomic and quality traits. The Yd2 gene was introgressed into a BYDV‐susceptible background through two cycles of marker‐assisted backcrossing. BC₂ F₂‐derived lines, either carrying or not carrying the YLM allele associated with resistance, were compared in the presence and absence of BYDV. The YLM marker was shown to be effective in the introgression of Yd2. Lines carrying the YLM allele associated with resistance produced significantly fewer leaf symptoms and showed a reduction in yield loss when infected with BYDV. There were no deleterious effects associated with the introgression of Yd2 on grain yield, grain size or malting quality. The implications of marker‐assisted selection for Yd2 on barley improvement are discussed.     

Mapping and validation of the quantitative trait loci for leaf stay‐green‐associated parameters in maize

Functional stay‐green is generally regarded as a desirable trait of varieties in major crops including maize. In this study, we used an F_3:4 recombinant inbred line population with 165 lines from a cross between a stay‐green inbred line (Zheng58) and a model inbred line (B73) using 211 polymorphic simple sequence repeat markers to map quantitative trait loci for three stay‐green‐associated parameters, chlorophyll content, photosystem II photochemical efficiency and stay‐green area, at maturity stage, detected a total of 23 quantitative trait loci (QTL) on nine chromosomes. Single QTL explained 3.7–13.5% of the phenotypic variance. Additionally, we validated some important stay‐green QTL using a heterogeneous inbred family approach and found that the stay‐green‐associated parameters were significantly correlated with the plant yield. This study may contribute to a better insight into the regulatory mechanism behind leaf stay‐green in maize and a novel development of elite maize varieties with delayed leaf senescence through molecular marker‐assisted selection.     

Selection of soybean elite cultivars based on phenotypic and genomic characters related to lodging tolerance

Soybean lodging can result in serious yield reduction. Detecting the quantitative trait loci (QTL) associated with lodging tolerance for their further application in marker‐assisted selection (MAS) has the potential to enhance soybean breeding efficiency. In this study, a genome‐wide association analysis (GWAS) was performed to identify soybean accessions that could potentially be used to produce lodging‐tolerant varieties, based on the comprehensive evaluation of lodging scores (LS) obtained for the parental cultivar “Tokachi nagaha” and its 137 derived cultivars. Results showed that genotype, environment and genotype × environment interaction significantly influenced LS. Of the 31 significant SNPs identified, 22 were consistently detected in two or more environments and 27 SNPs were located in or close to agronomically important QTL mapped by linkage analysis. Best linear unbiased predictors (BLUPs) of LS tend to decrease with the elite alleles contained by accessions increasing. Some excellent accessions, with lower BLUPs and D_i (stability coefficients) values and more elite alleles, were selected. This study contributed to understand the genetic mechanism of lodging, providing genetic and phenotypic information for MAS.     

QTL for maize grain yield identified by QTL mapping in six environments and consensus loci for grain weight detected by meta‐analysis

Grain yield is the most important and complicated trait in maize. In this study, a total of 498 recombinant inbred lines (RIL) derived from a biparental cross of two elite inbred lines, 178 and P53, were grown in six different environments. Quantitative trait locus (QTL) mapping was conducted for three grain yield component traits (100 grain weight, ear weight and kernel weight per plant). Subsequently, meta‐analysis was performed after a comprehensive review of the research on QTL mapping for grain weight (100, 300 and 1000) using BioMercator V4.2. In total, 62 QTLs were identified for 100 grain weight, ear weight and kernel weight per plant in six environments. Forty‐three meta‐QTLs (MQTLs) were detected by meta‐analysis. A total of 13 candidate genes homologous to eight functionally characterized rice genes were found, and four candidate genes were located in the two hot spot regions of MQTL1.5 and MQTL2.3. Our results suggest that the combination of literature collection, meta‐analysis and homologous blast searches can offer abundant information for further fine mapping, marker‐assisted selection (MAS) breeding and map‐based cloning for maize.     

Crop breeding for salt tolerance in the era of molecular markers and marker‐assisted selection

Crop salt tolerance (ST) is a complex trait affected by numerous genetic and non‐genetic factors, and its improvement via conventional breeding has been slow. Recent advancements in biotechnology have led to the development of more efficient selection tools to substitute phenotype‐based selection systems. Molecular markers associated with genes or quantitative trait loci (QTLs) affecting important traits are identified, which could be used as indirect selection criteria to improve breeding efficiency via marker‐assisted selection (MAS). While the use of MAS for manipulating simple traits has been streamlined in many plant breeding programmes, MAS for improving complex traits seems to be at infancy stage. Numerous QTLs have been reported for ST in different crop species; however, few commercial cultivars or breeding lines with improved ST have been developed via MAS. We review genes and QTLs identified with positive effects on ST in different plant species and discuss the prospects for developing crop ST via MAS. With the current advances in marker technology and a better handling of genotype by environment interaction effects, the utility of MAS for breeding for ST will gain momentum.     

Efficient development of Haynaldia villosa chromosome 6VS‐specific DNA markers using a CISP‐IS strategy

Development of effective molecular markers linked to Pm21 deriving from Haynaldia villosa is critical for wheat breeding of powdery mildew resistance. In this study, we designed 12 pairs of conserved‐intron scanning primers (CISPs), using intron‐containing conserved genes located on the short arm of Brachypodium distachyon chromosome 3 (3BdS) aligned with cDNA or expressed sequence tags (ESTs) of Triticeae crops. Of 12 CISP primer pairs, 11 amplified DNA both in H. villosa and in wheat, and four displayed H. villosa chromosome 6VS‐specific polymorphisms. Six non‐polymorphic DNAs were further sequenced for designing internal primers, and five additional 6VS‐specific markers were obtained. Of the total nine 6VS‐specific co‐dominant markers, six could effectively trace Pm21 in F₂ population derived from the hybrid between the T6AL.6VS line and ‘Yangmai 158’. This study demonstrated that Brachypodium genomic information could be powerfully utilized to develop molecular markers in H. villosa or other Triticeae species.     

Development of a kompetitive allele‐specific PCR marker for selection of the mutated Wx‐D1d allele in wheat breeding

Waxy (Wx) protein is a key enzyme for synthesis of amylose in endosperm. Amylose content in wheat grain influences the quality of end‐use products. Seven alleles have been described at the Wx‐D1 locus, but only two of them (Wx‐D1b, Wx‐D1e) were genotyped with codominant markers. The waxy wheat line K107Wx1 developed by treating ‘Kanto 107’ seeds with ethyl methanesulphonate carries the Wx‐D1d allele. However, no molecular basis supports this nomenclature. In the present study, DNA sequence analysis confirmed that a single nucleotide polymorphism in the sixth exon of Wx‐D1 changed tryptophan at position 301 into a termination codon. Based on this sequence variation, a PCR‐based KASP marker was developed to detect this point mutation using 68 BC₈F₁ plants and 297 BC₈F₂ lines derived from the cross ‘Ningmai 14’*9/K107Wx1. Combined with codominant markers for the Wx‐A1 and Wx‐B1 alleles, waxy and non‐waxy near‐isogenic lines were distinguished. The KASP marker was efficient in identifying the mutant allele and can be used to transfer waxiness to elite lines.     

Marker‐assisted introgression of rare allele of β‐carotene hydroxylase (crtRB1) gene into elite quality protein maize inbred for combining high lysine,tryptophan and provitamin A in maize

Vitamin A deficiency in humans is a widespread health problem. Quality protein maize (QPM) is a popular food rich in lysine and tryptophan, but poor in provitamin A (proA). Here, we report the improvement of an elite QPM inbred, HKI1128Q for proA using marker‐assisted introgression of crtRB1‐favourable allele. HKI1128 was one of the parental lines of three popular hybrids in India and was converted to QPM in our earlier programme. Severe segregation distortion for crtRB1 was observed in BC₁F₁, BC₂F₁ and BC₂F₂. Background selection by 100 SSRs revealed mean recovery of 91.07% recurrent parent genome varying from 88.78% to 93.88%. Across years, introgressed progenies possessed higher mean β‐carotene (BC: 9.22 µg/g), β‐cryptoxanthin (BCX: 3.05 µg/g) and provitamin A (proA: 10.75 µg/g) compared to HKI1128Q (BC: 2.26 µg/g, BCX: 2.26 µg/g and proA: 3.38 µg/g). High concentration of essential amino acids, viz. lysine (mean: 0.303%) and tryptophan (0.080%) in endosperm, was also retained. Multi‐year evaluation showed that introgressed progenies possessed similar grain yield (1,759–1,879 kg/ha) with HKI1128Q (1,778 kg/ha). Introgressed progenies with higher lysine, tryptophan and proA hold immense potential as donors and parents in developing biofortified hybrids.     

Rapid and convenient gel‐free screening of SCAR markers in wheat using SYBR green‐based melt‐profiling

Sequence characterized amplified region (SCAR) markers that are highly desirable in crop breeding for marker‐assisted selection (MAS) are routinely analysed by gel‐based methods that are low‐throughput, time‐consuming and laborious. In this study, we showed a rapid and convenient method for analysis of SCAR markers in a gel‐free manner. Seven SCAR markers, linked to rust resistance genes (Sr24, Sr26 and Sr31) and seed quality traits (Pina, Pinb and Glu‐D1) in wheat (Triticum aestivum), were amplified on a real‐time PCR machine using custom reaction mixture. Subsequently, melting curve analysis was performed, to assess the specificity of amplicons. Using the amplicon‐specific melt‐profiles, the presence/absence of SCAR markers was analysed in fifteen genotypes and five F₂ populations. Unlike the fluorescence‐based in‐tube detection methods, the present method used the amplicon‐specific melt‐profiles to evaluate the status of the SCAR markers, thus eliminating the need for gel‐based analysis. Results also showed feasibility of multiplex analysis of two markers with well‐separated melting profiles. Overall, the approach is a rapid, convenient and cost‐effective method for high‐throughput screening of SCAR markers.     

Detection of QTLs for bread‐making quality in wheat using a recombinant inbred line population

Whereas gluten fraction accounts for 30–60% of the variation in wheat bread‐making quality, there remains substantial variation determined by non‐gluten factors. The objective of this study was to detect new loci for wheat quality. The genetics of sodium dodecyl sulphate‐sedimentation volume (Ssd), grain hardness (GH), grain protein content, wet gluten content (WGC) and water absorption (Abs) in a set of 198 recombinant inbred lines derived from two commercial varieties was studied by quantitative trait loci (QTL) analysis. A genetic map based on 255 marker loci, consisting of 250 simple sequence repeat markers and five glutenin loci, Glu‐A1, Glu‐B1, Glu‐D1, Glu‐B3 and Glu‐D3, was constructed. A total of 73 QTLs were detected for all traits. A major QTL for GH was detected on chromosome 1B and its relative contribution to phenotypic variation was 27.7%. A major QTL for Abs on chromosome 5D explained more than 30% of the phenotypic variation. Variations in Ssd were explained by four kinds of genes. Some QTLs for correlated traits mapped to the same regions forming QTL clusters or indicated pleiotropic effects.     

Breeding cowpea for resistance to Striga gesnerioides in the Nigerian dry savannas using marker‐assisted selection

Historically, conventional breeding has been the primary strategy used to develop a number of Striga‐resistant varieties currently grown in the Sahel of Western Africa. In this study, we have successfully developed and applied a marker‐assisted selection strategy that employs a single backcross programme to introgress Striga resistance into farmer preferred varieties of cowpea for the Nigeria savannas. In this strategy, we have introduced the Striga resistance gene from the donor parent IT97K‐499‐35 into an elite farmer preferred cowpea cultivar ‘Borno Brown’. The selected 47 BC₁F₂ populations confirmed the recombinants with desirable progeny having Striga resistance gene(s). The 28 lines selected in the BC₁F_2:4 generation with large seed size, brown seed coat colour and carrying marker alleles were evaluated in the field for resistance to Striga resistance. This led to the selection of a number of desirable improved lines that were immune to Striga having local genetic background with higher yield than those of their parents and standard varieties.     

Haplotype structure around the nud locus in barley and its association with resistance to leaf stripe (Pyrenophora graminea)

The naked/hulled kernel trait is controlled in barley by a single gene called nud, on chromosome 7H. The first aim of this work was use bulked segregant analysis to find, new PCR‐based markers linked to nud for marker‐assisted selection (MAS). A new SCAR marker (sJ14) was developed, which is useful for introgressing the naked trait. This, and three other SCARs, were placed on the ‘Proctor’ × ‘Nudinka’ map to detail a 0.9‐cM fragment tagging nud. In order to evaluate the haplotypes around the nud locus, a phenotypically differentiated collection of naked/hulled genotypes was characterized by means of the above markers. Eight different marker haplotypes were found in the breeding germplasm, and a new allele for the marker sKT7 was found. The same barley collection has been surveyed for resistance/susceptibility to leaf stripe (Pyrenophora graminea), in order to investigate any possible association between this and other traits. The naked/hulled seed trait was not associated with resistance/susceptibility to the fungus.