Pistil-specific Expression Research Articles

Mapping quantitative trait loci associated with self-(in)compatibility in goji berries (Lycium barbarum)

BackgroundGoji (Lycium barbarum L.) is a perennial deciduous shrub widely distributed in arid and semiarid regions of Northwest China. It is highly valued for its medicinal and functional properties. Most goji varieties are naturally self-incompatible, posing challenges in breeding and cultivation. Self-incompatibility is a complex genetic trait, with ongoing debates regarding the number of self-incompatible loci. To date, no genetic mappings has been conducted for S loci or other loci related to self-incompatibility in goji.ResultsWe used genome resequencing to create a high-resolution map for detecting de novo single-nucleotide polymorphisms (SNP) in goji. We focused on 229 F1 individuals from self-compatible ‘13–19’ and self-incompatible ‘new 9’ varieties. Subsequently, we conducted a quantitative trait locus (QTL) analysis on traits associated with self-compatibility in goji berries. The genetic map consisted of 249,327 SNPs distributed across 12 linkage groups (LGs), spanning a total distance of 1243.74 cM, with an average interval of 0.002 cM. Phenotypic data related to self-incompatibility, such as average fruit weight, fruit rate, compatibility index, and comparable compatibility index after self-pollination and geitonogamy, were collected for the years 2021–2022, as well as for an extra year representing the mean data from 2021 to 2022 (2021/22). A total of 43 significant QTL, corresponding to multiple traits were identified, accounting for more than 11% of the observed phenotypic variation. Notably, a specific QTL on chromosome 2 consistently appeared across different years, irrespective of the relationship between self-pollination and geitonogamy. Within the localization interval, 1180 genes were annotated, including Lba02g01102 (annotated as an S-RNase gene), which showed pistil-specific expression. Cloning of S-RNase genes revealed that the parents had two different S-RNase alleles, namely S1S11 and S2S8. S-genotype identification of the F1 population indicated segregation of the four S-alleles from the parents in the offspring, with the type of S-RNase gene significantly associated with self-compatibility.ConclusionsIn summary, our study provides valuable insights into the genetic mechanism underlying self-compatibility in goji berries. This highlights the importance of further positional cloning investigations and emphasizes the importance of integration of marker-assisted selection in goji breeding programs.

Rapid evolution of T2/S-RNase genes in Fragaria linked to multiple transitions from self-incompatibility to self-compatibility

The T2/RNase gene family is widespread in eukaryotes, and particular members of this family play critical roles in the gametophytic self-incompatibility (GSI) system in plants. Wild diploid strawberry (Fragaria) species have diversified their sexual systems via self-incompatible and self-compatible traits, yet how these traits evolved in Fragaria remains elusive. By integrating the published and de novo assembled genomes and the newly generated RNA-seq data, members of the RNase T2 gene family were systematically identified in six Fragaria species, including three self-incompatible species (Fragaria nipponica, Fragaria nubicola, and Fragaria viridis) and three self-compatible species (Fragaria nilgerrensis, Fragaria vesca, and Fragaria iinumae). In total, 115 RNase T2 genes were identified in the six Fragaria genomes and can be classified into three classes (I–III) according to phylogenetic analysis. The identified RNase T2 genes could be divided into 22 homologous gene sets according to amino acid sequence similarity and phylogenetic and syntenic relationships. We found that extensive gene loss and pseudogenization coupled with small-scale duplications mainly accounted for variations in the RNase T2 gene numbers in Fragaria. Multiple copies of homologous genes were mainly generated from tandem and segmental duplication events. Furthermore, we newly identified five S-RNase genes in three self-incompatible Fragaria genomes, including two in F. nipponica, two in F. viridis, and one in F. nubicola, which fit for typical features of a pistil determinant, including highly pistil-specific expression, highly polymorphic proteins and alkaline isoelectric point (pI), while no S-RNase genes were found in all three self-compatible Fragaria species. Surprisingly, these T2/S-RNase genes contain at least one large intron (>10 kb). This study revealed that the rapid evolution of T2/S-RNase genes within the Fragaria genus could be associated with its sexual mode, and repeated evolution of the self-compatible traits in Fragaria was convergent via losses of S-RNase.

Identification and fine mapping of a stigma exsertion mutant gene (Bolsem) in ornamental kale (Brassica oleracea var. acephala)

Stigma exsertion is a plant trait wherein stigmas emerge from flower buds before anthesis. It is a key determinant of the ornamental kale mating system, greatly increases the efficiency of hybrid seed production, and represents a technique to promote crossing pollination. The present study investigated a stigma exsertion mutant (‘Y009-10-5’) in ornamental kale and determined that the trait was determined by a recessive loss-of-function allele at the Bolsem (B. oleracea stigma exsertion mutant) locus. Then, using linkage analysis, with 350 F2 progeny and 98 SSR markers, the Bolsem locus was mapped to a 65.64-kb region of Chromosome 7, between the markers SSRCWS5 and SSRLS3. Sequence analysis and Brassica Database analysis identified two genes in this region: Bol005242, a gene of unknown function, and Bol005241, an ortholog of the Arabidopsis gene ABAP1, which controls cell proliferation and was, therefore, considered a potential candidate for Bolsem. Further sequence analysis identified two single-nucleotide polymorphisms (SNPs) in Bol005241. One SNP was found in the third exon, with the ‘Y009-10-5’ sequence yielding a TAA stop codon; therefore, it is predicted as the significant effects on stigma exsertion. Bol005241 also fully co-segregated with the exon SNP, and quantitative real-time PCR indicated that Bol005241 exhibited pistil-specific expression. Pollination experiments were performed to determine the effect of pollination timing on hybrid seed production. The results of this study provide a basis for elucidating the molecular mechanism underlying stigma exsertion, which could significantly improve hybrid seed production in ornamental kale.

Two Genes (ClS1 and ClF-box) Involved the Self-Incompatibility of ”Xiangshui” Lemon (Citrus limon (L.) Burm. f.)

Gametophytic self-incompatibility (GSI) is an important factor affecting fruit development and is one of the important mechanisms of seedlessness in citrus. Although many studies have investigated the mechanisms of SI, this process remains unclear in “Xiangshui” lemon (C. limon). In this study, we cloned one S-RNase homologous gene and one SLF homologous gene from the “Xiangshui” lemon, designated as ClS1 (pistil-specific expression) and ClF-box (pollen-specific expression), which contain open reading frames (ORFs) of 534 and 810 bp, respectively. The expression pattern of these two genes was analyzed by quantitative real-time polymerase chain reaction, and the results showed that the expression pattern of these two genes was significantly upregulated after self-pollination compared to that after cross-pollination. Through pollination in transgenic tobacco, we found that the pollen tubes grew slowly and became twisted after cross-pollination of ClS1♀ and ClF-box ♂ transgenic tobaccos, but the pollen tubes of control, self-pollination ClS1 and cross-pollination ClS1♂, and ClF-box ♀ grew normally. Furthermore, significantly fewer pollen tubes of ClS1♀ and ClF-box ♂ entered the ovules than that in other combinations, and the seed number of cross-pollination ClS1♀ and ClF-box ♂ transgenic tobaccos was significantly reduced. These results indicated that the ClS1 and ClF-box genes contribute to the self-incompatibility of the “Xiangshui” lemon.

Cloning, characterization and promoter analysis of S-RNase gene promoter from Chinese pear (Pyrus pyrifolia)

The 5′‐flanking region of the S12-, S13-, S21-RNase with a length of 854bp, 1448bp and 1137bp were successfully isolated by TAIL-PCR from genomic DNA from ‘Jinhua’, ‘Maogong’ (Pyrus pyrifolia) and ‘Yali’ (Pyrus bretschneideri) genomic DNA. Sequence alignment and analysis of S13-, S12-, S21-RNase gene promoter sequences with S2-, S3-, S4-, S5-RNase 5′-flanking sequences indicated that a homology region of about 240bp exists in the regions just upstream of the putative TATA boxes of the seven Chinese/Japanese pear S-RNase genes. Phylogenetic tree suggests that the homology region between the Chinese/Japanese pear and apple S-RNase gene promoter regions reflects the divergence of S-RNase gene was formed before the differentiation of subfamilies. Full length and a series of 5′-deletion fragments-GUS fusions were constructed and introduced into Arabidopsis thaliana plants. GUS activity were detected in S12-pro-(1 to 5)-GUS‐pBll01.2 transgenic pistils and progressively decreased from S12-pro-1-GUS-pBI l01.2 to S12-pro-5-GUS-pBll01.2. No GUS activity was detected in S12-pro-6-GUS-pBll01.2 transgenic pistil and other tissues of non-transformants and all transgenic plants. The result suggested S12-RNase promoter is pistil specific expression promoter.

New microsome-associated HT-family proteins from Nicotiana respond to pollination and define an HT/NOD-24 protein family

HT-family proteins have been identified in Nicotiana, Solanum, and Petunia. HT-B-type proteins are implicated in S-RNase-based self-incompatibility, but the functions of other family members are unknown. Screening for cDNA sequences with an expression pattern similar to HT-B in Nicotiana alata revealed a new group of small HT-family proteins, designated HT-M. HT-M proteins resemble HT-B in several respects: their pistil-specific expression pattern is indistinguishable from HT-B, they pellet with a microsome fraction, and their abundance decreases after pollination. Unlike HT-B, there is no S-specificity to this response, and RNAi experiments show that HT-M proteins are not necessary for self-incompatibility. Identification of a third group of pistil-specific HT-family proteins helps better define the characteristics of the family and allowed identification of putative new family members. By searching the databases with only the most conserved HT-family sequence elements, the signal sequence and cysteine motifs, we identified nodulin-24-like proteins and several small glycine-rich proteins as putative HT-family members. Like HT-M and HT-B, nodulin-24 is membrane associated. We propose that the conserved features in HT-family proteins are important for targeting or modification and refer to the broader family that includes both HT- and nodulin-24-like proteins as the HT/NOD-24-family.

CDNA microarray analysis of gene expression changes during pollination, pollen-tube elongation, fertilization, and early embryogenesis in rice pistils

During the processes of pollen recognition, pollen-tube guidance, fertilization, and early embryogenesis, a complicated gene network is highly regulated in plants. To visualize the profile of gene expression during these processes, we performed cDNA microarray analysis with RNA isolated from rice pistils at several stages. We identified 32 cDNA clones, in 21 non-redundant groups, with pistil-specific expression patterns. These genes could be clustered into five groups based on expression patterns, and included pathogenesis-related genes, transcription factors, and genes related to pollen germination, pollen tube elongation, and starch degradation. The microarray analysis identified several previously undescribed genes that are expressed in a pistil-specific manner. These results provide new clues to the molecular mechanisms that underlie plant reproduction.

Characterization of the S-RNase promoters from sweet cherry (Prunus avium L.).

Genomic DNA fragments containing the S(3)-, S(4)-, and S(6)-RNase genes were isolated from the sweet cherry (Prunus avium L.) and sequenced. Comparison of the 5'-flanking sequences of these three S-RNases indicated that a highly conserved region (designated CR) existed just upstream from the putative TATA boxes. We postulate that CR contains cis-regulatory element(s) involved in pistil expression. To examine the activity of the isolated S-RNase promoters of sweet cherry in the pistil, we transiently introduced approximately 650-bp fragments of the S(4)- and S(6)-RNase promoters fused to beta-glucuronidase (GUS) gene into the pistil of the petunia using a particle bombardment technique. Histochemical analysis showed that the 5'-flanking region of each S-RNase was active in the pistil. This suggests that cis-regulatory element(s) for pistil-specific expression may exist(s) within the 650-bp region upstream from the TATA box in the sweet cherry S-RNase promoter.

CIS-REGULATORY ELEMENTS FOR PISTIL SPECIFIC EXPRESSION IN S-RNASE PROMOTER REGION OF JAPANESE PEAR (PYRUS PYRIFOLIA NAKAI)

The pistil-specific and temporal expression of Japanese pear S-RNase gene is regulated by S-RNase promoter region. To identify the cis-regulatory elements on the S-RNase promoter of Japanese pear, we analysed GUS activities in transgenic tobacco plants carrying 9 sequential deletions of S3-RNase promoter fused to the -glucuronidase (GUS) gene. GUS activity was detected quantitatively in the pistils from the transgenic plants carrying the promoter deleted from -747 to -310. GUS activity was localized in the stigmatic secretory zone, and the transmitting tissues in the pistils but not in other tissues; pollen, anthers, petals, sepals, leaves and roots. GUS activities in the pistils increased steadily from 4 days before flowering. No transgenic pistils with deletions carrying -255 or shorter promoters displayed GUS activity, indicating that the critical cis-regulatory elements for pistil specific expression were present between -310 and -255 of S3-RNase promoter region. Furthermore, deletions from -552 to -356 led to the drastic decrease of GUS activity in the transgenic pistil, which assumes that enhancer-like element for the high level of expression in pistils are present within this region.

Multiple elements of the S2-RNase promoter from potato (Solanum tuberosum L.) are required for cell type-specific expression in transgenic potato and tobacco.

A functional analysis of the promoter of the S2-RNase gene from potato was performed in transgenic potato and tobacco plants, using a deletion series of S2-RNase promoter GUS fusions. A detailed histochemical and quantitative analysis of the transgenic tobacco plants revealed that S2 promoter fragments ranging in size from 5.6 kb in length down to 0.2 kb mediate a weak developmentally regulated expression in the pistil, and strong ectopic expression in pollen. In the pistil, different expression patterns were seen depending on the transformant, the predominant one being characterized by expression in the stigma and the transmitting tract of the style, whereas a few plants showed expression exclusively either in the stigma or in the stylar transmitting tissue. All transformants also showed GUS expression in the placental epidermis of the ovary. Two sequences that are conserved between the potato S1-RNase and S2-RNase promoters, termed motif and motif III, are located in a fragment of the S2 promoter extending from position of -200 to bp -100, and motif II, located between by -498 and -480, was identified on the basis of sequence comparisons between pistil-specific promoters. Motif II was found to be dispensible for pistil-specific and for pollen-specific expression. Two submotifs, A and B, were identified with the motif I. Both were essential for expression in the pistil but only B was necessary for expression in pollen. Although motif III has a similar bipartite structure and sequence to motif I, it was not sufficient to confer-either pollen- or pistil-specific expression. However, deletion of motif III abolished pollen-specific expression in transient expression experiments, suggesting that an interaction between the two sequence motifs may be needed to specify cell type-specific expression. In transgenic potato the S2-RNase promoter also mediates expression in pollen and in the pistil; however, significantly fewer plants showed expression than in tobacco, with most plants also exhibiting GUS expression in other issues.

A promoter directing high level expression in pistils of transgenic plants.

The promoter of the potato (Solanum tuberosum L.) SK2 gene, encoding a pistil-specific basic endochitinase, was cloned. Various fragments of the SK2-promoter, from 1 kb down to 0.23 kb in length, were fused to the GUS reporter gene. Chimaeric SK2 promoter-GUS fusion constructs were transformed into potato by Agrobacterium tumefaciens-mediated transformation. The SK2-GUS transgenic potato plants exhibited a highly specific GUS activity in the pistil. Expression in the pistil was shown to be developmentally regulated. In addition to the GUS activity in pistils, transgenic plants also showed a much weaker ectopic expression in anthers. In other tissues no systematic expression was detectable. All SK2 promoter fragments analysed conferred pistil-specific expression without significant qualitative or quantitative differences, demonstrating that the regulatory elements mediating this expression pattern are located within a 230 bp SK2 promoter fragment. The SK2 promoter may be used to engineer high levels of expression in pistils of transgenic plants.