Construction of High Quality Quinoa Genome Based on Three - generation Sequencing + Optical Atlas + Hi - C

Research background: mention quinoa Chenopodium quinoa (quinoa), many people are relatively unfamiliar, but as early as 7000 years ago in South America and the Andean mountains, quinoa has been human cultivation and in centuries after the Inca empire known as the " Mother's grain ". Quinoa has adapted to the Andean plateau environment (above 3500 meters above sea level) and has evolved the ability to adapt to a variety of abiotic adversities, which are considered important crops that can improve world food security. Quinoa has a global concern because of its nutritional value. It contains no gluten, low glycemic index and contains very balanced nutrients such as amino acids, fiber, fat, carbohydrates, vitamins and minerals. And can be planted on land that is not suitable for the growth of major food crops. Although quinoa has agricultural potential, it is still not being fully utilized. People continue to work to improve their important agricultural traits to expand quinoa on a global scale. In order to accelerate its improvement, the authors report the heterotetraploid quinoa genome and found genes that may regulate saponin content in seeds.

Comparison of research strategies and results:

The authors reported that they used the PacBio three-generation sequencing, Bionano optical map, Hi-C technology combined with the genetic assemblage of the high quality chromosome grade quinoa reference genomic sequence and published in the latest Nature magazine.

In fact, in 2016, there was also a Japanese research team sequencing the quinoa genome using a short read-length second-generation sequencing technique combined with low-depth PacBio sequencing data, so that only the draft was drafted, not complete Of the reference genome, the article only in the DNA Research (IF: 5.267) magazine. After a lapse of six months, why the sequencing of quinoa can be sent to Nature (IF: 38.138) article Here we come to specifically understand what the difference between the two articles?

The genome of Chenopodium quinoa (2n = 4x = 36) is expected to be 1.45-1.50Gb. In this paper, the genome sequence of 1.39Gb was obtained by PacBio three-generation sequencing + Bionano optical spectrum + Hi-C classical strategy, A total of 3,486 scaffolds, Scaffold N50 size of 3.84Mb, 90% of the genome included in 439 scaffold. And previously published in DNA Research, based on short read length sketch obtained more than 24,000 scaffold, lost more than 25% of the sequence.

For the complex heterotetraploid genome such as quinoa, 64% of the sequence is a repeating sequence containing a large number of long terminal repeat (LTR) transposable factors. The assembly strategy based on PacBio + Bionano + Hi-C is far superior to the short-length sequencing technology, which has a significant improvement in genomic coverage, such as Contig N50 and Scaffold N50. In the case of a second-generation sequencing of published genomic sketches, it can still be published in the journal Nature.

Evolution history of quinoa:

Quinoa is from the ancestral source of A and B diploid varieties of hybridization. Previous studies of single gene sequencing were identified in the germplasm bank, and two diploids in North America and Eurasia were the candidate sources for the A subgenomic and B subgenomic groups, and later hybridized in North America. In order to further understand the genomic structure and evolution of quinoa, the authors were sequenced, assembled and annotated for the diploid B. pallidicaule and B genome diploid C. suecicum. A large proportion of homologous gene pairs in quinoa showed similar synonymous substitution rates at each synonymous site, indicating replication events across the genome. The authors estimate that the reorganization occurred about 3.3-6.3 million years ago. Evolutionary tree analysis shows that North America C. berlandieri is a basic member of species synthesis. Quinoa is considered to be acclimatized from C. hircinum in a single event. The authors' sequencing data suggest that quinoa may be acclimated independently in the plateau and coastal environments, respectively. A total of 7,809,381 SNPs were found from these registered samples and the reference quinoa genome, including 2,668,694 quinoa-specific SNPs. This will help to assess its genetic diversity, as well as identify valuable traits related to genomic regions.

Analysis of subgenomic characteristics:

By comparing the results of the sequencing from C. pallidicaule and C. suecicum onto the scaffold assembly of quinoa, BLASTN compares each diploid to quaffold of quinoa and found that there were 156 and 410 quinoa scaffold Compared to the A genome and the B genome (total 202.6Mb and 646.3Mb). The localization of 5,807 homologous genes on chromosomes revealed a high degree of collinearity with the A and B subgenomes.

The potential mechanism of saponin synthesis:

A. Saponin mass spectrometry in the skin of quinoa seed

B. Accumulation of saponins was measured by total acid count during seed development

C. The difference in the frequency of the allele between the sweet descendants and the bitter descendants

D. Saponin biosynthetic pathway, indicating the enzymes that catalyze each step of the pathway and the gene ID that encodes each enzyme

E. TSARL1 gene pattern in bitter and sweet varieties

Quinoa seeds contain saponins. Although this is beneficial for plant growth (such as preventing herbivores), it must be removed for human consumption, because it has hemolytic and bitter taste. But to remove saponins, the cost is too high, water consumption, but also reduce the nutritional value of seeds. The authors found that saponins were concentrated in the peel of 20 to 24 days after flowering and eventually accounted for 4% (w / w) of the total seed mass.

The naturally occurring sweet quinoa contains very low levels of saponins, although the underlying regulatory genes are not yet clear. To find these genes, the authors performed linkage maps of two hybrid isolates and BSA analysis: Kurmi (sweet) × 0654 (bitter), and Atlas (sweet) × Carina Red (bitter). As with the results obtained in other groups, the separation ratio in these populations indicates that the presence of saponins in the seeds is controlled by a single gene. The presence or absence of saponins is associated with differences in seed coat thickness. Bitter varieties have a thicker seed coat than sweet varieties.

Transcript analysis:

The upper part of the figure shows the sequencing results of the PacBio Iso-Seq transcript, and the middle part shows the Illumina RNA-Seq sequencing results. The gray lines on the upper two parts represent the intron subdomain. The bottom shows the chromosomal location of the AUR62017258 gene. It can be seen that PacBio's long read-length technique does not require splicing for full-length transcript sequencing and can be completely covered from a 5 'untranslated region, all exons and 3' untranslated regions in a reads. While short read long technology needs to be assembled.

summary:

Quinoa is a new crop because it is nutritious and resistant to a variety of abiotic stresses and is considered to have great potential for improving global food security. High quality genomic assembly results will accelerate its genetic improvement. PacBio three generations of sequencing combined with Bionano optical spectrum technology and Hi-C technology can perfectly assemble complex genomes. At the same time, PacBio long read long reads can easily get the full transcript length without splicing. This paper once again confirms its advantages in genome assembly, transcript selective cutting, and the like.

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