Concurrent Disruption of Genetic Interference and Increase of Genetic Recombination Frequency in Hybrid Rice Using CRISPR/Cas9

Manipulation of the distribution and frequency of meiotic recombination events to increase genetic diversity and disrupting genetic interference are long-standing goals in crop breeding. However, attenuation of genetic interference is usually accompanied by a reduction in recombination frequency and subsequent loss of plant fertility. In the present study, we generated null mutants of the ZEP1 gene, which encodes the central component of the meiotic synaptonemal complex (SC), in a hybrid rice using CRISPR/Cas9. The null mutants exhibited absolute male sterility but maintained nearly unaffected female fertility. By pollinating the zep1 null mutants with pollen from indica rice variety 93-11, we successfully conducted genetic analysis and found that genetic recombination frequency was greatly increased and genetic interference was completely eliminated in the absence of ZEP1. The findings provided direct evidence to support the controversial hypothesis that SC is involved in mediating interference. Additionally, the remained female fertility of the null mutants makes it possible to break linkage drag. Our study provides a potential approach to increase genetic diversity and fully eliminate genetic interference in rice breeding.

Von Genen zu Chromosomen: Pflanzenzüchtung mit CRISPR-CAS

Using the CRISPR-Cas system, it has been possible to introduce different kinds of mutations in single or multiple genes for trait improvement in crops. Last year, for the first time, the CRISPR-Cas-mediated induction of different kinds of targeted heritable chromosomal rearrangements has been achieved in plants. This novel application has the potential to revolutionize plant breeding as genetic exchange and linkage drag are now becoming controllable in a targeted manner.

A gap-free tomato genome built from complementary PacBio and Nanopore long DNA sequences reveals extensive linkage drag during breeding

The assembly and scaffolding of plant crop genomes facilitates the characterization of genetically diverse cultivated and wild germplasm. The cultivated tomato has been improved through the introgression of genetic material from related wild species, including resistance to pandemic strains of Tobacco Mosaic virus (TMV) from Solanum peruvianum. Here we applied PacBio HiFi and ONT nanopore sequencing to develop independent, highly contiguous and complementary assemblies of an inbred TMV-resistant tomato variety. We merged the HiFi and ONT assemblies to generate a long-read-only assembly where all twelve chromosomes were represented as twelve contiguous sequences (N50=68.5 Mbp). The merged assembly was validated by chromosome conformation capture data and is highly consistent with previous tomato assemblies that made use of genetic maps and HiC for scaffolding. Our long-read-only assembly reveals that a complex series of structural variants linked to the TMV resistance gene likely contributed to linkage drag of a 64.1 Mbp region of the S. peruvianum genome during tomato breeding. We show that this minimal introgression region is present in six cultivated tomato hybrid varieties developed in three commercial breeding programs. Our results suggest that complementary long read technologies can facilitate the rapid generation of near complete genome sequences.

Mapping Recombination Landscape and Basidial Spore Number in the Button Mushroom Agaricus bisporus

The button mushroom Agaricus bisporus is represented mainly by two varieties, a secondarily homothallic variety with predominantly two heterokaryotic spores per basidia and a heterothallic variety with predominantly four homokaryotic spored basidium. Both varieties also differ in their recombination landscape with the former showing crossovers (CO) predominantly at chromosome ends whereas the latter has a more evenly distribution of CO over the chromosomes. The two varieties are compatible, and this has been used to study segregation of the basidial spore number (BSN) and the genomic positions of recombination, i.e., the CO landscape, in order to find the underlying genetic determinants. Knowledge on genes controlling CO positions might facilitate either the conservation of favorable allele combinations or the disruption of unwanted allele combinations to reduce linkage drag. For BSN, in total seven QTL were found with the major QTL on chromosome 1 explaining ca. 55% of the phenotypic variation. It appeared, however, difficult to map the recombination landscape. This phenotype can only be assessed in the meiotic offspring of an intervarietal hybrid which is a laborious and difficult task. Nevertheless, this was done, and we were able to map three QTLs for this trait, two on chromosome 1 and one on chromosome 2 not overlapping with the QTL for BSN. The hurdles encountered are discussed and a new strategy is proposed that can solves these. We propose to use two genetically unrelated mapping populations both offspring of a cross between a var. bisporus and a var. burnettii homokaryon and thus segregating both for CO and BSN. Homokaryotic offspring of both populations can be intercrossed without limitation of mating incompatibility and marker homozygosity and the hybrid mushrooms directly used to map BSN. Homokaryotic offspring of these hybrid mushrooms can be genotypes to assess CO positions using next generation sequencing technologies that will solve marker problems encountered, especially for genotyping chromosome ends. This new approach can be a useful strategy for a more efficient breeding strategy for mushrooms in general.

Favorable pleiotropic loci for fiber yield and quality in upland cotton (Gossypium hirsutum)

Upland cotton (Gossypium hirsutum L.) is an important economic crop for renewable textile fibers. However, the simultaneous improvement of yield and fiber quality in cotton is difficult as the linkage drag. Compared with breaking the linkage drag, identification of the favorable pleiotropic loci on the genome level by genome-wide association study (GWAS) provides a new way to improve the yield and fiber quality simultaneously. In our study restriction-site-associated DNA sequencing (RAD-seq) was used to genotype 316 cotton accessions. Eight major traits in three categories including yield, fiber quality and maturation were investigated in nine environments (3 sites × 3 years). 231 SNPs associated with these eight traits (− log10(P) > 5.27) were identified, located in 27 genomic regions respectively by linkage disequilibrium analysis. Further analysis showed that four genomic regions (the region 1, 6, 8 and 23) held favorable pleiotropic loci and 6 candidate genes were identified. Through genotyping, 14 elite accessions carrying the favorable loci on four pleiotropic regions were identified. These favorable pleiotropic loci and elite genotypes identified in this study will be utilized to improve the yield and fiber quality simultaneously in future cotton breeding.

Evaluation and Selection of Interspecific Lines of Groundnut (Arachis hypogaea L.) for Resistance to Leaf Spot Disease and for Yield Improvement

Early and late leaf spot are two devastating diseases of peanut (Arachis hypogaea L.) worldwide. The development of a fertile, cross-compatible synthetic amphidiploid, TxAG-6 ([A. batizocoi × (A. cardenasii × A. diogoi)]4x), opened novel opportunities for the introgression of wild alleles for disease and pest resistance into commercial cultivars. Twenty-seven interspecific lines selected from prior evaluation of an advanced backcross population were evaluated for resistance to early and late leaf spot, and for yield in two locations in Ghana in 2006 and 2007. Several interspecific lines had early leaf spot scores significantly lower than the susceptible parent, indicating that resistance to leaf spot had been successfully introgressed and retained after three cycles of backcrossing. Time to appearance of early leaf spot symptoms was less in the introgression lines than in susceptible check cultivars, but the opposite was true for late leaf spot. Selected lines from families 43-08, 43-09, 50-04, and 60-02 had significantly reduced leaf spot scores, while lines from families 43-09, 44-10, and 63-06 had high pod yields. One line combined both resistance to leaf spot and high pod yield, and several other useful lines were also identified. Results suggest that it is possible to break linkage drag for low yield that accompanies resistance. However, results also suggest that resistance was diluted in many of the breeding lines, likely a result of the multigenic nature of resistance. Future QTL analysis may be useful to identify alleles for resistance and allow recombination and pyramiding of resistance alleles while reducing linkage drag.

Characterization and elimination of linkage-drag associated with Fusarium wilt race 3 resistance genes

Key message Reducing the size of the I-3 introgression resulted in eliminating linkage-drag contributing to increased sensitivity to bacterial spot and reduced fruit size. The I-7 gene was determined to have no effect on bacterial spot or fruit size, and germplasm is now available with both the reduced I-3 introgression and I-7. Abstract Tomato (Solanum lycopersicum) production is increasingly threatened by Fusarium wilt race 3 (Fol3) caused by the soilborne fungus, Fusarium oxysporum f. sp. lycopersici. Although host resistance based on the I-3 gene is the most effective management strategy, I-3 is associated with detrimental traits including reduced fruit size and increased bacterial spot sensitivity. Previous research demonstrated the association with bacterial spot is not due to the I-3 gene, itself, and we hypothesize that reducing the size of the I-3 introgression will remedy this association. Cultivars with I-7, an additional Fol3 resistance gene, are available but are not widely used commercially, and it is unclear whether I-7 also has negative horticultural associations. To characterize the effect of I-3 on fruit size, segregating populations were developed and evaluated, revealing that the large I-3 introgression decreased fruit size by approximately 21%. We reduced the I-3 introgression from 5 to 140 kb through successive recombinant screening and crossing efforts. The reduced I-3 introgression and I-7 were then separately backcrossed into elite Florida breeding lines and evaluated for effects on bacterial spot sensitivity and fruit size across multiple seasons. The reduced I-3 introgression resulted in significantly less bacterial spot and larger fruit size than the large introgression, and it had no effect on these horticultural characteristics compared with Fol3 susceptibility. I-7 was also found to have no effect on these traits compared to Fol3 susceptibility. Together, these efforts support the development of superior Fol3-resistant cultivars and more durable resistance against this pathogen.

Using wild relatives and related species to build climate resilience in Brassica crops

Climate change will have major impacts on crop production: not just increasing drought and heat stress, but also increasing insect and disease loads and the chance of extreme weather events and further adverse conditions. Often, wild relatives show increased tolerances to biotic and abiotic stresses, due to reduced stringency of selection for yield and yield-related traits under optimum conditions. One possible strategy to improve resilience in our modern-day crop cultivars is to utilize wild relative germplasm in breeding, and attempt to introgress genetic factors contributing to greater environmental tolerances from these wild relatives into elite crop types. However, this approach can be difficult, as it relies on factors such as ease of hybridization and genetic distance between the source and target, crossover frequencies and distributions in the hybrid, and ability to select for desirable introgressions while minimizing linkage drag. In this review, we outline the possible effects that climate change may have on crop production, introduce the Brassica crop species and their wild relatives, and provide an index of useful traits that are known to be present in each of these species that may be exploitable through interspecific hybridization-based approaches. Subsequently, we outline how introgression breeding works, what factors affect the success of this approach, and how this approach can be optimized so as to increase the chance of recovering the desired introgression lines. Our review provides a working guide to the use of wild relatives and related crop germplasm to improve biotic and abiotic resistances in Brassica crop species.

Validating a segment on chromosome 7 of japonica for establishing low-cadmium accumulating indica rice variety

Cadmium (Cd) contamination of rice is a serious food safety issue that has recently been gaining significant public attention. Therefore, reduction of Cd accumulation in rice grains is an important objective of rice breeding. The use of favourable alleles of Cd accumulating genes using marker-assisted selection (MAS) is theoretically feasible. In this study, we validated a segment covering OsHMA3-OsNramp5-OsNramp1 on chromosome 7 of japonica for establishing low-cadmium accumulating indica rice variety. The OsHMA3-OsNramp5-OsNramp1jap haplotype significantly decreased grain Cd concentration in middle-season indica genetic background. The improved 9311 carrying the OsHMA3-OsNramp5-OsNramp1jap haplotype with recurrent parent genome recovery of up to 91.6% resulted in approximately 31.8% decrease in Cd accumulation in the grain and with no penalty on yield. There is a genetic linkage-drag between OsHMA3-OsNramp5-OsNramp1 jap and the gene conditioning heading to days (HTD) in the early-season indica genetic background. Because the OsHMA3-OsNramp5-OsNramp1-Ghd7jap haplotype significantly increases grain Cd concentration and prolongs growth duration, the linkage-drag between OsHMA3-OsNramp5-OsNramp1 and Ghd7 should be broken down by large segregating populations or gene editing. A novel allele of OsHMA3 was identified from a wide-compatibility japonica cultivar, the expression differences of OsNramp1 and OsNramp5 in roots might contribute the Cd accumulating variation between japonica and indica variety.

Enhancing backcross programs through increased recombination

Background Introgression of a quantitative trait locus (QTL) by successive backcrosses is used to improve elite lines (recurrent parent) by introducing alleles from exotic material (donor parent). In the absence of selection, the proportion of the donor genome decreases by half at each generation. However, since selection is for the donor allele at the QTL, elimination of the donor genome around that QTL will be much slower than in the rest of the genome (i.e. linkage drag). Using markers to monitor the genome around the QTL and in the genetic background can accelerate the return to the recurrent parent genome. Successful introgression of a locus depends partly on the occurrence of crossovers at favorable positions. However, the number of crossovers per generation is limited and their distribution along the genome is heterogeneous. Recently, techniques have been developed to modify these two recombination parameters. Results In this paper, we assess, by simulations in the context of Brassicaceae, the effect of increased recombination on the efficiency of introgression programs by studying the decrease in linkage drag and the recovery of the recurrent genome. The simulated selection schemes begin by two generations of foreground selection and continue with one or more generations of background selection. Our results show that, when the QTL is in a region that initially lacked crossovers, an increase in recombination rate can decrease linkage drag by nearly ten-fold after the foreground selection and improves the return to the recurrent parent. However, if the QTL is in a region that is already rich in crossovers, an increase in recombination rate is detrimental. Conclusions Depending on the recombination rate in the region targeted for introgression, increasing it can be beneficial or detrimental. Thus, the simulations analysed in this paper help us understand how an increase in recombination rate can be beneficial. They also highlight the best methods that can be used to increase recombination rate, depending on the situation.