Traditionally, the most popular sentences used to describe the arbuscular mycorrhizal symbiosis sound like: “AM fungi form one of the most widespread root symbioses, associating with 80% of land plants. In this symbiosis, the fungus provides the plant host with mineral nutrients, especially phosphate, receiving in turn carbohydrates.” In the last years, the mycorrhiza research field has witnessed a big step forward in the knowledge of the physiology and the mechanisms governing this important symbiosis, that helped plants colonizing the lands more than 400 MYA. The huge expansion of the -omics studies produced the first results on the fungal side, with genomes and transcriptomes of AM fungi being published. In parallel, the need for more sustainable agricultural practices has boosted the research in the field of the plant symbioses, with the final aim of improving plant productivity employing symbiotic microbes as bioinoculants. Beside all the other (positive) effects that mycorrhizal fungi exert on plants, the nutrient exchange is considered as the keystone, and the core mechanism governing this symbiosis. This review will focus on the molecular determinants underneath this exchange, both on the fungal and the plant side. Coming back to the sentence that claims this symbiosis as based on phosphate provided to the plant in return to carbohydrate, we will find that some concepts of this view still stand, while some others have been partly revolutionized.

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To trade in the field: the molecular determinants of arbuscular mycorrhiza nutrient exchange

Bulk segregation analysis (BSA) utilizes a strategy of pooling individuals with extreme phenotypes to conduct economical and rapidly linked marker screening or quantitative trait locus (QTL) mapping. With the development of next-generation sequencing (NGS) technology in the past 10 years, BSA methods and technical systems have been gradually developed and improved. At the same time, the ever-decreasing costs of sequencing accelerate NGS-based BSA application in different species, including eukaryotic yeast, grain crops, economic crops, horticultural crops, trees, aquatic animals, and insects. This paper provides a landscape of BSA methods and reviews the BSA development process in the past decade, including the sequencing method for BSA, different populations, different mapping algorithms, associated region threshold determination, and factors affecting BSA mapping. Finally, we summarize related strategies in QTL fine mapping combining BSA. 

Bulk segregation analysis in the <scp>NGS</scp> era: a review of its teenage years

A male sterile mutant was created by 60Co γ-rays of microspores isolated from Chinese cabbage DH line ‘FT’. A candidate gene for the male sterile trait was identified as Bra010198. Male sterility is used for hybrid seed production in Chinese cabbage. In this study, we derived a male sterile mutant (ftms) from Chinese cabbage DH line ‘FT’ by irradiating microspores with 60Co γ-rays and realized the rapid trait transformation from male fertility to sterility for creating valuable breeding materials. Genetic analysis indicated that the male sterile trait is controlled by a single recessive nuclear gene, ftms. Microspore development in mutant ftms was aborted at the tetrad stage and associated with severely retarded degeneration and vacuolation of tapetum. Using BSR-seq analysis, the candidate region for ftms was mapped on chromosome A05. A large F2 population was created, and the region was narrowed to approximately 1.7-Mb between markers Indel20 and Indel14 via linkage analysis. The recombination frequency was extremely suppressed because the region was located on the chromosome A05 centromere. Whole-genome resequencing of mutant ftms and wild-type ‘FT’ aligned only one nonsynonymous SNP to Bra010198; this gene is a homolog of Arabidopsis KNS4/UPEX1, which encodes a putative β-(1,3)-galactosyltransferase that controls pollen exine development. Comparative sequencing verified the SNP position on the fifth exon of Bra010198 in mutant ftms. Further genotyping revealed that the male sterile phenotype was fully co-segregated with this SNP. Quantitative real-time PCR indicated that Bra0101918 specifically expressed in stamen. The data presented herein suggested that Bra010198 is a strong candidate gene for ftms. Hence, we developed a male sterile line for potential application in breeding and expanded the knowledge about the molecular mechanism underlying male sterility in Chinese cabbage.

Mapping of the male sterile mutant gene ftms in Brassica rapa L. ssp. pekinensis via BSR-Seq combined with whole-genome resequencing

Leaf shape is an important trait that influences the utilization rate of light, and affects quality and yield of pea (Pisum sativum). In the present study, a joint method of high-density genetic mapping using specific locus amplified fragment sequencing (SLAF-seq) and bulked segregant analysis (BSA) was applied to rapidly detect loci with leaf shape traits. A total of 7,146 polymorphic SLAFs containing 12,213 SNP markers were employed to construct a high-density genetic map for pea. We conducted quantitative trait locus (QTL) mapping on an F2 population to identify QTLs associated with leaf shape traits. Moreover, SLAF-BSA was conducted on the same F2 population to identify the single nucleotide polymorphism (SNP) markers linked to leaf shape in pea. Two QTLs (qLeaf_or-1, qLeaf_or-2) were mapped on linkage group 7 (LG7) for pea leaf shape. Through alignment of SLAF markers with Cicer arietinum, Medicago truncatula, and Glycine max, the pea LGs were assigned to their corresponding homologous chromosomal groups. The comparative genetic analysis showed that pea is more closely related to M. truncatula. Based on the sequencing results of two pools with different leaf shape, 179 associated markers were obtained after association analysis. The joint analysis of SLAF-seq and BSA showed that the QTLs obtained from mapping on a high-density genetic map are convincing due to the closely associated map region with the BSA results, which provided more potential markers related to leaf shape. Thus, the identified QTLs could be used in marker-assisted selection for pea breeding in the future. Our study revealed that joint analysis of QTL mapping on a high-density genetic map and BSA-seq is a cost-effective and accurate method to reveal genetic architecture of target traits in plant species without a reference genome.

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QTL Mapping Combined With Bulked Segregant Analysis Identify SNP Markers Linked to Leaf Shape Traits in Pisum sativum Using SLAF Sequencing

Male sterility (induced or natural) is a potential tool for commercial hybrid seed production in different crops. Despite numerous endeavors to understand the physiological, hereditary, and molecular cascade of events governing CMS in cotton, the exact biological process controlling sterility and fertility reconstruction remains obscure. During current study, RNA-Seq using Ion Torrent S5 platform is carried out to identify 'molecular portraits' in floral buds among the Cytoplasmic Genic Male Sterility (CGMS) line, its near-isogenic maintainer, and restorer lines. A total of 300, 438 and 455 genes were differentially expressed in CGMS, Maintainer, and Restorer lines respectively. The functional analysis using AgriGo revealed suppression in the pathways involved in biogenesis and metabolism of secondary metabolites which play an important role in pollen and anther maturation. Enrichment analysis showed dearth related to pollen and anther's development in sterile line, including anomalous expression of genes and transcription factors that have a role in the development of the reproductive organ, abnormal cytoskeleton formation, defects in cell wall formation. The current study found aberrant expression of DYT1, AMS and cytochrome P450 genes involved in tapetum formation, pollen development, pollen exine and anther cuticle formation associated to male sterility as well as fertility restoration of CGMS. In the current study, more numbers of DEGs were found on Chromosome D05 and A05 as compared to other chromosomes. Expression pattern analysis of fourteen randomly selected genes using qRT-PCR showed high concurrence with gene expression profile of RNA-Seq analysis accompanied by a strong correlation of 0.82. The present study provides an important support for future studies in identifying interaction between cyto-nuclear molecular portraits, to accelerate functional genomics and molecular breeding related to cytoplasmic male sterility studies in cotton.

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Transcriptome analysis identified aberrant gene expression in pollen developmental pathways leading to CGMS in cotton (Gossypium hirsutum L.).

Cytoplasmic male sterility (CMS) is a maternally inherited trait failing to produce functional pollen. It plays a pivotal role in the exploitation of crop heterosis. The specific locus amplified fragment sequencing (SLAF-seq) as a high-resolution strategy for the identification of new SNPs on a large-scale is gradually applied for functional gene mining. The current study combined the bulked segregant analysis (BSA) with SLAF-seq to identify the candidate genes associated with fertility restorer gene (Rf) in CMS cotton. Illumina sequencing systematically investigated the parents. A segregating population comprising of 30 + 30 F2 individuals was developed using 3096A (female parent) as sterile and 866R (male parent) as a restorer. The original data obtained by dual-index sequencing were analyzed to obtain the reads of each sample that were compared to the reference genome in order to identify the SLAF tag with a polymorphism in parent lines and the SNP with read-associated coverage. Based on SLAF tags, SNP-index analysis, Euclidean distance (ED) correlation analysis, and whole genome resequencing, the hot regions were annotated. A total of 165,007 high-quality SLAF tags, with an average depth of 47.90× in the parents and 50.78× in F2 individuals, were sequenced. In addition, a total of 137,741 SNPs were detected: 113,311 and 98,861 SNPs in the male and female parent, respectively. A correlation analysis by SNP-index and ED initially located the candidate gene on 1.35 Mb of chrD05, and 20 candidate genes were identified. These genes were involved in genetic variations, single base mutations, insertions, and deletions. Moreover, 42 InDel markers of the whole genome resequencing were also detected. In this study, associated markers identified by super-BSA could accelerate the study of CMS in cotton, and as well as in other crops. Some of the 20 genes’ preliminary characteristics provided useful information for further studies on CMS crops.

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Physical mapping and candidate gene prediction of fertility restorer gene of cytoplasmic male sterility in cotton

The utility model relates to a special incubator of drought resisting resistant salt appraisal of plant, including incubator and stock solution bucket, the lateral wall of incubator is provided with the transparent observation window, has laid rough stone bed course, first non -woven fabrics layer, thin rock block cushion layer, second non -woven fabrics layer and vermiculite layer in the incubator in proper order, is provided with the drain pipe outside extending to the incubator in the rough stone bed course, is provided with a plurality of wash ports on the pipe wall of the district's section inside the drain pipe is located the incubator and wraps up having and prevent blockking up the non -woven fabrics, be provided with the water pump in the stock solution bucket, the water pump is connected and is used for the delivery pipe in culture solution pump sending to the incubator in the stock solution bucket. The utility model discloses a transparent observation window can audio -visually see that crops coerced the real -time performance of back root system, contains the nutrient solution of handling the composition and circulates through setting up liquid storage pot and water pump and the wet return that is equipped with the conductivity meter, making, can detection composition concetration change, and processing nutrient solution composition is mended in people's wage adjustment, also can replenish partial oxygen among the while cyclic process.

Special incubator of drought resisting resistant salt appraisal of plant

The invention discloses a method for simultaneously detecting the emergence rate and insect resistance purity of transgenic Bt insect-resistant cottonseeds. The method is characterized by comprising the following steps of: (1) preparing a kanamycin sulfate aqueous solution with concentration between 0.4 and 0.6 percent; (2) preparing wet sand; (3) sowing; and (4) culturing. Under a selective pressure applicable to antibiotics, the method adopts a mechanism that a product which is encoded with a neomycin phosphotransferase (npt II) gene carried by a transgenic Bt plant as a label can inactivate kanamycin sulfate and other aminoglycoside antibiotics to grow normally, but a non-transgenic Bt plant does not contain an npt II gene serving as a label and cannot grow normally, so that the insect resistance purity can be detected while the emergence rate of the transgenic Bt insect-resistant cottonseeds is detected. The method is easy and practical and is low in cost, and the detection accuracy of the method can be 100 percent.

Method for simultaneously detecting emergence rate and insect resistance purity of transgenic Bt insect-resistant cottonseeds

The invention discloses a method for extracting total RNA of a polysaccharide polyphenol plant. Plant tissues are lysed by using a lysate for the polysaccharide polyphenol plant and a beta-mercaptoethanol solution instead of a Trizol solution, water-saturated phenol is added to decrease involved DNA, and meanwhile the beta-mercaptoethanol is added to reduce the oxidative damage of oxygen to cells.Compared with a CTAB method, the preparation and sterilization process of the solution is omitted, the time is greatly shortened, the total RNA degradation of sugars and phenols in the plant can be effectively avoided, and meanwhile the protein and DNA mixed in RNA can be completely cleared away. The total RNA extracted by adopting the method can achieve the study on Micro RNA and its target genes.

Method for extracting total RNA of polysaccharide polyphenol plant

The invention discloses genes and method for identifying gossypium hirsutum linn. variety verticillium wilt resistance by a fluorescence quantitative polymerase chain reaction (PCR) technique; the genes have the sequences shown in SEQ ID No:1 and SEQ ID No:2 in a sequence table. The method comprises the identification steps: (1) culturing seedlings of a to-be-tested cotton variety in a nutrition pot in a culture room; (2) culturing verticillium wilt pathogenic bacteria; (3) dipping roots, and inoculating; (4) extracting total RNA; (5) synthesizing cDNA; (6) synthesizing fluorescence quantitative PCR specific primers of a gossypium hirsutum linn. resistance related key gene and an internal reference gene GhACTIN14; (7) carrying out fluorescence quantitative PCR detection; and (8) analyzing a result. The genes and the method can identify resistance to verticillium wilt in a six-leaf stage of the gossypium hirsutum linn. variety, the identification result is reliable, the accuracy rate can reach 100%, and the operation is simple and easy to implement.

Guo Baosheng

Papers

Physical mapping and candidate gene prediction of fertility restorer gene of cytoplasmic male sterility in cotton

Special incubator of drought resisting resistant salt appraisal of plant

Method for simultaneously detecting emergence rate and insect resistance purity of transgenic Bt insect-resistant cottonseeds

Method for extracting total RNA of polysaccharide polyphenol plant

Genes and method for identifying gossypium hirsutum linn. variety verticillium wilt resistance by fluorescence quantitative PCR technique