SUMMARY: 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.

Lycium Linn. (Solanaceae) is a genus of economically important plants producing fruits and leaves with high nutritional value and medicinal benefits. However, genetic analysis of this plant and molecular breeding for quality improvement are limited by the lack of sufficient molecular markers. In this study, two parental strains, ‘Ningqi No. 1’ (Lycium barbarum L.) and ‘Yunnan Gouqi’ (Lycium yunnanense Kuang et A.M. Lu), and 200 F1 hybrid individuals were resequenced for genetic analysis. In total, 8,507 well-selected SNPs were developed, and a high-density genetic map (NY map) was constructed with a total genetic distance of 2,122.24 cM. A consensus genetic map was established by integrating the NY map and a previously published genetic map (NC map) containing 15,240 SNPs, with a total genetic distance of 3,058.19 cM and an average map distance of 0.21 cM. The 12 pseudochromosomes of the Lycium reference genome were anchored using this consensus genetic map, with an anchoring rate of 64.3%. Moreover, weak collinearities between the consensus map and the pepper, potato, and tomato genomes were observed. Twenty-five stable QTLs were identified for leaf- and fruit-related phenotypes, including fruit weight, fruit longitude, leaf length, the fruit index, and the leaf index; these stable QTLs were mapped to four different linkage groups, with LOD scores ranging from 2.51 to 19.37 and amounts of phenotypic variance explained from 6.2% to 51.9%. Finally, 82 out of 188 predicted genes underlying stable QTLs for fruit-related traits were differentially expressed according to RNA-seq analysis. A chromosome-level assembly can provide a foundation for further functional genomics research for wolfberry. The genomic regions of these stably expressed QTLs could be used as targets for further fine mapping and development of molecular markers for marker-assisted selection (MAS). The present study provided valuable information on saturated SNP markers and reliable QTLs for map-based cloning of functional genes related to yield and morphological traits in Lycium spp.

Summary: Wolfberry is a plant with medicinal and food values. However, its bioactive ingredients and the corresponding genetic bases have not been determined. Here, we de novo generated a chromosome‐level genome assembly for wolfberry, yielding a genome sequence of ~1.77 Gb with contig N₅₀ of 50.55 Mb and 39 224 predicted gene models. A variation map, using 307 re‐sequenced accessions, was called based on this genome assembly. Furthermore, the fruit metabolome of these accessions was profiled using 563 annotated metabolites, which separated Lycium barbarum L. and non‐L. barbarum L. The flavonoids, coumarins, alkaloids and nicotinic acid contents were higher in the former than in the latter. A metabolite‐based genome‐wide association study mapped 156 164 significant single nucleotide polymorphisms corresponding to 340 metabolites. This included 19 219 unique lead single nucleotide polymorphisms in 1517 significant association loci, of which three metabolites, flavonoids, betaine and spermidine, were highlighted. Two candidate genes, LbUGT (evm.TU.chr07.2692) and LbCHS (evm.TU.chr07.2738), with non‐synonymous mutations, were associated with the flavonoids content. LbCHS is a structural gene that interacts with a nearby MYB transcription factor (evm.TU.chr07.2726) both in L. barbarum and L. ruthenicum. Thus, these three genes might be involved in the biosynthesis/metabolism of flavonoids. LbSSADH (evm.TU.chr09.627) was identified as possibly participating in betaine biosynthesis/metabolism. Four lycibarbarspermidines (E–G and O) were identified, and only the lycibarbarspermidines O content was higher in L. barbarum varieties than in non‐L. barbarum varieties. The evm.TU.chr07.2680 gene associated with lycibarbarspermidines O was annotated as an acetyl‐CoA‐benzylalcohol acetyltransferase, suggesting that it is a candidate gene for spermidine biosynthesis. These results provide novel insights into the specific metabolite profile of non‐L. barbarum L. and the genetic bases of flavonoids, betaine and spermidine biosynthesis/metabolism.

Functional genome research, including gene transcriptional and posttranslational modifications of histones, can benefit greatly from a high-quality genome assembly. Histone modification plays a significant role in modulating the responses to abiotic stress in plants. However, there are limited reports on the involvement of dynamic changes in histone modification in cold stress response in upland cotton. In this study, the genome of an elite accession, YM11, with considerable cold stress tolerance was de novo assembled, which yielded a genome of 2343.06 Mb with a contig N50 of 88.96 Mb, and a total of 73,821 protein-coding gene models were annotated. Comparisons among YM11 and five Gossypium allopolyploid cotton assemblies highlighted a large amount of structural variations and presence/absence variations. We analyzed transcriptome and metabolome changes in YM11 seedlings subjected to cold stress. Using the CUT&Tag method, genome-wide H3K4me3 and H3K9ac modification patterns and effect of histone changes on gene expression were profiled during cold stress. Significant and consistently changing histone modifications and the gene expressions were screened, of which transcription factors (TFs) were highlighted. Our results suggest a positive correlation between the changes in H3K4me3, H3K9ac modifications and cold stress-responsive gene activation. This genome assembly and comprehensive analysis of genome-wide histone modifications and gene expression provide insights into the genomic variation and epigenetic responses to cold stress in upland cotton.

Wild germplasm resources are crucial for gene mining and molecular breeding because of their special trait performance. Haplotype-resolved genome is an ideal solution for fully understanding the biology of subgenomes in highly heterozygous species. Here, we surveyed the genome of a wild walnut tree from Gongliu County, Xinjiang, China, and generated a haplotype-resolved reference genome of 562.99 Mb (contig N50 = 34.10 Mb) for one haplotype (hap1) and 561.07 Mb (contig N50 = 33.91 Mb) for another haplotype (hap2) using PacBio high-fidelity (HiFi) reads and Hi-C technology. Approximately 527.20 Mb (93.64%) of hap1 and 526.40 Mb (93.82%) of hap2 were assigned to 16 pseudochromosomes. A total of 41039 and 39744 protein-coding gene models were predicted for hap1 and hap2, respectively. Moreover, 123 structural variations (SVs) were identified between the two haplotype genomes. Allele-specific expression genes (ASEGs) that respond to cold stress were ultimately identified. These datasets can be used to study subgenome evolution, for functional elite gene mining and to discover the transcriptional basis of specific traits related to environmental adaptation in wild walnut.

Golden pompano (Trachinotus ovatus) is economically significant important for offshore cage aquaculture in China and Southeast Asian countries. Lack of high-quality genomic data and accurate gene annotations greatly restricts its genetic breeding progress. To decode the mechanisms of sex determination and rapid growth in golden pompano and facilitate the sex- and growth-aimed genetic breeding. Genome assemblies of male and female golden pompano were generated using Illumina, PacBio, BioNano, genetic maps and Hi-C sequencing data. Genomic comparisons, whole genome re-sequencing of 202 F1 individuals, QTL mapping and gonadal transcriptomes were used to analyze the sex determining region, sex chromosome evolution, SNP loci, and growth candidate genes. Zebrafish model was used to investigate the functions of growth candidate gene. Female (644.45 Mb) and male (652.12 Mb) genomes of golden pompano were assembled and annotated at the chromosome level. Both genomes are highly conserved and no new or highly differentiated sex chromosomes occur. A 3.5 Mb sex determining region on LG15 was identified, where Hsd17b1, Micall2 and Lmx1a were putative candidates for sex determination. Three SNP loci significantly linked to growth were pinpointed, and a growth-linked gene gpsstr1 was identified by locus BSNP1369 (G → C, 17489695, Chr23). Loss of sstr1a (homologue of gpsstr1) in zebrafish caused growth retardation. This study provides insights into sex chromosome evolution, sex determination and rapid growth of golden pompano.