Maize participatory breeding in Portugal: Comparison of farmer's and breeder's on‐farm selection

“VASO” is a Portuguese participatory maize breeding project (1984), where several maize landraces such as “Pigarro” have been selected both by a farmer's (phenotypic recurrent selection) and a breeder's approach (S2 lines recurrent selection). The objectives of this study were to determine the phenotypic and genotypic responses to participatory selection using these two different approaches, to clarify to which extent both selection methods preserve genetic diversity, and conclude what is the preferred method to apply in sustainable farming systems. The results, obtained via ANOVA, regression analyses and molecular markers, indicate that for both selection methods, genetic diversity was not significantly reduced, even with the most intensive breeder's selection. Although there were some common outputs, such as the determinated versus indeterminated ears, cob and ear weight ratio per ear and rachis 2, specific phenotypic traits evolved in opposite directions between the two selection approaches. Yield increase was only detected during farmer selection, indicating its interest on PPB. Candidate genes were identified for a few of the traits under selection as potential functional markers in participatory plant breeding.     

User‐friendly markers linked to Fusarium wilt race 1 resistance Fw gene for marker‐assisted selection in pea

Fusarium wilt is one of the most widespread diseases of pea. Resistance to Fusarium wilt race 1 was reported as a single gene, Fw, located on linkage group III. The previously reported AFLP and RAPD markers linked to Fw have limited usage in marker‐assisted selection due to their map distance and linkage phase. Using 80 F₈ recombinant inbred lines (RILs) derived from the cross of Green Arrow × PI 179449, we amplified 72 polymorphic markers between resistant and susceptible lines with the target region amplified polymorphism (TRAP) technique. Marker–trait association analysis revealed a significant association. Five candidate markers were identified and three were converted into user‐friendly dominant SCAR markers. Forty‐eight pea cultivars with known resistant or susceptible phenotypes to Fusarium wilt race 1 verified the marker–trait association. These three markers, Fw_Trap_480, Fw_Trap_340 and Fw_Trap_220, are tightly linked to and only 1.2 cM away from the Fw locus and are therefore ideal for marker‐assisted selection. These newly identified markers are useful to assist in the isolation of the Fusarium wilt race 1 resistance gene in pea.     

Biometrical analysis of alternative plot types for selection in rye

Summary Maximizing the efficiency of selection for yield and baking quality of winter rye (Secale cereale L.) requires reliable knowledge on the pertinent population parameters. This study reports estimates of variances, heritabilities and genetic correlations from a) large (5 m²) drilled (LD) plots, b) micro drilled (MD) plots, and c) one-row plots of 4 spaced plants (SP). Thirty eight single crosses of rye were grown at two locations for two years in LD, MD and SP plots replicated 2, 4, and 6 times, respectively. Genotypic differences were significant in all plot types for all agronomic and quality traits, although estimates of genetic variances were smaller in LD than MD or SP plots for grain yield, 32-spike weight, and kernels per spike. Heritabilities on an entry mean basis (0.62 to 0.95) were similar among plot types, but on a single plot basis estimates were lower for SP than for MD or LD plots. Genetic correlations were high between all three plot types, with correlations between LD and SP plots being approximately 1.0 for falling number, thousand-kernel weight, and bloom date, 0.90 for grain yield, kernels per spike, test weight and height, and 0.70 for tiller number. Based on the high heritabilities achieved in small plots and the close agreement between MD- or SP- with LD-plot performance we conclude that greater use should be made of small plot types when selecting for yield and quality in winter rye.     

Evaluation of a semi‐controlled test as a selection tool for frost tolerance in durum wheat (Triticum durum)

Most durum wheat (Triticum durum) varieties possess only low winter hardiness due to their frost susceptibility. In North America and Central Europe, durum wheat is therefore typically sown in spring to circumvent the local winter conditions. However, the yield potential of durum in these regions could be much better exploited if durum varieties with increased frost tolerance were available, which could be sown in autumn. A factor limiting breeding for increased frost tolerance is the variation in the occurrence of frost stress across years. The ‘Weihenstephaner Auswinterungsanlage’ is a semi‐controlled test that exposes the plants to all weather conditions. Snow coverage of the plants, serving as frost protection, is prevented by the movable glass lid of the semi‐controlled test. In this study, different scorings for frost tolerance based on this semi‐controlled test were evaluated and compared with frost tolerance data in the field. Our results illustrate the potential of the ‘Weihenstephaner Auswinterungsanlage’ as an indirect selection tool for frost tolerance in durum breeding programmes, especially when regular frost tolerance data from the field are not available.     

Generating genetic variation in narrow‐leafed lupin (Lupinus angustifolius L.) for plant architecture by ethyl methanesulfonate mutagenesis

Narrow‐leafed lupin (NLL) is a source of protein and fibre with exceptional functional properties useful for a variety of purposes. In adapted NLL germplasm selected for low alkaloid contents, the genetic base is narrow, thus limiting fast breeding progress. In an EMS‐based mutagenesis programme, we have treated 40,000 seeds of cv. ‘Boruta’ and obtained a variety of phenotypic mutant lines expressing novel growth types with altered plant height and/or branching. In 3‐year field trials at two locations, three of these lines exhibited significantly improved grain yield components compared to the cv. ‘Boruta’. Growth types of these promising lines were shown to be inherited in a monogenic recessive manner and should, thus, be easily fixable by breeding.     

Evaluation of the Ph‐3 gene‐specific marker developed for marker‐assisted selection of late blight‐resistant tomato

Late blight caused by Phytophthora infestans is one of the most destructive diseases of tomato (Solanum lycopersicum L.) that mainly occurs in cool and wet environments. With the spread of the A2 mating type and new clonal lineages, fewer fungicides provide effective control of the disease, which has increased its worldwide threat. Host resistance could contribute significantly to sustainable disease control. Ph‐3 is a race‐specific late blight resistance gene commonly used in commercial tomato breeding. Availability of precise and easy to use gene‐based markers would facilitate selection. In this study, a Ph‐3 on‐gene cleaved amplified polymorphic sequence (CAPS) marker, Ph3.gsm/HincII, was developed based on the published gene sequence of Ph‐3. The effectiveness of the marker was evaluated along with other published Ph‐3 markers using an F₉ recombinant inbred line (RIL) population derived from NC 23E‐2(93) × L3708. Markers Ph3.gsm/HincII and TG328/BstNI accurately genotyped the RIL population for Ph‐3. In addition, Ph3.gsm/HincII was able to differentiate variable susceptible alleles. This reliable codominant DNA marker would be very useful in marker‐assisted selection, particularly for resistance gene pyramiding.     

Oligogenic control of resistance to soil‐borne viruses SBCMV and WSSMV in rye (Secale cereale L.)

Rye production in European growing areas is constrained by the soilborne cereal mosaic virus (SBCMV) and the wheat spindle streak mosaic virus (WSSMV). To date, no European rye cultivars are known to exhibit resistance against these viruses. In this study, we pursued a quantitative trait locus (QTL) mapping strategy to identify genomic regions for resistance to SBCMV and WSSMV in rye. Three populations, each comprising 100 lines segregating for resistance to SBCMV and/or WSSMV, were evaluated for disease response at two years in three locations in Germany where soils are naturally infested with SBCMV and WSSMV. In the combined analysis across environments, one QTL for SBCMV resistance on chromosome 5R explained 31.9% of the phenotypic variation in one of the populations. For WSSMV resistance, one QTL explaining up to 64.0% of the phenotypic variation was detected on chromosome 7R in each of the three populations. On the Triticeae homoeologous group 5, we found evidence for synteny of the major QTL for SBCMV resistance between the wheat and rye genomes.     

Marker‐assisted selection for rice blast resistance genes Pi2 and Pi9 through high‐resolution melting of a gene‐targeted amplicon

The use of host resistance (R) genes is considered the most cost‐effective option to control the rice blast disease. The two allelic R genes Pi2 and Pi9 confer very broad‐spectrum resistance against blast isolates collected worldwide. However, the two genes have not yet been widely deployed in rice breeding programmes. Availability of specific markers for them would facilitate incorporating the two R genes into new rice lines through marker‐assisted selection. Herein, we report the development and utilization of a robust and specific marker for the Pi2 and Pi9. This marker was derived from polymorphisms within the target gene, and achieved simultaneously distinguish Pi2 and Pi9 from other alleles through high‐resolution melting of a small amplicon. With the marker, we were able to transfer the Pi2 into an elite restorer line through marker‐assisted backcrossing, successfully obtained effective resistance to blast disease, and we were also able to, respectively, incorporate the Pi2 and Pi9 with two other R genes. As the additive effect, blast resistance in these stacking lines harbouring three R genes were significantly improved.     

Comparative Study of CaMsrB2 Gene Containing Drought‐Tolerant Transgenic Rice (Oryza sativa L.) and Non‐Transgenic Counterpart

Concerns regarding the safety of transgenic foods have been raised because of possibility of undesirable effects development during genetic engineering. Analysis of phenotypic traits can increase the likelihoods of identifying those unintended effects in dietary composition of the GM crops. Objective of this study was to compare the transgenic lines with their non‐transgenic counterpart. Different vegetative and reproductive traits as well as antioxidant properties were considered to evaluate the transgenic (HV8 and HV23) lines containing CaMsrB2 gene and their non‐transgenic (Ilmi) parent line. Grain size and weight, seed germination, root length, root and shoot dry weight, length and width of flag leaf, plant height, and ligule, stamen and carpel length were not significantly different. Onset and completion of heading in each line occurred almost during the same period. The antioxidant properties in terms of DPPH (1,1‐diphenyl‐2‐picrylhydrazyl) radical scavenging activity and polyphenol content were not statistically different under same treatment condition. The results suggested that the transgenic rice lines containing CaMsrB2 gene were equivalent to their non‐transgenic counterpart without any visible unintended effects.     

RNAi‐based Bean golden mosaic virus‐resistant common bean (Embrapa 5.1) shows simple inheritance for both transgene and disease resistance

Bean golden mosaic virus (BGMV) is the causal agent of bean golden mosaic of common beans. A transgenic bean line that has been developed based on RNA interference to silence the BGMV rep gene showed immunity to the virus. Crosses were done between the transgenic line and six bean cultivars followed by four backcrosses to the commercial cultivars ‘Pérola’ and ‘BRS Pontal’. The transgene locus was consistently inherited from the crosses analysed in a Mendelian fashion in the segregating populations. The disease resistance reaction co‐inherited with the transgene. Nevertheless, the expression of disease resistance displayed a dosage effect phenomenon in the F₁ generation. The analysis of the homozygous near‐isogenic lines in field conditions, under high BGMV disease incidence, indicated that the transgenic lines were completely resistant. These results show the strength of the disease resistance obtained, the stability of the trait across generations and its usefulness in the management of a disease for which there is no reported Phaseolus germplasm with immunity.     

Sexual hybridization between Capsella bursa‐pastoris (L.) Medik (♀) and Camelina sativa (L.) Crantz (♂) (Brassicaceae)

The development of transgenic oilseed Camelina sativa (2n = 40) and the potential for hybridization with its weedy relative Capsella bursa‐pastoris (2n = 36) necessitates a careful evaluation of the reproductive compatibility between the species. Here, we conducted over 1800 crosses (emasculation and manual pollination) to examine the ability of 10 Canadian C. bursa‐pastoris (♀) accessions to hybridize with five accessions of C. sativa (♂). Seven hybrids were confirmed among 586 putative hybrids screened with species‐specific markers, indicating a hybridization rate of 1.5 hybrids per 10 000 ovules pollinated. All seven hybrids had intermediate DNA content compared to their parents, were morphologically distinct, had low (1.9%) pollen fertility and failed to produce selfed or backcrossed seed. Given the abundance of C. bursa‐pastoris along field margins, hybrids will likely be generated in the wild, but they will be unable to establish lineages unless fertility is restored. The large number of crosses and the diversity captured by the use of multiple accessions resulted in strong statistical power and a high degree of confidence in the estimated hybridization rate.     

Inheritance of resistance to the peach root‐knot nematode (Meloidogyne floridensis) in interspecific crosses between peach (Prunus persica) and its wild relative (Prunus kansuensis)

The peach root‐knot nematode, Meloidogyne floridensis (MF), infects majority of available nematode‐resistant peach rootstocks which are mostly derived from peach (Prunus persica) and Chinese wild peach (P. davidiana). Interspecific hybridization of peach with its wild relative, Kansu peach (P. kansuensis), offers potential for broadening the resistance spectrum in standard peach rootstocks. We investigated the inheritance of resistance to MF in segregating populations of peach (‘Okinawa’ or ‘Flordaguard’) × P. kansuensis. A total of 379 individuals from 13 F₂ and BC₁F₁ families were challenged with a pathogenic MF isolate “MFGnv14” and were classified as resistant (R) or susceptible (S) based on root galling intensity. Segregation analyses in F₂ progeny revealed the involvement of a major locus with a dominant or recessive allele determining resistance in progeny segregating 3R:1S and 1R:3S, respectively. Testcrosses with a homozygous‐susceptible peach genotype (‘Flordaguard’ or ‘UFSharp’) confirmed P. kansuensis as a source of new resistance and the heterozygous allelic status of P. kansuensis at the locus conferring resistance to MF. We propose a single‐locus dominant/recessive model for the inheritance of resistance.     

Multiplex PCR genotyping for five bacterial blight resistance genes applied to marker‐assisted selection in rice (Oryza sativa)

Bacterial blight (BB) is the most economically damaging disease of rice in Asia and other parts of the world. In this study, a multiplex PCR genotyping method was developed to simultaneously identify genotypes of five BB resistance genes, Xa4, xa5, Xa7, xa13 and Xa21. The resistance R alleles were amplified using five functional markers (FMs) to generate amplicons of 217, 103, 179, 381 and 595 bp in IRBB66. Amplicons of 198, 107, 87, 391 and 467 bp corresponded to susceptible alleles in Taiwanese japonica rice cultivars. In backcross breeding programmes, the multiplex PCR assay was integrated into selection from a population using BB resistance donor IRBB66 crossed to rice cultivar ‘Tainung82’. Two plants with homozygosity for Xa4, xa5, Xa7, xa13 and Xa21 were selected from 1100 BC₂F₂ plants. In addition, the five BB resistance genes were also accurately identified in F₂ populations. This multiplex PCR method provides a rapid and efficient method for detecting various BB resistance genes, which will assist in pyramiding genes to improve durability of BB resistance in Taiwanese elite rice cultivars.     

Genome‐Wide Identification of MicroRNAs Responsive to High Temperature in Rice (Oryza sativa) by High‐Throughput Deep Sequencing

MicroRNAs (miRNAs) are known to play important roles in plant growth and stress response. Heat stress is a severe abiotic stresses by adversely affecting plant growth and yield. To identify heat‐responsive miRNAs at the genome‐wide level in rice (Oryza sativa), we constructed two small RNA libraries from young panicles treated or not with heat conditions. Ion torrent sequencing of the two libraries identified 294 known miRNAs and 539 novel miRNAs. Differential expression analysis showed that 26 miRNAs were downregulated and 21 miRNAs were upregulated in response to heat stress. Among them, five heat‐responsive miRNAs, including miR162b, miR529a‐p5, PC‐5P‐62245‐9, miR171b and miR169n, were validated by quantitative real‐time polymerase chain reaction. A total of 44 target genes of the differentially expressed miRNAs were predicted. These target genes are most significantly overrepresented in the cell growth process. The results demonstrated that rice miRNAs play critical roles in the heat stress response. This study opens up a new avenue for understanding the regulatory mechanisms of miRNAs involvement in the heat stress response in rice.     

Pyramiding three rust‐resistance genes confers a high level of resistance in soybean (Glycine max)

Pyramiding Asian soybean rust (ASR) resistance (Rpp) genes in a single genotype has been shown to increase ASR resistance in soybean. However, it remains unclear which combinations of Rpp genes are superior. Therefore, here, we developed six new Rpp‐pyramided lines carrying different combinations of Rpp genes and evaluated their resistance against 13 Bangladeshi rust (Phakopsora pachyrhizi) isolates (BdRPs) alongside seven previously developed Rpp‐pyramided lines. We found that lines carrying one, two and three Rpp genes had high ASR resistance without sporulation in 13.8%, 35.2% and 73.1% of the assays, respectively. Among the new lines that were developed, those with Rpp3 + Rpp4 and Rpp3 + Rpp4 + Rpp5 had high levels of ASR resistance, while the line containing Rpp2 + Rpp4 + Rpp5 showed immunity phenotype at two weeks after inoculation by the BdRP‐22 infection. Thus, pyramiding larger numbers of Rpp genes confers soybean with a higher level of resistance to ASR pathogens and can produce an immunity phenotype at two weeks after inoculation.     

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 broad‐spectrum bacterial blight resistance into thermo‐sensitive genic male sterile lines

Two‐line hybrid rice technology is an effective way to increase rice production and improve rice quality. In this study, three bacterial blight (BB) resistance genes, Xa7, Xa21 and Xa23, were introgressed separately into C815S, a popular thermo‐sensitive genic male sterile (TGMS) line to develop five BB‐resistant lines (Hua1005S, Hua1002S, Hua1009S, Hua1006S and Hua1001S) to be resistant against seven races of Xanthomonas oryzae pv. oryzae (Xoo). The two‐line hybrids with heterozygous Xa23 were resistant against seven Xoo strains. But, the hybrids with heterozygous loci for both Xa7 and Xa21 were only resistant against three Xoo strains and were moderately susceptible to the other four strains indicating the role of modifiers influencing the poor expression of dominant BB resistance genes under heterozygous state. Among them, Hua1006S was found to be a promising TGMS line with its higher degree of disease resistance level on account of broad‐spectrum resistance gene Xa23 besides possessing better plant type and rice grain quality features.     

Identification and characterization of sources for photo‐ and thermo‐insensitivity in Vigna species

Cultivation of the same varieties of mungbean and blackgram across different seasons and locations is constrained by their photo‐ and thermo‐sensitive behaviour. Developing insensitive genotypes, which can fit well across all seasons, requires robust donors which would provide genes imparting this trait. This study was undertaken to identify such donors in the Vigna species. Forty‐eight accessions belonging to 13 Vigna species and eight released cultivars were evaluated under natural field conditions. Among these, two accessions, viz. V. umbellata (IC251442) and V. glabrescens (IC251372) were found photo‐ and thermo‐insensitive as these were able to flower and set pods at temperatures as high as 43.9°C and as low as 2.7°C. Pollen viability studies indicated viable pollen (>75% at 2.7°C and >85% at 41.9°C) and normal pollen tube growth at both the extremes of temperature. The identified V. glabrescens accession has long, constricted pods and dark green, mottled seeds while V. umbellata has smooth, curved pods and shining, oval, large seeds. Both these accessions can be utilized in developing photo–thermo insensitive genotypes in cultivated Vigna species.     

QTL mapping of a partial resistance to the corn delphacid‐transmitted viruses in Lepidopteran‐resistant maize line Mp705

A partial resistance to maize mosaic virus (MMV) and maize stripe virus (MStV) was mapped in a RILs population derived from a cross between lines MP705 (resistant) and B73 (susceptible). A genetic map constructed from 131 SSR markers spanned 1399 cM with an average distance of 9.6 cM. A total of 10 QTL were detected for resistance to MMV and MStV, using composite interval mapping. A major QTL explaining 34–41% of the phenotypic variance for early resistance to MMV was detected on chromosome 1. Another major QTL explaining up to 30% of the phenotypic variation for all traits of resistance to MStV was detected in the centromeric region of chromosome 3 (3.05 bin). After adding supplementary SSR markers, this region was found to correspond well to the one where a QTL of resistance to MStV already was located in a previous mapping study using an F₂ population derived from a cross between Rev81 and B73. These results suggested that these QTL of resistance to MStV detected on chromosome 3 could be allelic in maize genome.     

S‐locus diversity and cross‐compatibility of wild Prunus avium for timber breeding

Prunus avium is primarily cultivated for its fruit, sweet cherries. However, it is also used to produce high‐quality timber. In a P. avium seed orchard, gametophytic self‐incompatibility is a restriction for free pollen flow and should be considered when establishing basic forest materials. In this study, S‐locus diversity and cross‐incompatibility of wild cherry individuals in clonal banks established for breeding for timber production were investigated. Wild cherry trees (140) with outstanding forest growth habit, collected in northern Spain, grafted and planted in two clonal banks, were genotyped at the S‐locus. The self‐incompatibility S‐locus genes, S‐RNase and SFB, were analysed by PCR. Twenty‐two S‐haplotypes, resulting in 72 different S‐genotypes, were identified. The genotypes were grouped into 33 incompatibility groups and 39 unique genotypes. This initial S‐locus analysis revealed large genetic diversity of wild cherry trees from the Spanish northern deciduous forest, and provides useful information for seed orchard design. Wild P. avium displays significantly more genetic diversity than what is detected in local cultivars, revealing a narrowing of genetic diversity during local domestication.