Identification of Self-Incompatibility Alleles by Specific PCR Analysis and S-RNase Sequencing in Apricot.

Self-incompatibility (SI) is one of the most efficient mechanisms to promote out-crossing in plants. However, SI could be a problem for fruit production. An example is apricot (Prunus armeniaca), in which, as in other species of the Rosaceae, SI is determined by an S-RNase-based-Gametophytic Self-Incompatibility (GSI) system. Incompatibility relationships between cultivars can be established by an S-allele genotyping PCR strategy. Until recently, most of the traditional European apricot cultivars were self-compatible but several breeding programs have introduced an increasing number of new cultivars whose pollination requirements are unknown. To fill this gap, we have identified the S-allele of 44 apricot genotypes, of which 43 are reported here for the first time. The identification of Sc in 15 genotypes suggests that those cultivars are self-compatible. In five genotypes, self-(in)compatibility was established by the observation of pollen tube growth in self-pollinated flowers, since PCR analysis could not allowed distinguishing between the Sc and S8 alleles. Self-incompatible genotypes were assigned to their corresponding self-incompatibility groups. The knowledge of incompatibility relationships between apricot cultivars can be a highly valuable tool for the development of future breeding programs by selecting the appropriate parents and for efficient orchard design by planting self-compatible and inter-compatible cultivars.

Self-(in)compatibility in apricot germplasm is controlled by two major loci, S and M.

BACKGROUND: Apricot (Prunus armeniaca L.) exhibits a gametophytic self-incompatibility (GSI) system and it is mostly considered as a self-incompatible species though numerous self-compatible exceptions occur. These are mainly linked to the mutated S C-haplotype carrying an insertion in the S-locus F-box gene that leads to a truncated protein. However, two S-locus unlinked pollen-part mutations (PPMs) termed m and m' have also been reported to confer self-compatibility (SC) in the apricot cultivars 'Canino' and 'Katy', respectively. This work was aimed to explore whether other additional mutations might explain SC in apricot as well. RESULTS: A set of 67 cultivars/accessions with different geographic origins were analyzed by PCR-screening of the S- and M-loci genotypes, contrasting results with the available phenotype data. Up to 20 S-alleles, including 3 new ones, were detected and sequence analysis revealed interesting synonymies and homonymies in particular with S-alleles found in Chinese cultivars. Haplotype analysis performed by genotyping and determining linkage-phases of 7 SSR markers, showed that the m and m' PPMs are linked to the same m 0-haplotype. Results indicate that m 0-haplotype is tightly associated with SC in apricot germplasm being quite frequent in Europe and North-America. However, its prevalence is lower than that for S C in terms of frequency and geographic distribution. Structures of 34 additional M-haplotypes were inferred and analyzed to depict phylogenetic relationships and M 1-2 was found to be the closest haplotype to m 0. Genotyping results showed that four cultivars classified as self-compatible do not have neither the S C- nor the m 0-haplotype. CONCLUSIONS: According to apricot germplasm S-genotyping, a loss of genetic diversity affecting the S-locus has been produced probably due to crop dissemination. Genotyping and phenotyping data support that self-(in)compatibility in apricot relies mainly on the S- but also on the M-locus. Regarding this latter, we have shown that the m 0-haplotype associated with SC is shared by 'Canino', 'Katy' and many other cultivars. Its origin is still unknown but phylogenetic analysis supports that m 0 arose later in time than S C from a widely distributed M-haplotype. Lastly, other mutants putatively carrying new mutations conferring SC have also been identified deserving future research.

Genetic variation for pseudo-self-compatibility in self-incompatible populations of Leavenworthia alabamica (Brassicaceae).

Self-incompatibility (SI) promotes outcrossing, but transitions to self-compatibility (SC) are frequent. Population genetic theory describing the breakdown of SI to SC suggests that, under most conditions, populations should be composed of either SI or SC individuals. Under a narrow range of conditions, theory suggests that SI may persist alongside reduced expression of SI (pseudo-SI, PSI) in mixed-mating populations. We studied genetic variation for PSI segregating in four SI populations of Leavenworthia alabamica by measurement of the heritability of pollen tube number after self-pollination. We tested for the role of the S-locus in this variation by sequencing seven S-alleles from plants with high pseudo-SC (PSC) and testing for the co-segregation of these alleles with PSC. We found a continuous distribution of PSC in all populations and 90% of plants exhibited PSC. The heritability ranged from 0.39 to 0.57. All seven S-alleles from plants with high PSC exhibited trans-specific polymorphism, and no stop codons were observed within the c. 600-bp region sequenced. One of these S-alleles was directly associated with the inheritance of PSC. We conclude that heritable variation in PSC is largely a result of genetic variation in the signaling cascade downstream of the S-locus reaction, together with the presence of one leaky S-allele.

Synergistic approach of PCR-based fragment length analysis and amplicon deep sequencing reveals rich diversity of S-alleles in sweet cherries from the Caucasian region of origin.

Introduction: The self-incompatibility system in sweet cherry (Prunus avium L.) prevents fertilization with own or genetically related pollen, and is genetically determined by the multi-allelic S-locus. Therefore, determining S-alleles is crucial for plant breeding and fruit production, as it enables the selection of compatible combinations of S-genotypes for successful pollination. Methods: In this study, S-alleles were identified in a total of 260 genotypes from the Caucasian region, the species' center of origin. S-allele genotyping was conducted using PCR fragment length analysis with the standard marker PaConsI-F/R2 and reference genotypes, complemented by sequence analysis through amplicon deep sequencing. Results and discussion: The genotypes collected from Azerbaijan and Turkey exhibit a high allelic richness at the S-locus, particularly compared to modern sweet cherry cultivars worldwide. Nine previously undescribed S-alleles were identified and designated as S45, S46, S47, S48, S49, S50, S51, S52 and S53. Given the expected high diversity for other traits, this plant material represents a valuable resource for further breeding research and introgression of new traits in future breeding programs. Furthermore, our results underscore that fragment length alone may not be sufficient for unambiguous assignment of S-alleles due to minimal length differences between different alleles. To address this issue, an S-allele reference ladder was developed using the rich diversity for precise assignment of the S-alleles. This tool can be applied in future experiments as a robust and cost-effective method for accurate S-genotyping across different runs and laboratories. Additionally, several selected S-genotypes were planted in a trial field and will be maintained as an S-allele reference collection.

Real-time PCR detection of PI*S and PI*Z alleles of SERPINA1 gene using SYBR green.

BACKGROUND: Alpha-1 antitrypsin deficiency is an underdiagnosed genetic condition that predisposes to pulmonary complications and is mainly caused by rs28929474 (PI*Z allele) and rs17580 (PI*S allele) mutations in the SERPINA1 gene. OBJECTIVE: Development of a homogeneous genotyping test for detection of PI*S and PI*Z alleles based on the principles of allele-specific PCR and amplicon melting analysis with a fluorescent dye. METHODS: Sixty individuals, which included all possible genotypes that result from combinations of rs28929474 and rs17580 single nucleotide variants, were assayed with tailed allele-specific primers and SYBR Green dye in a real-time PCR machine. RESULTS: A clear discrimination of mutant and wild-type variants was achieved in the genetic loci that define PI*S and PI*Z alleles. Specific amplicons showed a difference of 2.0 °C in melting temperature for non-S and S variants and of 2.9 °C for non-Z and Z variants. CONCLUSIONS: The developed genotyping method is robust, fast, and easily scalable on a standard real-time PCR platform. While it overcomes the handicaps of non-homogeneous approaches, it greatly reduces genotyping costs compared with other homogeneous approaches.

Self-Incompatibility in Apricot: Identifying Pollination Requirements to Optimize Fruit Production.

In recent years, an important renewal of apricot cultivars is taking place worldwide, with the introduction of many new releases. Self-incompatible genotypes tolerant to the sharka disease caused by the plum pox virus (PPV), which can severely reduce fruit production and quality, are being used as parents in most breeding programs. As a result, the self-incompatibility trait present in most of those accessions can be transmitted to the offspring, leading to the release of new self-incompatible cultivars. This situation can considerably affect apricot management, since pollination requirements were traditionally not considered in this crop and information is lacking for many cultivars. Thus, the objective of this work was to determine the pollination requirements of a group of new apricot cultivars by molecular identification of the S-alleles through PCR amplification of RNase and SFB regions with different primer combinations. The S-genotype of 66 apricot cultivars is reported, 41 for the first time. Forty-nine cultivars were considered self-compatible and 12 self-incompatible, which were allocated in their corresponding incompatibility groups. Additionally, the available information was reviewed and added to the new results obtained, resulting in a compilation of the pollination requirements of 235 apricot cultivars. This information will allow an efficient selection of parents in apricot breeding programs, the proper design of new orchards, and the identification and solution of production problems associated with a lack of fruit set in established orchards. The diversity at the S-locus observed in the cultivars developed in breeding programs indicates a possible genetic bottleneck due to the use of a reduced number of parents.

Patterns of Kdr-L995F Allele Emergence Alongside Detoxifying Enzymes Associated with Deltamethrin Resistance in Anopheles gambiae s.l. from North Cameroon.

Understanding how multiple insecticide resistance mechanisms occur in malaria vectors is essential for efficient vector control. This study aimed at assessing the evolution of metabolic mechanisms and Kdr L995F/S resistance alleles in Anopheles gambiae s.l. from North Cameroon, following long-lasting insecticidal nets (LLINs) distribution in 2011. Female An. gambiae s.l. emerging from larvae collected in Ouro-Housso/Kanadi, Be-Centre, and Bala in 2011 and 2015, were tested for susceptibility to deltamethrin + piperonyl butoxide (PBO) or SSS-tributyl-phosphoro-thrithioate (DEF) synergists, using the World Health Organization's standard protocol. The Kdr L995F/S alleles were genotyped using Hot Ligation Oligonucleotide Assay. Tested mosquitoes identified using PCR-RFLP were composed of An. arabiensis (68.5%), An. coluzzii (25.5%) and An. gambiae (6%) species. From 2011 to 2015, metabolic resistance increased in Ouro-Housso/Kanadi (up to 89.5% mortality to deltametnrin+synergists in 2015 versus <65% in 2011; p < 0.02), while it decreased in Be-Centre and Bala (>95% mortality in 2011 versus 42-94% in 2015; p < 0.001). Conversely, the Kdr L995F allelic frequencies slightly decreased in Ouro-Housso/Kanadi (from 50% to 46%, p > 0.9), while significantly increasing in Be-Centre and Bala (from 0-13% to 18-36%, p < 0.02). These data revealed two evolutionary trends of deltamethrin resistance mechanisms; non-pyrethroid vector control tools should supplement LLINs in North Cameroon.

Optimizing Production in the New Generation of Apricot Cultivars: Self-incompatibility, S-RNase Allele Identification, and Incompatibility Group Assignment.

Apricot (Prunus armeniaca L.) is a species of the Rosaceae that was originated in Central Asia, from where it entered Europe through Armenia. The release of an increasing number of new cultivars from different breeding programs is resulting in an important renewal of plant material worldwide. Although most traditional apricot cultivars in Europe are self-compatible, the use of self-incompatible cultivars as parental genotypes for breeding purposes is leading to the introduction of a number of new cultivars that behave as self-incompatible. As a consequence, there is an increasing need to interplant those new cultivars with cross-compatible cultivars to ensure fruit set in commercial orchards. However, the pollination requirements of many of these new cultivars are unknown. In this work, we analyze the pollination requirements of a group of 92 apricot cultivars, including traditional and newly-released cultivars from different breeding programs and countries. Self-compatibility was established by the observation of pollen tube behavior in self-pollinated flowers under the microscope. Incompatibility relationships between cultivars were established by the identification of S-alleles by PCR analysis. The self-(in)compatibility of 68 cultivars and the S-RNase genotype of 74 cultivars are reported herein for the first time. Approximately half of the cultivars (47) behaved as self-compatible and the other 45 as self-incompatible. Identification of S-alleles in self-incompatible cultivars allowed allocating them in 11 incompatibility groups, six of them reported here for the first time. The determination of pollination requirements and the incompatibility relationships between cultivars is highly valuable for the appropriate selection of apricot cultivars in commercial orchards and of parental genotypes in breeding programs. The approach described can be transferred to other woody perennial crops with similar problems.

Geographic distribution of human Blastocystis subtypes in South America.

Blastocystis is a cosmopolitan enteric protist colonizing probably more than 1 billion people. This protozoan exhibits genetic diversity and is subdivided into subtypes (STs). The aim of this study was to determine the distribution of Blastocystis STs in symptomatic and asymptomatic human samples from different countries of South America. A total of 346 fecal samples were genotyped by SSU rDNA showing ST1 (28.3%), ST2 (22.2%), ST3 (36.7%), ST4 (2%), ST5 (2.3%), ST6 (2%), ST7 (2.3%), ST8 (0.6%), ST12 (0.9%) and a novel ST (2.7%). These findings update the epidemiology of Blastocystis in South America and expand our knowledge of the phylogeographic differences exhibited by this stramenopile.

Life history mediates mate limitation and population viability in self-incompatible plant species.

Genetically controlled self-incompatibility systems represent links between genetic diversity and plant demography with the potential to directly impact on population dynamics. We use an individual-based spatial simulation to investigate the demographic and genetic consequences of different self-incompatibility systems for plants that vary in reproductive capacity and lifespan. The results support the idea that, in the absence of inbreeding effects, populations of self-incompatible species will often be smaller and less viable than self-compatible species, particularly for shorter-lived organisms or where potential fecundity is low. At high ovule production and low mortality, self-incompatible and self-compatible species are demographically similar, thus self-incompatibility does not automatically lead to reduced mate availability or population viability. Overall, sporophytic codominant self-incompatibility was more limiting than gametophytic or sporophytic dominant systems, which generally behaved in a similar fashion. Under a narrow range of conditions, the sporophytic dominant system maintained marginally greater mate availability owing to the production of S locus homozygotes. While self-incompatibility reduces population size and persistence for a broad range of conditions, the actual number of S alleles, beyond that required for reproduction, is important for only a subset of life histories. For these situations, results suggest that addition of new S alleles may result in significant demographic rescue.