Large-scale deployment of a rice 6 K SNP array for genetics and breeding applications

Background

Fixed arrays of single nucleotide polymorphism (SNP) markers have advantages over reduced representation sequencing in their ease of data analysis, consistently higher call rates, and rapid turnaround times. A 6 K SNP array represents a cost-benefit “sweet spot” for routine genetics and breeding applications in rice. Selection of informative SNPs across species and subpopulations during chip design is essential to obtain useful polymorphism rates for target germplasm groups. This paper summarizes results from large-scale deployment of an Illumina 6 K SNP array for rice.

Results

Design of the Illumina Infinium 6 K SNP chip for rice, referred to as the Cornell_6K_Array_Infinium_Rice (C6AIR), includes 4429 SNPs from re-sequencing data and 1571 SNP markers from previous BeadXpress 384-SNP sets, selected based on polymorphism rate and allele frequency within and between target germplasm groups. Of the 6000 attempted bead types, 5274 passed Illumina’s production quality control. The C6AIR was widely deployed at the International Rice Research Institute (IRRI) for genetic diversity analysis, QTL mapping, and tracking introgressions and was intensively used at Cornell University for QTL analysis and developing libraries of interspecific chromosome segment substitution lines (CSSLs) between O. sativa and diverse accessions of O. rufipogon or O. meridionalis. Collectively, the array was used to genotype over 40,000 rice samples. A set of 4606 SNP markers was used to provide high quality data for O. sativa germplasm, while a slightly expanded set of 4940 SNPs was used for O. sativa X O. rufipogon populations. Biparental polymorphism rates were generally between 1900 and 2500 well-distributed SNP markers for indica x japonica or interspecific populations and between 1300 and 1500 markers for crosses within indica, while polymorphism rates were lower for pairwise crosses within U.S. tropical japonica germplasm. Recently, a second-generation array containing ~7000 SNP markers, referred to as the C7AIR, was designed by removing poor-performing SNPs from the C6AIR and adding markers selected to increase the utility of the array for elite tropical japonica material.

Conclusions

The C6AIR has been successfully used to generate rapid and high-quality genotype data for diverse genetics and breeding applications in rice, and provides the basis for an optimized design in the C7AIR.

     

Identification of resistant germplasm containing novel resistance genes at or tightly linked to the <Emphasis Type="Italic">Pi2/9</Emphasis> locus conferring broad-spectrum resistance against rice blast

Background

The rice Pi2/9 locus harbors multiple resistance (R) genes each controlling broad-spectrum resistance against diverse isolates of Magnaporthe oryzae, a fungal pathogen causing devastating blast disease to rice. Identification of more resistance germplasm containing novel R genes at or tightly linked to the Pi2/9 locus would promote breeding of resistance rice cultivars.

Results

In this study, we aim to identify resistant germplasm containing novel R genes at or tightly linked to the Pi2/9 locus using a molecular marker, designated as Pi2/9-RH (Pi2/9 resistant haplotype), developed from the 5′ portion of the Pi2 sequence which was conserved only in the rice lines containing functional Pi2/9 alleles. DNA analysis using Pi2/9-RH identified 24 positive lines in 55 shortlisted landraces which showed resistance to 4 rice blast isolates. Analysis of partial sequences of the full-length cDNAs of Pi2/9 homologues resulted in the clustering of these 24 lines into 5 haplotypes each containing different Pi2/9 homologues which were designated as Pi2/9-A5, ?A15, ?A42, ?A53, and -A54. Interestingly, Pi2/9-A5 and Pi2/9-A54 are identical to Piz-t and Pi2, respectively. To validate the association of other three novel Pi2/9 homologues with the blast resistance, monogenic lines at BC₃F₃ generation were generated by marker assisted backcrossing (MABC). Resistance assessment of the derived monogenic lines in both the greenhouse and the field hotspot indicated that they all controlled broad-spectrum resistance against rice blast. Moreover, genetic analysis revealed that the blast resistance of these three monogenic lines was co-segregated with Pi2/9-RH, suggesting that the Pi2/9 locus or tightly linked loci could be responsible for the resistance.

Conclusion

The newly developed marker Pi2/9-RH could be used as a potentially diagnostic marker for the quick identification of resistant donors containing functional Pi2/9 alleles or unknown linked R genes. The three new monogenic lines containing the Pi2/9 introgression segment could be used as valuable materials for disease assessment and resistance donors in breeding program.

     

Updating the elite rice variety Kongyu 131 by improving the <Emphasis Type="Italic">Gn1a</Emphasis> locus

Background

Kongyu 131 is an elite japonica rice variety of Heilongjiang Province, China. It has the characteristics of early maturity, superior quality, high yield, cold tolerance and wide adaptability. However, there is potential to improve the yield of Kongyu 131 because of the relatively few grains per panicle compared with other varieties. Hence, we rebuilt the genome of Kongyu 131 by replacing the GRAIN NUMBER1a (Gn1a) locus with a high-yielding allele from a big panicle indica rice variety, GKBR. High-resolution melting (HRM) analysis was used for single nucleotide polymorphism (SNP) genotyping.

Results

Quantitative trait locus (QTL) analysis of the BC₃F₂ population showed that the introgressed segment carrying the Gn1a allele of GKBR significantly increased the branch number and grain number per panicle. Using 5 SNP markers designed against the sequence within and around Gn1a, the introgressed chromosome segment was shortened to approximately 430 Kb to minimize the linkage drag by screening recombinants in the target region. Genomic components of the new Kongyu 131 were detected using 220 SNP markers evenly distributed across 12 chromosomes, suggesting that the recovery ratio of the recurrent parent genome (RRPG) was 99.89%. Compared with Kongyu 131, the yield per plant of the new Kongyu 131 increased by 8.3% and 11.9% at Changchun and Jiamusi, respectively.

Conclusions

To achieve the high yield potential of Kongyu 131, a minute chromosome fragment carrying the favorable Gn1a allele from the donor parent was introgressed into the genome of Kongyu 131, which resulted in a larger panicle and subsequent yield increase in the new Kongyu 131. These results indicate the feasibility of improving an undesirable trait of an elite variety by replacing only a small chromosome segment carrying a favorable allele.

     

<Emphasis Type="Italic">OsLAP6/OsPKS1</Emphasis>, an orthologue of <Emphasis Type="Italic">Arabidopsis PKSA/LAP6</Emphasis>, is critical for proper pollen exine formation

Background

Male fertility is crucial for rice yield, and the improvement of rice yield requires hybrid production that depends on male sterile lines. Although recent studies have revealed several important genes in male reproductive development, our understanding of the mechanisms of rice pollen development remains unclear.

Results

We identified a rice mutant oslap6 with complete male sterile phenotype caused by defects in pollen exine formation. By using the MutMap method, we found that a single nucleotide polymorphism (SNP) variation located in the second exon of OsLAP6/OsPKS1 was responsible for the mutant phenotype. OsLAP6/OsPKS1 is an orthologous gene of Arabidopsis PKSA/LAP6, which functions in sporopollenin metabolism. Several other loss-of-function mutants of OsLAP6/OsPKS1 generated by the CRISPR/Cas9 genomic editing tool also exhibited the same phenotype of male sterility. Our cellular analysis suggested that OsLAP6/OsPKS1 might regulate pollen exine formation by affecting bacula elongation. Expression examination indicated that OsLAP6/OsPKS1 is specifically expressed in tapetum, and its product is localized to the endoplasmic reticulum (ER). Protein sequence analysis indicated that OsLAP6/OsPKS1 is conserved in land plants.

Conclusions

OsLAP6/OsPKS1 is a critical molecular switch for rice male fertility by participating in a conserved sporopollenin precursor biosynthetic pathway in land plants. Manipulation of OsLAP6/OsPKS1 has potential for application in hybrid rice breeding.

     

Conferring resistance to pre-harvest sprouting in durum wheat by a QTL identified in <Emphasis Type="Italic">Triticum spelta</Emphasis>

Pre-harvest sprouting (PHS) causes significant yield loss and degrade the end-use quality of wheat, especially in regions with prolonged wet weather during the harvesting season. Unfortunately, the gene pool of Triticum durum (tetraploid durum wheat) has narrow genetic base for PHS resistance. Therefore, finding out new genetic resources from other wheat species to develop PHS resistance in durum wheat is of importance. A major PHS resistance QTL, Qphs.sicau-3B.1, was mapped on chromosome 3BL in a recombinant inbred line population derived from ‘CSCR6’ (Triticum spelta), a PHS resistant hexaploid wheat and ‘Lang’, a PHS susceptible Australian hexaploid wheat cultivar. This QTL, Qphs.sicau-3B.1, is positioned between DArT marker wPt-3107 and wPt-6785. Two SCAR markers (Ph3B.1 and Ph3B.2) were developed to track this major QTL and were used to assay a BC₂F₈ tetraploid population derived from a cross between the durum wheat ‘Bellaroi’ (PHS susceptible) and ‘CSCR6’ (PHS resistant). Phenotypic assay and marker-assisted selection revealed five stable tetraploid lines were highly PHS resistant. This study has successfully established that PHS-resistance QTL from hexaploid wheat could be efficiently introgressed into tetraploid durum wheat. This tetraploid wheat germplasm could be useful in developing PHS resistant durum cultivars with higher yield and good end-use quality.     

Identification of favorable alleles in the <Emphasis Type="Italic">non</Emphasis>-<Emphasis Type="Italic">yellow coloring 1</Emphasis> gene by association mapping in maize

Association mapping was conducted to explore favorable alleles of the chlorophyll-related non-yellow coloring 1 (NYC1) gene under light and dark using an association panel of 146 maize inbred lines. A total of 14 polymorphic sites were identified to be significantly associated with at least one of the chlorophyll-related traits at the seedling stage. Four single nucleotide polymorphisms (SNPs) (S320, S2951, S3901, and S3355) from the NYC1 gene were respectively strongly associated with chlorophyll b (chlb), the ratio of chlorophyll a to chlorophyll b (chl_ratio), chlorophyll a degradation (chla_deg), and total chlorophyll degradation (total_chl_deg). SNPs S320 (C/A) in exon 1, and S2951 (A/G) in intron 8 was related to chlb, with 6.01 and 8.89% of phenotypic variation under light treatment, respectively. Under dark treatment, SNP S3901 (C/T), located in 3′ untranslated region (3′UTR), was associated with chl_ratio, explaining 7.01% of the observed phenotypic variation, whereas SNP S3355 (C/G) in intron 9 explained 6.48 and 5.18% of phenotypic variations in chla_deg and total_chl_deg, respectively. Taken together, these results indicated that the NYC1 gene plays an important role in chlorophyll content and other related traits, and different sites act on chlorophyll metabolism under different light intensities in maize seedlings. Furthermore, these findings improve our understanding of the genetic basis of chlorophyll metabolism under different light conditions.     

Development of male-specific markers and identification of sex reversal mutants in papaya

Papaya is a productive and nutritious fruit grown in tropical and sub-tropical regions worldwide. It is polygamous with three sex types: female, male and hermaphrodite. Sex determination in papaya is controlled by an XY sex chromosome system with two slightly different Y chromosomes, Y for males and Y^h for hermaphrodites. Comparative analysis of the hermaphrodite-specific region of Y^h chromosome (HSY) and male-specific region of Y chromosome (MSY) revealed 99.6% sequence identity, which explains why DNA markers that amplify for both males and hermaphrodites have easily been developed, but not for the male trait specifically. We examined the 0.4% sequence differences, and found 1887 indels and 21,088 SNPs between MSY and HSY. The vast majority of indels are single nucleotide or few base pairs. A large male-specific retrotransposon insertion of 8396 bp was used to develop two papaya male-specific markers, PMSM1 and PMSM2 that amplify 585 and 548 bp fragments, respectively. These two markers were tested in 11 gynodioecious and four dioecious varieties along with autosomal DNA marker 71E and male/hermaphrodite marker W11, and the results showed clear separation of male from hermaphrodite and female. PMSM1 and PMSM2 were also used to test the sex type of six sex male-to-hermaphrodite reversal mutants which are crucial materials for validating candidate genes for sex determination in papaya. Our result showed all six mutants were positive for the male-specific markers. These male-specific markers can be used to distinguish gynodioecious and dioecious cultivars in papaya seed market, and facilitate genetic and genomic research for papaya improvement.     

Polymorphisms of the <Emphasis Type="Italic">FT</Emphasis> gene as a tool to identify underground rhizome types of bamboos

The Flowering Locus T (FT)-like genes of angiosperms are highly conserved. The FT-encoded proteins include a phosphatidylethanolamine-binding domain that is involved in the control of the shoot apical meristem identity and flowering time. In the present study, FT genes were investigated in 20 bamboo species that are grouped into sympodial, mixed and scattered bamboos based on their morphology. All examined orthologous FT genes consisted of four exons and three introns. Their encoded protein sequences contained the critical amino acid residues Tyr85, Glu109, Leu128, Tyr134, Trp138, Arg139, Gln140 and Asn152, of which each possesses a biological function. The DNA sequences were rich in single nucleotide polymorphism (SNP) sites. The SNP frequency was 1 SNP/16.8 bp, and the nucleotide diversity (π) equaled 0.265. Some SNPs altered restriction enzyme sites or resulted in changes in amino acid contents. The correlation analysis showed that several SNPs were informative in relation to the underground rhizome types of bamboos. Therefore, FT polymorphisms could be used as a tool to identify the underground rhizome types of bamboos. The phylogenetic tree constructed based on the FT gene sequences showed that the obtained clustering was consistent with the underground rhizome types. The SNP markers developed in the present study will provide information on the genetic diversity of bamboos and they can aid taxonomic study as well.