Validation of M/P markers in different embryonic genotypes
The M/P marker was designed by Shimada et al . M-marker implied to monoembryonic alleles with 0.75 kb fragments and P-marker implied to polyembroynic alleles with 1.0 kb fragments. By the genotyping analysis of 95 traditional and breeding varieties, they found that P-marker could be detected in all polyembryonic varieties and only M-marker could be detected in monoembryonic varieties. However, inconsistent results were observed in our study.
Firstly, P marker were detected in three monoembryonic sweet orange varieties (Li2, TD, MS) and their M/P patterns were the same as two polyembryonic sweet orange varieties (Tarocco blood orange and Hamlin sweet orange). Only M-marker were detected in monoembryonic Banpeiyu pummelo and clementine mandarin (Fig. 1). Sweet orange is the most planted citrus variety and most sweet orange varieties produce polyembryonic seeds, but monoembryonic lines were occasionally found around the world also. Our monoembryonic sweet orange varieties were found during our breeding process and citrus germplasm collection and evaluation in China. They should be somatic mutation origin. According to the results, the CitRWP of monoembryonic sweet orange should have the MITE insertions also. It is contradictory with conclusion “polyembryonic alleles with a MITE insertion in the upstream region and monoembryonic alleles without it” proposed by Shimada et al .
Subsequently, only M-marker was detected in different Poncirus genotypes, a close related genus of Citrus (Fig. 2). A reasonable deduction might be that the CitRWP exists in Poncirus and the gene does not contain the MITE insertion or a huge mutation occurred in upstream. The marker gave a different scenario in offsprings of W-Murcott × Flying dragon trifoliate orange (Fig. 2). Only M-marker was detected in all 7 progenies, P-marker together with M-marker were detected in 9 progenies. But a different band pattern was found in both P-marker and M-marker progenies. 4 polyembryonic genotype offsprings produced the same band pattern as the seed parent W-Murcott with a stronger M-marker band and a weaker P-marker band, and 3 polyembryonic progenies produced new band pattern. The new band pattern included a stronger and longer band than the P-marker band and M-marker band.
CitRWP expression in different materials
Nucellar embryo initiates and develops in the ovule, and the flowering stage is the critical period for the development of nucellar embryo, so gene expression analysis was done in ovules of different embryogenic varieties at flowering stage. CitRWP expression was detected by RT-PCR with Actin as the reference gene. Under same mRNA extraction and reverse transcription procedure and PCR conditions, RT-PCR revealed that CitRWP expressed in all varieties (Fig. 3). Judged by the strength of band, the expression level of monoembryonic Longan pummelo was the lowest, followed by polyembryonic Flying dragon trifoliate orange and Ponkan, and all the remaining samples showed very similar expression strength. No obvious difference could be discriminated between monoembryonic sweet orange varieties and polyembryonic sweet orange varieties.
CitRWP expression in ovule was further checked by qRT-PCR in two consecutive years. REV (Relative expression values) of different embryonic varieties were computed with Banpeiyu pummelo as control. The REV of Ponkan was similar with polyembryonic sweet orange Hamlin and Tarocco blood orange, while the expression value of W-Murcott was similar to those of monoembryonic sweet orange (Fig. 4). In the 2019 CitRWP expression test, the REV of Flying dragon trifoliate orange was 5.5 times that of monoembryonic Banpeiyu pummelo, and the REV of polyembryonic sweet orange Hamlin and Tarocco blood orange was 73.1 and 46.9 times that of Banpeiyu pummelo, respectively. The REV of monoembryonic sweet orange Licheng No. 2, TD and MS was 21.7 times, 32.14 times, and 42.9 times that of Banpeiyu pummelo, respectively. Notably, the REV of CitRWP in the polyembryonic Flying dragon trifoliate orange was much lower than that of the monoembryonic sweet orange, and slightly higher than that of the monoembryonic Banpeiyu pummelo, such as the REV of monoembryonic sweet orange MS was 7.7 times that of polyembryonic Flying dragon trifoliate orange. In addition, The CitRWP expression value was not significantly different between monoembryonic and polyembryonic sweet oranges. REV of Hamlin (the highest in polyembryonic sweet orange) was 3.36 times that of Licheng No.2 (the lowest REV in monoembryonic sweet oranges), the REV of Tarocco blood orange was only 1.1 times that of monoembryonic MS sweet orange (Fig. 4). These results showed that the expression of CitRWP might not directly related to embryonic phenotypes.
The ovule in citrus and its close relative genus is very tiny during flowering time and young fruit stage. It is difficult to get enough pure ovule for experiment quickly and safely. So we carried out a correlation analysis on the expression of ovule and ovary at the same period. The result showed that the CitRWP expression of ovary was correlated with the ovule positively significantly (Fig. 6). Therefore, the expression in ovary was detected in the offspring population of W-Murcott × Flying dragon trifoliate orange. The analysis found that the CitRWP REV in W-Murcott was 6.43 times that of Flying dragon trifoliate orange, and the REV of Wangcang daye trifoliate orange and 74 − 1 trifoliate orange were similar to that of Flying dragon trifoliate orange. In the hybrid offspring population, the REV of CitRWP gene was an extreme two-stage differentiation, there were 5 high-expressing progenies, 4 progenies of which were higher than their female parent W-Murcott, while 9 low-expressing progenies lower than their male parent Flying dragon trifoliate orange. The REV of high-expressing progenies was ten or even hundreds of times that of low-expression. Among the tested progenies, the high-expressing lines had been recorded as polyembryony in the embryony investigation, and the low-expression lines was monoembryonic, which indicated that the REV of CitRWP might have correlation with embryonic phenotype in the W-Murcott × Flying dragon trifoliate orange hybrid offspring population (Fig. 7).
In addition to the test of CitRWP expression in ovule, we also tested the expression of different tissues or organs such as young stem, young leaf, old leaf, and ovary of mono/polyembryony sweet orange. Among the polyembryonic Hamlin and Tarocco blood orange, the young leaf had the highest expression value which was slightly lower than its ovule. However, the expression value of leaf decreased significantly after maturity. REV in young leaves of Hamlin and Tarocco blood orange was 2.5, 3.2 times that of its old leaves, respectively. Among the monoembryonic sweet orange, the ovule was the part with highest expression, and the young leaf expression value had no significant difference with the young stem, old leaf, and ovary (Fig. 8). CitRWP expressed in vegetative tissue, especially the young leaf in polyembryonic orange had the highest REV, indicating that CitRWP was not specifically expressed in reproductive tissue.
Cloning and sequencing of citRWP from different embryonic materials
cDNAs of CitRWP were cloned and sequenced from ovules of 3 polyembryonic Poncirus cultivars, 2 mono/polyembryonic sweet orange selections and 2 monoembryonic pummelos. CDS sequence of Cs4g05960 in sequenced sweet orange  was used as standard to compare with the cloned citRWP sequences. Three types of major variations were observed in the obtained sequences of polyembryonic Poncirus (Fig. 9). These variations appeared in almost all Poncirus cultivars with a high rate (Table. 1). The type I variation was the deletion of a 106 bp fragment at the 406 bp position (relative to the translation start codon, the same hereinafter) and this deletion caused the CDS length to change from 1065 bp to 959 bp. The primers used for RT-PCR were found to locate in this region, so it might be one of the reason that caused a lower REV in polyembryonic Poncirus. 8 in 18 clones presented the 106 bp fragment deletion mutation. The type II variation was a 5-base (-TGCAG-) insertion mutation at the 774 bp position, which caused the CDS length changed from 1065 bp to 1070 bp. This variation presented in 8 sequenced clones. Two types of the variations coexisted in 2 sequenced clones and the CDS length of these sequences was 964 bp. The length of these three CDS sequences was not integral multiple of triplet codons, so they could not be translated normally. The type III variation was mutations in initiation codon and termination codon. The key codon changed also lead to mistranslation of functional protein. In general, the 18 sequenced samples of Poncirus, only 3 sequenced clones could be normally translated, and the normal translation rate was only 16.7%.
Less variations were found in cloned citRWP cDNA sequences from 2 sweet orange cultivars and 2 pummelo cultivars. The type I variation of a 106-base deletion was found only in one sequenced clone from Banpeiyu pummelo. The type II variation of a 5-base (-TGCAG-) insertion was found only in four sequenced clones, one from polyembryonic Tarocco blood orange and three from monoembryonic sweet orange TD. No type III variation in initiation codon and termination codon was found. The overall normal translation rate was 69.6% in sweet orange and pummelo, which was higher than that in Poncirus. In addition, compared with Cs4g05960, there are 5 SNP sites in the sequenced pummelo clone, but they are all synonymous mutations and do not change the encoded amino acids.